Mutational Profile of Human Papilloma Virus (HPV) Induced and Non-HPV Induced Head and Neck Squamous Cell Carcinoma

Abstract

Head and Neck cancer accounts for approximately 900,000 cases and over 400,000 deaths annually worldwide. The primary risk factors associated with Head and Neck cancer include usage of tobacco, alcohol consumption, Human Papillomavirus (HPV) infection and Epstein-Barr virus (EBV) infection. Few subsites of Head and Neck Squamous Cell Carcinoma (HNSCC) are associated with Human Papilloma Virus (HPV) while others remain non-associated. The anatomical, physiological, genetic, protein profile and epigenetic changes that occur in both HPV-positive and HPV-negative HNSCC has been discussed in this chapter. The mutational profile plays a crucial role in the treatment of the HNSCC patients as the HPV-positive HNSCC patients have a better prognosis compared to the HPV-negative HNSCC patients. This chapter mainly focusses on the mutational profile of both HPV-associated and non-HPV associated HNSCC tumours.

1. Introduction

Head and Neck Squamous Cell Carcinoma (HNSCC) contribute to substantial morbidity and mortality worldwide, with an estimated 526,481 incident cases annually [1]. HNSCC arise from the mucosal epithelium of oral cavity, pharynx and larynx. . Apart from the prime etiologic factors like environmental carcinogens and carcinogenic viruses, genetic predisposition plays a risk-modulating role [2] in which the large burden of mutations lead to the heterogeneity of the tumour. Human Papilloma Virus (HPV) is a well-known risk factor for malignant transformation and is increasingly associated with the majority (60–70%) of the recently diagnosed oropharyngeal cancer incidences. Majority of the HPV-induced Oropharyngeal cancers harbour high risk HPV16 primarily and to a lesser extent HPV18 and other strains of HPV [3]. In Human papilloma virus induced tumourigenesis, HPV derived oncoproteins E6 and E7 inactivate the tumour suppressor genes p53 and pRb (retinoblastoma), resulting in the onset and eventual progression to malignancy [4]. HPV-associated HNSCC cells are poorly differentiated, non-keratinizing, and have a distinct ‘basaloid’ appearance in contrast to the non-HPV associated HNSCC which are usually moderately differentiated and keratinizing [5]. As the HPV DNA integrates into the host cell genome in a large proportion of HPV associated HNSCC, the tumours of HPV positive HNSCC differ at their genetic level [6]. Compared to the HPV negative HNSCC, HPV- associated HNSCCs manifest lower levels of chromosomal mutations [7]. Southern blotting, Polymerase Chain Reaction (PCR) and its variations like Reverse transcriptase and Real Time PCR, in situ hybridization, immunohistochemical staining for p16, immunostaining with anti E6, E7 antibosies, PCR in situ hybridization (PISH) are some of the techniques used for the detection of HPV in the Head and Neck cancers [8]. HPV positive HNSCC patients with lymph node metastases exhibit improved loco-regional control showing regression more quickly. They are more likely to resolve better after treatment when compared to the lymph node metastases of HPV negative HNSCC patients. Patients with HPV associated HNSCC have a better survival rate over HPV negative HNSCC patients with a 58% reduction in mortality risk [9].

2. Differences between HPV-positive and HPV-negative Head and Neck Squamous Cell Carcinoma (HNSCC)

2.1 Anatomical differences

The Head and Neck cancers arise from the tumours of mucosal epithelium in the oral cavity (lips, hard palate, buccal mucosa, floor of mouth, anterior tongue, and retromolar trigone), nasopharynx, oropharynx (palatine tonsils, lingual tonsils, soft palate, base of tongue, uvula and posterior pharyngeal wall), hypopharynx and larynx. Tumour growth in the oral cavity, hypopharynx and larynx is due to tobacco consumption and continuous alcohol abuse whereas, cancers in the pharynx (from the palatine and lingual tonsils of the oropharynx) are increasingly attributed to infection with Human Papillomavirus (HPV), primarily HPV-16, 18 and also other HPV strains. Therefore, the Head and Neck Squamous Cell Carcinoma (HNSCC) can be grouped into HPV-negative and HPV-positive [10]. The primary site of HPV-positive groups is the oropharynx (51%) and various other sites like larynx (11%), oral cavity (9%), nasopharynx (9%), and pharynx (5%) also encompass HPV positive tumours [11]. HPV is an epithelium-specific infection that does not spread though the bloodstream. Consequently, a limitation of HPV serology is that it does not specify the anatomic site of HPV infection. Hence, the elevated odds of oral cancer observed in association with oral HPV infection are considered more strong evidence for a direct relationship between HPV infection and oral cancer. The data obtained from risk factors associated with sexual behaviour, HPV exposure and oral HPV detection indicate that sexually acquired oral HPV infection is the principal risk factor for many cancers arising from the oral cavity. Based on the research findings, there is apparent evidence on the HPV transformation within the oral cavity, the tonsillar crypt epithelium, ectopic tonsillar tissue in the lateral posterior tongue or floor of mouth. This is approximated to occur in 0.4 per 100,000 individuals. These findings prove that the oral cavity is an intended site for HPV positive tumours [12, 13]. An international case-control study has estimated that HPV plays a part in approximately 3% of oral cavity cancers [14].

