HNSCC have complex karyotypes, with the most common change being tetraploidization, and an average of 15 aberrations including deletions, translocations, isochromosomes, and less frequently amplifications.⁴³ Losses of genomic material are more frequent than gains and affect 3p, 9p, 21q, 5q, 13q, 18q, and 8p.⁴⁴ Chromosomal gains affect 3q, 8q, 9q, 20q, 7p, 11q13, and 5p. Tumor suppressor genes or oncogenes have been associated with some of those alterations.³⁴ Loss of 3p is the most frequent genomic loss and involves FHIT and RASSF1A. Other examples are losses of 9p21 (CDKN2A) and 17p13 (TP53). Specific sites of loss of heterozygocity (LOH) have been associated with a risk to develop HNSCC in patients with oral leukoplakias (OLs).⁴⁵ They are discussed in the Prevention and Risk Assessment section of this chapter. Gain in 3q is among the most frequent gain and includes TP63, an epithelial developmental gene, and PIK3CA. Other examples are gains in 7p (EGFR), 8q (MYC oncogene), and l1q13 (CCND1). Dysplasias and carcinoma in situ have fewer imbalances compared with OSCC (3.2 vs. 11.9).⁴⁶ An increasing trend of microsatellite instability, which reflects a reduced DNA repair capacity by mismatch repair proteins, has also been reported in hyperplasia (6%), dysplasia (15%), and HNSCC (30%).⁴⁷ The “Vogelgram” proposed by Califano et al. for HNSCC is the following³⁵ : The loss of 9p, leading to inactivation of CDKN2A, is a very early event and found in 70% of HNSCC⁴⁸; it is frequently followed by the loss of 3p (FHIT, RASSF1) and 17p (TP53) in transition to dysplasia.⁴⁹

Two important studies published recently provided new information on the mutational landscape of HNSCC using whole exome-sequencing technology.^4,5 Far fewer genes were found to be mutated per tumor in HPV-associated tumors (5/tumor) compared to HPV-negative ones (20/tumor), independent of smoking status. More mutations were identified in tumors from patients with a smoking history (22/tumor) as compared with those from never smokers (10/tumor). Among smokers, tumors with the highest fraction of G > T transversions showed a trend toward increase in overall mutation rates. Interestingly, several outliers were observed in tumors from never smokers with low fraction of G > T transversions but very high mutation rates, including mutations in some DNA repair genes, which may explain this paradox. Most frequent somatic mutations were found in TP53, NOTCH1, CDKN2A, PIK3CA, and HRAS were seen in 47% to 62%, 14% to 15%, 9% to 12%, 6% to 8%, and 4% to
5% of patients, respectively. The most frequently mutated genes were often affected by copy number alterations. LOH of NOTCH1 were observed in tumors with mutations in the same genes, and most NOTCH1 mutations were predicting truncated mutations or deleting critical functional residues. These observations support its role as a tumor suppressor gene in HNSCC. Less frequent mutations included PTEN, MLL2, TP63, EZH2, NOTCH3, NOTCH2, DICER1, and RB1. A significant proportion of mutated genes are involved in epidermal development and squamous differentiation. Similar studies using next-generation sequencing technology remain to be done to characterize premalignant lesions.

The specific case of TP53 tumor suppressor gene is interesting. The gene encodes a nuclear protein with transcription factor activity that is a necessary component of the G1-S transition checkpoint, thereby contributing to cell cycle control, DNA repair and synthesis, and genomic integrity.^50,51 The wild-type p53 protein can induce either programmed cell death or growth arrest at the G1 phase of the cell cycle. TP53 inactivation is nearly universal in HNSCC, although the mechanism of loss of function depends on the etiology: inactivating TP53 mutations are identified in 78% of the HPV-negative tumors that are usually associated with an alcohol and smoking history; in the setting of HPV infection, TP53 gene is typically none mutated but p53 is inactivated by binding to the E6 viral protein.^5,52,53 TP53 mutation and binding to other proteins can lead to overexpression of p53 protein that is reported in 19% of adjacent normal mucosa, 29% of hyperplastic lesions, 46% of dysplastic lesions, and 58% of HNSCC. Increase in p53 expression has been associated with increased genomic instability.⁵⁴ The timing of p53 inactivation remains controversial, with some reports demonstrating aberrant expression in premalignant lesions, but the majority suggesting mutation only late in tumor evolution.^35,55,56,57