2.1.1 Physiological factors in HPV positive and negative HNSCC

The HPV positive HNSCC is made up of highly malignant cells that have a high nuclear to cytoplasmic ratio and exhibit little or no keratinization. These cells differ from the non-neoplastic squamous epithelium that lines the oral cavity. The HPV related cancers mostly arise in the reticulated epithelium-the specialized epithelium lining the tonsillar crypts. So, the HPV-related oropharyngeal cancers remain highly differentiated. The HPV negative HNSCC are of a heterogeneous group of benign and malignant lesions characterized by small tumour cells with round or ovoid nuclei surrounded by a thin rim of cytoplasm. On the other hand, HPV-related HNSCC encompasses basaloid cells. These cells exhibit lobular growth with dense hyperchromatic nuclei and a high nuclear to cytoplasmic ratio [15]. A recent study has shown that the “basaloid” subtype is, in fact, composed of a mixed group of HPV-positive and HPV-negative cancers that widely differ with respect to clinical behaviour [16].

2.1.2 Risk factors

Various epidemiological studies have revealed a diverse range of HNSCC associated risk factors. These risk factors include tobacco consumption, alcohol abuse, exposure to environmental pollutants and infection with viral agents namely, Human Papilloma Virus and Epstein Barr Virus (EBV). Certain risk factors show geographical, cultural and habitual prevalence [10]. Among the Asian population, oral cavity cancer is attributed to chewing of areca nut products including ‘betel quid’-variety of customized mixtures comprising areca nut (Areca catechu, the carcinogen source), betel leaf (the leaf of Piper betel), slaked lime and/or tobacco, as well as spices according to local custom [17]. In common, the high male to female ratios for HPV-negative HNSCC incidence reflects the sex-specific patterns of variable risk behaviours, including the use of the aforesaid tobacco, smokeless tobacco, areca nut, betel quid and alcohol [17]. The additional risk factors that contribute to HNSCC include ageing, poor oral hygiene and diets lacking in vegetables [18]. In terms of the infectious agents that causes HNSCC, continuous infection with HPV and EBV can cause a rise in the cancers of Oropharynx and Nasopharynx [10]. The HPV infection that leads to HNSCC is mainly transmitted by oral sex and the occurrence of HPV-positive HNSCC continues to increase, especially in populations that are not vaccinated against HPV prior to HPV exposure [19]. Certain genetic factors have also been reviewed to contribute to HNSCC risk. Individuals with Fanconi anaemia, a rare, inherited genetic disease characterized by impaired DNA repair, have a 500- to 700-fold increased risk of developing HNSCC, primarily in the oral cavity [10].

2.1.3 Genetic differences

Frequent loss of chromosome arms 3p, 9p and amplification of 11q13 chromosomal region are observed in HNSCC. The key genes which are reported to be mutated by comprehensive genomic sequencing studies for Head and Neck squamous cell carcinoma are TP53, PIK3CA, PTEN, FBXW7, HRAS. The composition of chromosomal aberrations and mutations differs between HPV-positive and HPV-negative HNSCC. The TP53gene is the most frequently mutated gene (41%), and this gene was not detected in the HPV-positive subgroup. PIK3CApathway is the frequently mutated oncogenic pathway in Head and Neck squamous cell carcinoma. Mutations in the PIK3CApathway are slightly higher in the HPV-positive Head and squamous cell carcinoma. Mutations in PIK3CAand PTENgene occur in both HPV-positive and HPV-negative patients but with slightly higher rates in HPV-positive patients [11]. Certain chromosomal aberrations include loss of 16q or 16q24.3, 5q35.1 or 17p12, 3q26.3. Loss of 9p21 containing the tumour suppressor gene CDKN2Ais discerned in HPV negative HNSCC. The amplification of chromosome 7 is mutually exclusive for HPV-negative tumours [12]. CpG transversions are observed in HPV-negative HNSCC while TpC mutations are identified in HPV-positive HNSCC.

2.1.4 Protein expression

The protein expression alterations in HPV- positive and HPV-negative groups showed a low expression of biomarker proteins such as MGMT, EGFR, and PD1-positive TILs in HPV positive and negative patients. Overexpression of EGFR protein is reported in HNSCC resulting in treatment resistance, aggressive clinical behaviour, and poor prognosis. The immunomodulatory protein PD-1 positivity occurs with highest frequency in pharyngeal cancers and PDL1 levels are detected in higher levels in nasopharyngeal cancers [11]. Patients with HPV positive tumours ensues abrogation of p53 and retinoblastoma (Rb) genes. The downregulation of the Rb gene results in the upregulation of p16 oncoprotein. The p16 oncoprotein is considered a biomarker for HPV-related HNSCC, where it is overexpressed. The minichromosomal maintenance protein 7 (MCM7) is expressed in high levels in HPV-positive head and neck cancer [19]. The p21 protein expression is identified in the HPV related tonsillar cancer. Reduced expression of p21 results in E6-mediated p53 inactivation. Outcomes from other studies indicate that E7 bypasses the inhibitory effect of p21 on cell cycle progression [20, 21]. Survivin (Baculoviral IAP repeat-containing protein 5) is negatively regulated by p53. This protein represses apoptosis and plays a role in cell division. Nuclear survivin expression is associated with a poor disease-free survival rate and negative HPV status in OPSCC [22]. Thioredoxin (TRX) (redox mediator promoting cell survival) and epidermal fatty acid binding protein (E-FABP) involved in keratinocyte differentiation and other cellular signaling processes were perceived to be upregulated in HPV-related tumours and their role in HPV-related OSCC. Several cell surface glycoprotein molecular biomarkers such as CD44, CD133, ALDH1 occurs in elevated level in HNSCC. The HNSCC cells with high levels of CD44 glycoproteins are capable of self-renewal. CD44 levels in HNSCC tumours are associated with metastasis and a poor prognosis [23, 24]. CD133 glycoproteins results in increased invasiveness and metastasis in HNSCC. The increased levels of ALDH1 causes self-renewal, invasiveness and metastasis in HNSCC (Figures 1–3 and Table 1) [25].

Figure 1.

The Genomic Map of HPV 16.

Figure 2.

Molecular events in HPV carcinogenesis.

Figure 3.

HPV transformation via Tonsillar crypt.

HPV proteins	Functions
Early proteins
E1	Initiation of viral genome replication.
E2	Viral DNA replication and transcription. Segregation of viral genomes.
E4	Viral genome packing. Maturation of viral particles.
E5	Oncoprotein. Participates in host cell transformation and blocks apoptosis in late events of HPV carcinogenesis.
E6	Major oncoprotein. Inactivates p53 protein. Block apoptosis. Interacts with many host proteins with PDZ domains.
E7	Major oncoprotein. Inactivates pRb protein. Promotes host DNA synthesis and proliferation. Interacts with many host proteins
Late proteins
L1	Major capsid protein, Viral replicating proteins
L2	Minor capsid protein. Viral replicating proteins

Table 1.

HPV proteins and functions.

2.2 Mutational profile of head and neck squamous cell carcinoma (HNSCC)

2.2.1 Common genetic alterations in HNSCC

The common cytogenetic changes observed in head and neck cancers are losses of segments of 3p, 5q, 8p, 9p, 10p, and 18q and gains of segments within 3q, 5p, 7p, 8q, distal 1q, and 11q13–23 regions.

Amplifications of 11q13 and 7p11 regions encoding Cyclin D1and EGFRrespectively are noted in HNSCC [26]. Telomerase Reverse Transcriptase (TERT) found on chromosome 5p and MYConcogene on 8q region of the chromosome exhibits additional amplification in both HPV positive and HPV negative HNSCC [27]. Portions of chromosomes 3P and 8P which encompasses the tumour suppressor genes FHITand CSMD1respectively are deleted in HNSCC [28].

The microRNA let-7c, a cell cycle regulator, is frequently inactivated in both HPV negative and HPV positive HNSCC. Decreased expression of let7-c is linked with increased expression of CDK4, CDK6, E2F1 and PLK1 kinases and translational regulators important for advancement through the cell cycle [29].

2.2.2 Genetic alterations in HPV associated HNSCC

Molecular heterogeneity has been found to exist within HPV (+) tumours themselves. High rate of proliferation and increase in genomic instability is associated with HPV integration [30]. Human papilloma virus induced tumourigenesis occurs predominantly in the oropharynx region (tonsil or base of tongue), where HPV acquired oncoproteins E6 and E7 inactivate the tumour suppressor genes p53and pRb(retinoblastoma), resulting in the inception and eventual progression to malignancy [31].

HPV positive HNSCC are characterized by wild-type TP53. High-risk types HPV encode two viral oncoproteins namely E6 and E7 that aid tumour progression by inactivating the two well-characterized tumour suppressor proteins TP53 and RB1, respectively. Un-phosphorylated RB1 plays a crucial role in the negative regulation of cell proliferation, generating cell cycle arrest in mid to late G₁. Wild-type TP53 behaves as a cell cycle checkpoint after DNA damage and induces G₁ arrest or apoptosis, essential to conserve the genomic stability [32]. However, HPV-associated cancers normally do not manifest TP53mutations

PIK3CA(protein kinase C), an anti-apoptotic kinase and transcription factors TP63and SOX2located on chromosome 3q are among the most frequently amplified regions in HPV associated Head and Neck Squamous Cell Carcinoma(HNSCC) [33]. PIK3CAencodes the p110α catalytic subunit of phosphinositol-3-kinase. Regulation of signal from multiple input sources including many of the receptor tyrosine kinases (RTKs) relevant to HNSCC is advocated by PIK3CAthrough phosphorylation of AKT1. Mutated PIK3CAhas been shown to impair apoptotic signals and support tumour invasion. Additionally, mutational turned on PIK3CAhas been shown to assist cyclin D activity, further emphasizing cell cycle deregulation in head and neck cancers [34].

Meagre or no EGFRgene amplification and EGFRprotein expression has been observed in HPV-positive HNSCC [35]. HPV (+) tumours manifest infrequent mutations in TP53gene.