The inactivation of individual tumor suppressor genes such as CDKN2A, CDH1, DAPK1, RASSF1, RAR-β, DCC, MGMT, NDRG2, and DLEC1 by promoter DNA methylation has been reported in HNSCC.^58,59,60,61 Using high-throughput DNA promoter methylation profiles, it has been shown that the overall pattern of epigenetic alteration can significantly distinguish tumor from normal head and neck epithelial tissues more effectively than specific gene methylation events. Tobacco smoking and alcohol exposures are associated with specific DNA methylation profiles.⁵⁸ Another important feature of malignant tumors is global hypomethylation, which is measured by the degree of methylation of repetitive sequences across the genome such as LINE1, and has been associated with genome instability. Poage et al. found that global methylation was different according to tumor site and LINE1 methylation was associated with HPV16 E6 serology. The authors identified a distinct subset of CpG loci with significant hypermethylation associated with LINE1 hypomethylation.⁶² Few studies have looked at methylation levels of individual genes in preneoplastic lesion. RARβ- silencing by methylation has been shown to be an early event in HNSCC carcinogenesis; 66% of the HNSCC showed hypermethylation of RAR-β versus 53% of OLs and 27% in adjacent normal tissue.⁶³ CDKN2A methylation was never detected in normal individuals, but in 5% of leukoplakia with hyperplasia, 18% of leukoplakia with mild dysplasia, 10% of leukoplakia with moderate dysplasia, 22% of leukoplakia with moderate dysplasia, and in 50% or more of OSCC.⁶⁴ In patients with betel-quid chewing in Sri Lanka, a high frequency of hypermethylation of p14, p15, and p16 was detected in precancerous lesions, although no hypermethylation was found in normal epithelium.⁶⁵

All three pathways represent potential targets for chemoprevention, although only EGFR pathway has been studied in premalignant lesions. EGFR activation by its ligand transforming growth factor-alpha (TGFa), activates STAT3 which increases proliferation, suppresses apoptosis, and decrease differentiation. EGFR and TGFα are upregulated in HNSCC, constituting an autocrine activation loop.^{66,67,68,69,70,71,72,73} EGFRvIII, a variant detected in 42% of HNSCC, is associated with increased proliferation in vitro and in vivo and with decreased apoptosis in response to cisplatin and cetuximab.⁷⁴ High EGFR expression and high EGFR gene copy number have been associated with poor survival in patients with HNSCC.^56,68,75 Cetuximab, an anti-EGFR antibody, is now approved in the treatment of locally or regionally advanced HNSCC or as a single agent for the treatment of patients with recurrent or metastatic disease for whom prior platinum-based therapy has failed.^76,77 EGFR is overexpressed in malignant, premalignant, and normal-appearing tissues from HNSCC patients. EGFR expression increases progressively with increasing degree of dysplasia and is markedly elevated in more than 90% HNSCC.^67,68 We have reported that increased EGFR gene copy number in OLs was frequent and associated with an increased risk of developing OSCC.⁷⁸ Erlotinib has been shown to decrease the incidence of OSCC in a chemically induced mouse model.⁷⁹ The use of EGFR tyrosine kinase inhibitor (TKI) in the chemoprevention setting is discussed below.

PI3K/AKT/mTOR pathway has been shown to be activated in approximately 50% of HNSCC.^80,81,82 Mechanisms of this activation include activation of upstream tyrosine kinase receptors (EGFR, IGF-1R…), frequent amplification of PIK3CA (3q26) that is associated with poor prognosis, PI3KCA activating mutations that have been reported in up to 10% of the cases, and PTEN downregulation in rare cases.