Truncating mutations are observed in TNF receptor-associated factor 3 (TRAF3) gene which is implicated in anti-viral responses (innate and acquired) of Human Papilloma Virus (HPV) [36]. TRAF3region is absolutely lost in about 20% of HPV-associated tumours. Tumour necrosis factor Receptor-Associated Factor 3 (TRAF3, encoded on 14q32.32) is involved in the innate and acquired antiviral immune response in HPV positive HNSCC. Deletion or mutations in TRAF3genes is over-represented in HPV-related HNSCC. As genes coding for HLA I components are frequently mutated and higher numbers of CD56-positive natural killer cells have been reported for HPV-related Oropharyngeal Squamous Cell Carcinoma recently innate immunity seems to be eloquent for HPV-related HNSCC [37].

Amplification of E2F1 region which is necessary for cell cycle initiation and proliferation and an intact 9p21.3 region containing the CDKN2A gene are observed in the HPV positive HNSCC [38].

TpC mutations were observed predominantly in HPV associated HNSCC patients during the whole exome sequence analysis. These TpC mutations lead to APOBEC mutational signature in HPV positive HNSCC [38]. Overexpression of APOBEC enzymes in HPV-associated tumours may be linked to increased cytosine deaminase mutation [39]. Genes encoding HLA I components are frequently mutated in HPV positive Oropharyngeal Squamous Cell Carcinoma (OPSCC) [39]. Sewell et al. [40] in 2014 screened eleven DNA repair proteins which included BRCA2, PARP-1, and MSH2 ATMand observed all of them to be upregulated in HPV positive OPSCC samples compared to the HPV negative OPSCC samples.

2.2.3 Inactivating mutations in HPV-positive HNSCC

Segregation of four genes are observed as inactivating mutations in HPV positive tumours of which two genes CDKN2Aand TP53are associated with survival and cell cycle and two genes FAT1and AJUBAwith Wnt/b-catenin signalling [41]. A higher rate of TP53 mutations are observed in HPV positive HNSCC compared to non-HPV associated HNSCC.

2.2.4 Genetic alterations in non-HPV associated HNSCC

HPV negative HNSCC tumours features novel co-amplifications of 11q13 (CCND1, FADDand CTTN) and 11q22 (BIRC2and YAP1), which also contain genes implicated in cell death/NF-κB and Hippo pathways. They also emphasize novel focal deletions in the nuclear set domain gene (NSD1) and tumour suppressor genes like FAT1, NOTCH1, SMAD4and CDKN2A. Recurrent focal amplifications in receptor tyrosine kinases like EGFR, ERBB2and FGFR1also predominate in HPV negative HNSCC tumours [38].

Cyclin-dependent kinase inhibitor 2A (also known as p16 INK4A) that is encoded by CDKN2Agene located at 9p21, is frequently inactivated viacopy number loss among HPV-negative head and neck cancer patients and is involved in the HNSCC pathogenesis. CDKN2Aregulates cell cycle progression by obstructing the activity of CCND1(cyclin D1) and its related kinases, CDK6 and CDK4. These kinases are involved in the phosphorylation and inactivation of the tumour suppressor gene RB1 CDKN2Ahinders cell cycle progression at the G1 to S check point by preventing the phosphorylation of the retinoblastoma protein (RB1). Deletion, mutation or hypermethylation of CDKN2Ais frequent in HPV negative HNSCC and is associated with worse prognosis in these Head and Neck cancers [34]. On the other hand, CDKN2Aoverexpression has been correlated with improved outcome in Oropharyngeal Squamous Cell Carcinoma. This occurs as an outcome of functional inactivation of RB1by the HPV E7 protein, resulting in the upregulation of CDKN2A[42]. Thus, HPV positive HNSCC are characterized by high expression of CDKN2A, implying that CDKN2Apositivity may be a biomarker for tumours harboring HPV infections [42]. Inactivation of the CDKN2Agene has been found in 57% of HPV negative HNSCC cases in the TCGA cohort [38].

The transmembrane receptor protein NOTCH1is involved in cell proliferation, differentiation, cell fate determination and self-destruction Additionally, Notch plays a decisive role in angiogenesis, crucial for the maintenance and progression of tumourigenesis. NOTCH1 inactivation has been inferred in about 15% of the HNSCC tumours. Most NOTCH1 mutations in HNSCC are considered inactivating, attributing its role as a tumour suppressor gene. On the contrary, cohorts from Asian HNSCC population have demonstrated activating NOTCH1 mutations. NOTCH activity in HNSCC is therefore circumstantial and NOTCH is considered to have a bimodal role as a tumour suppressor and an oncogene in HNSCC. HNSCC with NOTCH1 mutations have a worse prognosis than the NOTCH1 wild-type tumours. Most studies reveal that NOTCHpathway is upregulated in HNSCC and NOTCHexpression shows convincing relationship with the clinical stage [43]. The NOTCHpathway can play an influential role in HNSCC development, and anti-NOTCHtherapy can be attractive. NOTCH1is observed to be more mutated in HPV-negative HNSCC than in HPV-positive HNSCC [44]. Higher expression of NOTCH1is displayed in HPV-positive HNSCC compared to HPV-negative HNSCC.

The region of epidermal growth factor receptor (EGFR) gene is 7p12 and it encodes for a 170-kD transmembrane glycoprotein. It is a member of the receptor protein tyrosine kinase family with several extracellular growth factor ligands, comprising of epidermal growth factor (EGF) and transforming growth factor (TGF)-α. About 42–80% of HNSCC studied has overexpression of EGFR [26], and 30% of HNSCC tumours have been discerned to harbor EGFRgene amplification. Increased EGFR expression and gene copy number are associated with poorer patient outcomes in HNSCC.

Mutations in genes NFE2L2(encoding NRF2) and KEAP1occur exclusively in HPV-negative HNSCC. These two genes are known key regulators of oxidative stress. CpG transversions are frequent in HPV-negative HNSCC.

H-RASmutation is detected in about 35% of Indian oral cancer patients. This gene has been associated with betel nut chewing and so observed in HPV-negative HNSCC. Also, somatic mutation at codon 12 of K-rasgene makes the K-rasprotein hyper active, leading to uncontrolled signalling for cell division (Tables 2 and 3) [45].

TCGA [38]		Seiwert et al. [46]		Stransky et al. [47]		Agrawal et al. [43]
HPV (+ve)	HPV (−ve)	HPV (+ve)	HPV (−ve)	HPV (+ve)	HPV (−ve)	HPV (+ve)
E6/E7 (100%)	TP53 (84%, M)	E6/E7 (100%)	TP53 (81%, M)	E6/E7 (100%)	TP53 (73%, M)	E6/E7 (100%)
PIK3CA (56%, M/A)	CDKN2A (57%, M/D)	PIK3CA (22%, M)	CDKN2A (33%, M/D)	PIK3CA (27%, M)	CDKN2A (25%, M/D)	EPHB3 (25%, M)
TP63 (28%, A)	let-7c (40%, miRNA)	TP63 (16%, M/A)	MDM2 (16%, A)	RUFY1 (18%, M)	SYNE1 (22%, M)	UNC5D (25%, M)
TRAF3 (22%, M/D)	PIK3CA (34%, M/A)	PIK3CB (13%, M/A)	MLL2 (16%, M)	EZH2 (18%, M)	CCND1 (22%, A)	NLRP12 (25%, M)
E2F1 (19%, A)	FADD (32%, A)	FGFR3 (14%, M)	NOTCH 1 (16%, M)	CDH10 (18%, M)	MUC16 (19%, M)	PIK3CA (25%, M)
let-7c (17%, miRNA)	FAT1 (32%, M/D)	NF1/2 (12%, M)	CCND1 (13%, A)	THSD7A (18%, M)	USH2A (18%, M)	TM7SF3 (25%, M)
NOTCH1/3 (17%, M)	CCND1 (31%, A)	SOX2 (12%, A)	PIK3CA (13%, M)	FAT4 (18%, M)	FAT1 (14%, M)	ENPP1 (25%, M)
FGFR3 (11%, F/M)	NOTCH1/2/3 (29%, M/D)	ATM (10%, D)	PIK3CB (13%, M/A)	KMT2D (18%, M)	LRP1B (14%, M)	NRXN3 (25%, M)
HLA-A/B (11%, M/D)	TP63 (19%, A)	FLG (12%, M)	UBR5 (13%, M/D)	ZNF676 (18%, M)	ZFHX4 (14%, M)	MICAL2 (25%, M)
EGFR (6%, M)	EGFR (15%, M/A)	MLL3 (10%, M)	EGFR (12%, A)	MUC16 (18%, M)	NOTCH1 (13%, M)	—

Table 2.

Genes altered in HPV-positive and HPV-negative HNSCC.

M, mutation; A, amplification; D, deletion; F, fusion.

Lin et al. [48]	Pickering et al. [49]		Pickering et al. [50]
Nasopharyngeal cancer (NPC)	Tongue		Oral squamous cell carcinoma (OSCC)
	Young tongue	Old tongue
CDKN2A/B (13%, M/D)	TP53 (94%, M)	TP53 (57%, M)	CDKN2A (74%, D)
ARID1A (11%, M/D)	CSMD1 (25%,D)	CSMD1 (75%,D	TP53 (66%, M)
SYNE1 (8%, M)	PIK3CA (0%, M); (30%A)	PIK3CA (11%, M); (70%, A)	FAT1 (46%, M/D)
ATG13 (6%, M/D)	CDKN2A (6%, M); (55%, D)	CDKN2A (4%, M); (65%, D)	TP63 (26%, A)
MLL2 (6%, M)	FADD/CCND1 (40%, A)	FADD/CCND1 (65%, A)	CCND1 (23%, A)
PIK3CA (6%, M/A)	FAT1 (6%, M); (50%, D)	FAT1 (25%, M); (35%, D)	MAML1 (23%, D)
CCND1 (4%, A)	EGFR (20%, A)	EGFR (50%, A)	EGFR (17%, A)
NOTCH3 (4%, M)	NOTCH1 (25%, M)	NOTCH1 (18%, M)	TNK2 (17%, A)
FGFR2 (4%, M)	HLA-A (0%, M)	HLA-A (14%, M)	AKT1 (14%, A)
TP53 (17%, M/D)	CASP8 (6%, M)	CASP8 (11%, M)	SRC (14%, A)

Table 3.