MET pathway activation has been recently reported in HNSCC. Knowles et al. have shown that 80% of HNSCC express MET, its ligand HGF, or both.⁸³ Seiwert et al. have shown that 84% of HNSCC samples showed MET overexpression and 45% HGF overexpression.⁸⁴ MET gene copy number was increased in a significant proportion of HNSCC cell lines and tumors. The rate of MET tyrosine kinase domain mutations was 3 % and the rate of non-TK domain mutations was 9%. MET inhibition was associated with decreased proliferation, migration and motility, and angiogenesis and with a greater-than-additive inhibition of cell growth in combination with cisplatin or erlotinib. The functional role of TK domain mutations in HNSCC remains to be determined.

At least two isoenzymes, COX1 and COX2, have been described. COX1 is constitutively expressed in most tissues, while COX2 is inducible. COX2 has been shown to be frequently overexpressed in oral dysplasias and HNSCC.^{85,86,87,88,89,90} COX catalyzes the synthesis of prostaglandins from arachidonic acid. Levels of prostaglandin E2 (PGE2) are elevated in HNSCC as well and correlate with poor survival.⁹¹ COX2 inhibitors have shown to decrease proliferation and induce apoptosis in both in vitro and in vivo.⁹² In vitro, PGE2 causes dose-dependent stimulation of cellular growth of the nontumorigenic MSK-Leukl oral epithelial cell line, thus providing further evidence for a possible role COX and prostaglandins in oral carcinogenesis.⁹³ Using immortalized oral epithelial cells and HNSCC cell lines, Abrahao et al. have shown that COX2 overexpression in cancer and inflammatory cells induce PGE2 production in the tumor microenvironment that promotes HNSCC cell growth in an autocrine and paracrine fashion.⁹⁴ COX2 has been shown to be upregulated by Snails that
impedes terminal differentiation, invasion, and inflammation in oral keratinocytes.⁹⁵ COX inhibition has been shown to prevent premalignant lesions in preclinical and clinical studies, especially as it pertains to colonic polyps.⁹⁶ In murine models of oral carcinogenesis, COX2 inhibitors were found to have chemopreventive effects.^97,98,99

Transforming growth factor-beta (TGFβ) is a key regulator of epithelial homeostasis, immune function, and angiogenesis. TGFβ signaling dysregulation is common in HNSCC.³⁴ Defective TGFβ signaling in epithelial cells increases proliferation reduces apoptosis and increases genomic instability. The compensatory increase in TGFβ production by tumor epithelial cells further promotes tumor growth and metastases by increasing angiogenesis and inflammation in tumor stromal cells.¹⁰⁰ RAS mutations are rare in HNSCC in the Western countries, although its overexpression is common. Lu et al. have shown that combining TGFβRII loss with KRAS or HRAS overexpression in a murine model resulted in HNSCC tumor formation. Neither alteration alone resulted in a malignant phenotype.¹⁰¹

Retinoids are naturally occurring and synthetic vitamin A (retinol) metabolites and analogues. Retinoids are involved in many physiologic processes including cell differentiation that they promote, proliferation, and apoptosis that they decrease. Two families of retinoid receptors have been characterized: RAR and RXR. RAR is endogenously activated by all-trans-retinoic acid and 9-cis-retinoic acid; RXR is endogenously activated by 9-cis retinoic acid only. 13-cis-retinoic acid (13cRA) is readily converted to all-trans-retinoic acid, which in turn primarily regulates gene transcription through RAR. The “retinoid axis” also includes repressors and coactivators that form interacting complexes that ultimately define regulation of genes and cellular function.¹⁰² Cattle that are vitamin A deficient develop tumors of the aerodigestive tract and lung at a relatively high frequency.¹⁰³ Loss of nuclear RARβ is an early event observed in premalignant dysplastic lesions.¹⁰⁴ This has triggered evaluation of retinoids in chemoprevention trial that is discussed below.