Altered genes in different anatomic sites of HNSCC irrespective of HPV infection.

M, mutation; A, amplification; D, deletion; F, fusion.

2.3 Epigenetic alterations in HPV-positive and HPV-negative HNSCC

Epigenetic events of HNSCC include DNA methylation, chromatin remodelling, histone posttranslational covalent modifications and effects of non-coding RNA. Epigenetics sway silencing of tumour suppressor genes by promoter hypermethylation, regulate transcription by microRNAs and changes in chromatin structure, or induce genome instability through hypomethylation. Most of the HNSCC are caused by hypomethylation of the promoter genes or retrotransposons. Lower methylation of retrotransposons elements such as LINE (long interspersed elements) and SINE (short interspersed elements) causes the initiation of tumour in HNSCC. It is also reported that hypomethylation is concerned with tongue squamous cell carcinoma (TSCC) among the female gender [51]. The hypomethylation of Alu, one of the SINEs, is reported in the oral cancer patients of Asian population in the advanced stages of cancer [52]. Further, patients with severe malignant oral carcinogenesis are associated with hypomethylation of LINE sequences [53]. The hypermethylation in HNSCC implicate a high level of methylation in promoters of genes, which is a characteristic feature for epigenomes of cancer cells. Hypermethylation of certain genes such as CDKN2A, PTEN, DAPK, MGMT, ECADand RASSF1are frequently observed in HNSCC [54]. This increased methylation is associated with well-differentiated tumours and with patient age less than 50 years in OSCC among Indian population [55]. There occurs difference in methylation status among HPV-positive and HPV-negative patients. Studies have reported that HPV infection causes aberrant hyper or hypo methylation of genes. The study reports obtained from genome-wide methylation data from different cohorts showed that HPV infection affects DNA methylation in HNSCC across different anatomic sites. Only a few hypomethylated genes have been reported in HNSCC cases that are HPV infected. Two miRNAs, miR-875 and miR-3144 found in E6 gene, inhibit E6 oncogene expression, and in HPV16-positive cell lines inhibit the growth and promote apoptosis by high-level expression of both miRNAs (Table 4) [56].

Mirghani et al. [60]	Hui et al. [61]	Gao et al. [62]	Lajer et al. [63]	Gao et al. [64]
miR-324-5p	miR-324-5p		miR-324-5p
	miR-155	miR-155	miR-155
miR-107	miR-107
	miR-9	miR-9
	miR-145			miR-145
miR-99b-3p			miR-99b-3p
miR-18a-5p		miR-18a-5p
			miR-26b	miR-26b
			miR-363	miR-363
	miR-381			miR-381
	miR-101			miR-101

Table 4.

Deregulated miRNAs in HNSCC irrespective of HPV infection.

3. Pathways involved in HNSCC

3.1 EGFR pathway

In HNSCC, activation of EGFR is executed by binding of ligands such as EGF, amphiregulin, and transforming growth factor alpha-TGFα. Ligand binding provokes receptor dimerization (homo or hetero dimerization with other EGFR members), leading to phosphorylation of tyrosine residues. This leads to sequential activation of various signalling cascades like Ras/Raf/mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K)-Akt, signal transducer and activator of transcription pathways. Phosphorylated MAPK translocates into the nucleus, phosphorylating various transcription factors that trigger the expression of distinct target genes, which advocates proliferation, differentiation, migration, invasion, angiogenesis and metastasis in HNSCC cells. Aberration of EGFR signal activation can bring about disruption of cancer cell homeostasis [57, 58, 59].

3.2 PI3K-AKT mTOR pathway

Activated by the receptor-associated tyrosine kinases (RTKs) such as EGFR, the catalytic subunit phosphorylates phosphatidylinositol 1, 4-bisphosphate (PIP2) to phosphatidylinositol 1, 4, 5-triphosphate (PIP3). PIP3 recruits proteins like phosphoinositide-dependent protein kinase 1 (PDK1) and AKT to the plasma membrane, resulting in the phosphorylation of AKT by PDK1 and mammalian target of rapamycin complex 2 (mTORC2). Activated AKT and mTORC1 in turn activates the eukaryotic translation inhibition factor 4E-binding protein 1 (4E-BP1), resulting in cell growth, protein synthesis, and proliferation of HNSCC. The tumour suppressor phosphatase and tensin homology (PTEN) negatively regulates the cellular level of PIP3 by converting it to PIP2 through its lipid phosphatase activity thereby negating the activation of AKT and its downstream pathways. More than 80% of mutations occur in exon 9 (Helical domain) and exon 20 (Kinase domain) through gene amplification mechanism and increase in low-level copy number. More invasive forms of HNSCC have been proclaimed to harbour copy number increase in 3q26 region and engage in vascular invasion and lymph node metastasis. Oncogenic PIK3CA mutations are common particularly in HPV-positive head and neck cancers. PIK3CA mutations may combine with E6 and E7 proteins of HPV in the evolution of invasive OPSCC [57, 58, 59].

3.3 p53/Rb/CDKN2A/CCND1 pathway

In HNSCC, TP53 has been linked with the risk of progression from mild dysplasia to invasive carcinoma. P53 level is determined by MDM2, which by ubiquitination degrades p53. Contrarily, p14 and p16 encoded by CDKN2A inhibits MDM2 and shields p53 from degradation. RB inhibits E2F transcription factor from progressing into the cell cycle. Cyclin and cyclin-dependent kinases (CDK) like D1/CDK4/CDK6 are activated by mitotic signals which leads to the inactivation of RB via phosphorylation. p21 (CDKN1) and p16 (INK4A/MTS1/CDKN2) encoded by CDKN2A inhibits Cyclin D1-CDK4/6 complex. Phosphorylation of RB results in release of E2F and cell cycle progresses to S, G2 and M phases. Inactivation of p53, RB, p16 and p14 through mutation, deletion or epigenetic silencing and overexpression of cyclin D1 (CCND1 gene), MDM2 and CDK4 have been associated with tumorigenesis and reduced survival in HNSCC. HPV infection can inhibit the activation of p53 and RB in HNSCC. Seven early proteins (E1–E7) and two late capsid proteins (L1 and L2) are encoded by HPV genome.

HPV E6 combines with E6-associated protein (E6-AP) and endorses p53 ubiquitin proteasome degradation. For binding to RB, HPV E7 protein encounters with E2F. As RB acts as a negative regulator for the cyclin-dependent kinase inhibitor p16, overexpression of p16 has been established to be of great clinical value in determining the HPV-positive status of the tumours using immunohistochemistry (IHC) (Figure 4) [57, 58, 59].

Figure 4.

EGFR, PI3K-AKT- mTOR, p53/Rb/CDKN2A/CCND1 pathways.

3.4 NOTCH pathway

The NOTCH family consists of four receptors (NOTCH1-4) adhered to the cell membrane. They are activated by two families of ligands, namely, Delta-like (Dll1, DllL3, Dll4) and Jagged (Jag1 and Jag2). Binding of ligands to NOTCH receptors persuade NOTCH cleavage by TNFα-converting enzyme (TACE) (ADAM metalloprotease) and γ-secretase, which results in the release of NOTCH intracellular domain (NICD). NICD associates with CSL/MAM complex, binds to DNA and promotes transcription. NOTCH pathway is a conserved signal transduction cascade which alters cell function such as cell differentiation, survival and self-renewal capacity. Notch activity has been associated with the suppression of HPV E6 and E7 protein expression, leading to for loss of Notch in HPV+ HNSCC. NOTCH1 signalling stimulates terminal differentiation of keratinocytes and it is negatively regulated by EGFR pathway (Figure 5) [57, 58, 59].

Figure 5.

NOTCH pathway.

4. Conclusion

Based upon the research studies till date there is a clear evidence portraying that the high risk HPV types are well known for causing Head and neck squamous cell carcinoma. The studies have also proved the HPV Viral infection within the different anatomic sites; among the different anatomic sites the oropharyngeal region has a major impact of getting huge amount of viral load thus causing HPV infection. The infected virus further initiates transformation process within the oropharyngeal region such as the oropharynx (51%), pharynx (5%), and oral cavity (9%). The viral makeover within the oral cavity occurs in the tonsillar crypt epithelium and integrates within the human genome. Estimates have shown that there accounts huge amount of HPV viral-cellular entry within the tonsillar crypt epithelium. Several studies have revealed that there occurs physiological differences between HPV-positive and HPV-negative HNSCC, thus differing with respect to clinical behaviour. Certain risk factors influence HPV-positive and HPV-negative HNSCC. The HPV-negative oral cavity cancer is attributed to chewing of areca nut products, betel leaf (the leaf of Piper betel), slaked lime and/or tobacco. Smoking is also contributed to causing HPV-negative HNSCC. The HPV-positive risk factors include continuous infection with HPV and EBV which usually arise in the cancers of Oropharynx and Nasopharynx. HPV infections occur in higher rate mainly due to oral sex, and people who have not been vaccinated. Research findings have revealed that there occurs difference in genes being mutated in HPV-positive and HPV-negative HNSCC. The most common genes mutated within HPV-positive and HPV-negative HNSCC include TP53, PIK3CA, PTEN, FBXW7, HRAS. Among these the TP53has the highest mutations in HPV-negative HNSCC and in HPV-positive HNSCC the E6/E7 viral proteins has the highest integration in the human genome, the PIK3CAgene has a profound mutation levels in HPV-positive HNSCC. Studies on the epigenetic alterations in HPV-positive and HPV-negative HNSCC reveal differentially expressed miRNAs. The methylation characteristics of HNSCC illustrate a variation in hypermethylation and hypomethylation levels in HPV-positive and HPV-negative HNSCC. Further the methylation status differs among the anatomic sites of HNSCC. The integration of HPV within human genome causes an aberrant expression of proteins among the different anatomic sites. It has been reported that the viral proteins E6 suppresses p53 gene and E7 suppresses Rb gene. These two genes are involved in several normal regulatory cell cycles. Patients with HPV positive and Tobacco-associated HNSCC ensues abrogation of p53 and retinoblastoma (Rb) genes. There occurs other immunomodulatory proteins elevated in HPV-negative HNSCC and in HPV-positive HNSCC, PD-1 and PDL1 detected in higher levels in nasopharyngeal cancers, pharyngeal cancers. Thus the studies on the HPV-positive and HPV-negative HNSCC with regard to their anatomical, physiological, genetic, proteomic characteristics can bring out novel treatment strategies. Further molecular and genetic studies are required to bring out unknown facts within the HPV-positive and HPV-negative HNSCC. The so far obtained data can be implemented in future diagnostic and clinical applications.