Cell cycle regulation in HNSCC is altered through various mechanisms that are not exclusive to each other. One includes activation of CCND1 and MYC oncogene that are often amplified and/or overexpressed.¹⁰⁵ MYC overexpression has been associated with poor survival of patients with HNSCC.¹⁰⁶ Other mechanisms involve inactivation of TP53 and inactivation of the pl6/p14 ARF locus, both being frequent in HNSCC.¹⁰⁷ Both the inactivation of CDKN2A and overexpression of CCND1 increases CDK4/6 activity and RB phosphorylation that in turn promotes cell proliferation.^108,109 Soni et al. studied the expression of CD-KN2A, CCND1, and pRB using immunohistochemistry in normal-appearing oral mucosa, hyperplastic, and dysplastic lesions and in HNSCC.¹¹⁰ Alterations in all three genes were demonstrated in 11% hyperplastic and dysplastic lesions and in 18% of HNSCC. Loss of CDKN2A was the earliest event, loss of pRB was associated with the transition hyperplasia to dysplasia, and CCND1 or p53 overexpression was associated with the transition to HNSCC. Finally, a CCND1 polymorphism, thought to alter cyclin D1 expression, has been associated with HNSCC risk, tumor grade, tumor recurrence, and survival.^{111,112,113,114,115}

Reactivation of telomerase is a common feature of malignant tumors. Using a telomerase rapid amplification protocol (TRAP) assay, Patel et al. identified telomerase activation in 78% to 82% of HNSCC, 55% to 85% of premalignant lesions, and 39% to 53% of adjacent normal-appearing tissue.^116,117 A higher telomere length was associated with a poor survival.¹¹⁷ Thus, reactivation of telomerase appears to be an early molecular alteration. Interestingly, telomerase activity in peripheral blood mononuclear cells was correlated with higher T and N stages and was an independent predictor of survival.¹¹⁸

Angiogenesis contribution to HNSCC progression has been studied. Angiogenesis is required for the tumor growth and the metastatic spread. The majority of HNSCC overexpress the vascular endothelial growth factor (VEGF) or the VEGF-2 and VEGF-3 receptors.^119,120 A meta-analysis involving 1,002 patients showed that VEGF tumor overexpression evaluated by immunohistochemistry was associated with a decreased survival.¹²¹ The role of angiogenesis in progression of premalignant lesions has been recently outlined using a chemically induced mouse model and will be discussed below.¹²²

HPV-related HNSCC usually arise in the oropharynx (tonsillar crypts), they are infiltrated by lymphocytes, do not undergo significant keratinization, have basaloid morphology, affect younger individuals, and are usually stage Tx or T1-2 with early and large lymph node involvement with better outcome compared to HPV-negative HNSCC.¹⁸ After HPV integrates its DNA within the host nucleus, it overexpresses oncoproteins E6 and E7. E6 induces p53 degradation, and E7 binds and inactivates RB.¹²³ HPV-related HNSCC have also been shown to have a unique transcriptional profile for both mRNA and miRNAs, as well as a unique DNA methylation profile.^62,124,125 The genomic profile is also different as almost all HPV-associated tumors express wild-type TP53 and CDKN2A and have a lower rate of mutations as opposed to HPV-negative HNSCC.^4,5 This would suggest that HPV infection is an early event in head and neck squamous cell carcinogenesis. With regard to the prevention setting, they are not associated with dysplasia of surface epithelium which makes early detection challenging.¹²⁶ HPV positivity in normal adjacent tissues is considered a rare occurrence. In a recent study of 200 normal tonsils, the prevalence of HPV16 and 18 was found to be zero. Although there appears to be some link between HPV and OL, evidence supporting a causal role of HPV in oral squamous cell tumorigenesis is not convincing. The exact anatomical site is not always reported with the risk to include both oral cavity and oropharynx SCCs. The detection of HPV virus does not necessarily mean HPV integration in the host genome and E6/E7 oncoproteins activation. HPV serotypes are not available in all the studies and not all HPVs are oncogenic. Finally, the magnitude of association consistent with an infectious etiology is only achieved for SCC of the oropharynx.^{127,128,129,130,131}