Abbreviations

ADAM	a disintegrin and metalloproteinases
AJUBA	Ajuba LIM Protein
AKT1	v-akt murine thymoma viral oncogenes homolg 1
ALDH1	aldehyde dehydrogenase 1
APOBEC	apolipoprotein B mRNA-editing enzyme catalytic polypeptide
ARID1A	AT-rich interaction domain 1A
ATG13	autophagy-related protein 13
ATM	ataxia telangiectasia mutated
BIRC2	baculoviral IAP repeat containing 2
BRCA2	BReast CAncer gene 2
CASP8	cysteine-aspartic acid protease (caspase) family
CCND1	cyclin D1
CD133	cluster of differentiation 133
CD44	cluster of differentiation 44
CD56	cluster of differentiation 56
CDH10	Cadherin 10
CDK4	cyclin-dependent kinase 4
CDK6	cell division protein kinase 6
CDKN2A	cyclin-dependent kinase inhibitor 2A
CSMD1	CUB and Sushi multiple domains 1
CTTN	cortactin
DAPK	death-associated protein kinase 1
E2F1	E2F transcription factor 1
ECAD	epithelial cadherin (E-cadherin)
E-FABP	epidermal fatty acid binding protein
EGFR	epidermal growth factor receptor
ENPP1	ectonucleotide pyrophosphatase/phosphodiesterase 1
EPHB3	ephrin type-B receptor 3
ERBB2	receptor tyrosine-protein kinase erbB-2
EZH2	enhancer of Zeste 2 polycomb repressive complex 2 subunit
FADD	Fas associated via death domain
FAT1	FAT atypical cadherin 1
FBXW7	F-box and wd repeat domain containing 7
FGFR1	fibroblast growth factor receptor 1
FGFR3	fibroblast growth factor receptor 3
FHIT	fragile histidine triad
FLG	filaggrin
HLA I	human leukocyte antigen
HNSCC	Head and Neck Squamous Cell Carcinoma
HPV	Human Papillomavirus
HRAS	Harvey rat sarcoma viral oncogenes homolog
KEAP1	Kelch-like ECH-associated protein 1
KMT2D	lysine methyltransferase 2D
LINE	long interspersed elements
LRP1B	low-density lipoprotein receptor-related protein 1B
MCM7	minichromosomal maintenance protein 7
MDM2	mouse double minute 2 homolog
MGMT	O6-methylguanine DNA methyltransferase
MICAL2	Microtubule Associated Monooxygenase Calponin and LIM Domain Containing 2
MLL2	histone-lysine N-methyltransferase MLL2
MSH2	MutS homolog 2
MUC16	Mucin 16 cell surface associated
MYC	MYC proto-oncogene
NF1/2	neurofibromatosis type 1
NFE2L2	nuclear factor erythroid 2-related factor 2
NICD	NOTCH intracellular domain
NLRP12	NLR Family Pyrin Domain Containing 12
NOTCH1	Notch homolog 1 translocation-associated (Drosophila)
NRF2	nuclear factor erythroid 2-related factor 2
NRXN3	Neurexin 3
NSD1	nuclear receptor binding SET domain protein 1
OPSCC	Oropharyngeal Squamous Cell Carcinoma
PARP-1	poly [ADP-ribose] polymerase 1
PCR	Polymerase Chain Reaction
PD1	PDCD1; programmed cell death 1
PDL1	programmed cell death ligand 1
PI3KCA	phosphatidylinositol-45-bisphosphate 3-kinase catalytic subunit alpha
PIK3CB	phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit Beta
PISH	PCR in situ hybridization
PTEN	phosphatase and tensin homolog
RASSF1	Ras Association Domain Family Member 1
Rb	retinoblastoma
RTKs	receptor tyrosine kinases
RUFY1	RUN and FYVE domain containing 1
SINE	short interspersed elements
SMAD4	SMAD family member 4 mothers against decapentaplegic homolog 4
SOX2	sex determining region Y
SRC	proto-oncogene tyrosine-protein kinase sarcoma
SYNE1	spectrin repeat containing nuclear envelope protein 1
TERT	telomerase reverse transcriptase
THSD7A	thrombospondin type 1 domain containing 7A
TILs	Tumour infiltrating lymphocytes
TM7SF3	transmembrane 7 superfamily member 3
TNK2	tyrosine kinase non receptor 2
TP53	tumour protein p53
TRAF3	TNF receptor associated factor 3
TRX	thioredoxin
TSCC	tongue squamous cell carcinoma
UBR5	ubiquitin protein ligase E3 component N-recognin 5
UNC5D	Unc-5 netrin receptor D
USH2A	Usher syndrome type 2A
YAP1	yes-associated protein 1
ZFHX4	Zinc Finger Homeobox 4
ZNF676	zinc finger protein 676