Behind the Screen: The Emergence of New Evidence
Belayat H. Siddiquee1, *, Norhafiza Mat Lazim2
Head & Neck Squamous Cell Carcinoma (HNSCC) is a heterogeneous group of malignancies that collectively constitute a significant group of cancer worldwide. It affects not only the elderly patients but more so the middle age and the pediatric patient population. Around 90% of these tumors develop from the mucosal lining of the head and neck region i.e. Head & Neck Squamous Cell Carcinoma (HNSCC). These mainly include oral cavity carcinoma, oropharyngeal carcinoma, hypopharyngeal carcinoma, laryngeal carcinoma, sinonasal carcinoma, nasopharyngeal carcinoma, and salivary glands carcinomas. Different types of these carcinomas are prevalent at some geographic locations due to various environmental, dietary, social, and genetic factors. Head and neck cancers are critical as these affect many vital functions of the human being, such as breathing, eating, smell, hearing, and vision. Clinical and epidemiologic studies show aetio-pathological relation between chronic inﬂammation and cancer in several organs, including the Head and Neck region. A huge number of inflammatory mediators and markers have been identified and investigated in the current genomic era. Significant inflammatory biomarkers have a potential role not only in screening and prevention but also in treatment and assessing the prognosis of HNSCC. This chapter will highlight the recent facts, the discovery of evidence of the inflammation, and biomarkers for HNSCC.
* Corresponding author Belayat H. Siddiquee: Department of Otolaryngology-Head and Neck Surgery, Bangabandhu Sheikh Mujib Medical University (BSMMU), Dhaka-1000, Bangladesh; Tel: 01917374020; E-mail: email@example.com
Head and neck malignancies are increasing throughout the planet. It affects not only the elderly patients but more so the middle age and the pediatric population.
In the UK, HNSCC incidence has been rising by almost a quarter in the last decade, with an estimated annual burden of 11,400 new cases. Since the early 1990s, Oropharyngeal cancers (OPCs) have seen the biggest rise among head and neck squamous cell carcinoma (HNSCC), with incidence rates is doubling yearly. In contrast, there is a 20% decrease in the incidence of laryngeal cancer in the same period, though the rate has become steady more recently . Lifestyle factors play an important role in the etiology of these cancers. Around 75% of HNSCC have been attributed to the individual or combined effects of tobacco and alcohol consumption. Tobacco use is an established primary risk factor for HNSCC, which causes adverse outcomes, including increased overall and cancer-specific mortality and risk for developing a second primary cancer. Continued smoking is also strongly associated with treatment toxicity . Human papillomavirus (HPV), predominantly HPV-16 infection, is also recognized as a primary risk factor for oropharyngeal cancers (OPCs), especially in younger age groups. Despite an overall decline in mortality rates of HNSCC, survival remains poor. The overall 5- year survival rate is around 50% but ranges from 33% for hypopharyngeal cancers to 60% for laryngeal cancers. People with HPV positive oropharyngeal tumors have consistently demonstrated improved survival compared to their HPV negative counterparts, although they are frequently diagnosed at a later stage .
There is a marked difference in HNSCC incidence by gender, race, and geographical distribution. The development of HNSCC is the result of interaction among environmental factors and genetic inheritance and is, therefore, multifactorial. HNSCCs are molecularly and genetically heterogeneous diseases, encompassing a wide array of carcinogenicity involving tissue and organs of the head and neck. Over the last few decades better understanding of the etiological factors, that cause HNSCC contributes to the management of these serious diseases dramatically. Besides smoking and alcohol, other factors like viruses, chronic inflammation in the territory, nutritional elements deficiency, chronic irritations even the genetic pre-disposition drawing the attention of the researchers. Despite this lot of things are yet to be explored. Because this is often observed that having a risk factor or even several risk factors does not mean that one will get cancer, many people having an HNSCC may not have had any known risk factor exposure.
Biomarkers are biological molecules identified in the cancer tissue or blood. Research surrounding biomarkers help in opening a new horizon in the diagnosis and treatment of HNSCC. They used to correlate with the presence or absence of a disease, are prognostically related to the disease course, and sometimes predictive of a tumor’s response to a specific therapy. Biomarkers should be objective, independent, and require validation by clinical testing and patient outcome. Ideally, biomarkers should be easy to analyze, quantitative, affordable, and must be subjected to quality control and assurance .
Risk Factor Analysis
Tobacco is associated with HNSCC . More than 70 known carcinogens were found in cigarette smoke . Among the toxic and carcinogenic substances absorbing in the body from tobacco, exposure are tobacco-specific nitrosamines (TSNA) and polycyclic aromatic hydrocarbons (PAH), which are mostly studied with regard to carcinogenicity. TSNA found in tobacco and tobacco smoke, essentially formed during the curing and processing of tobacco. They are found both incombustible and smokeless forms of tobacco. TSNA can be formed through various chemical reactions . Seven TSNA have been identified in tobacco products and N′- nitrosonornicotine (NNN), 4(methylnitrosamino)-1- (3-pyridyl)-1-butanol (NNAL) are two of them . Two prospective cohorts, the Shanghai Cohort Study and the Singapore Chinese Health Study help us to gain valuable epidemiologic data regarding the use of NNN and NNAL to give information about cancer risk . Recently, a control study was performed to see tobacco-related carcinogens in HNSCC. Urinary levels of 1-HOP, NNN, and NNAL were measured in smokers with newly diagnosed cases of HNSCC and compared to smokers without cancer. Levels of 1-HOP and NNN were raised in the smokers with the HNSCC group compared to the control group who are matched on several other variables, including age, gender, number of cigarette sticks consumed per day .
Alcohol plays a role as a solvent to increase mucosal exposure to carcinogens and enhance cellular uptake of these. Acetaldehyde, a metabolite of alcohol can form DNA adducts that interfere with DNA synthesis and repair . The synergistic effect of tobacco and alcohol is supported by an analysis of combined data from 17 European and American studies. The population attributable to risk was 72%, which included 4% for alcohol alone, 33% for tobacco alone and 35% attributable to both alcohol and tobacco. This effect was more than multiplicative. Similar steep rises in the risk of HNSCC among alcohol and tobacco users, especially those using high amounts of each product, have been demonstrated by Dal Maso et al. . Consumption of tobacco along with alcohol increase the HNSCC risk. All smokers and alcohol users may not develop cancer, suggesting that individual variation in genetic susceptibility plays a critical role . In general, there is a strong association between alcohol and tobacco use and the combined use of these further increases the risk .
Although tobacco smoking and alcohol drinking are responsible for most new HNSCC cases, the prognostic role of smoking status and alcohol intake during cancer presentation is unclear, especially in HPV associated OPCs. Smoking and alcohol use both increase the mortality risk, but the magnitude of the effect is usually inconsistent. Moreover, it is not yet established whether smoking and alcohol use furnish any additional prognostic information other than the tumor, node, metastasis (TNM) staging system, which currently forms the basis for clinical decision making in HNSCC. Although there is a slight decline in HNSCC related mortality , the survival rate is still poor. The overall 5-year survival rate is around 50%, but ranges from 33% for hypopharyngeal cancers to 60% for laryngeal cancers . People with HPV-positive oropharyngeal tumors have consistently demonstrated better survival compared to their HPV-negative counterparts, despite the fact that they are frequently diagnosed at an advanced stage . This is because of improved therapeutic efficacy. People with HPV-positive oropharyngeal cancers have also distinct risk factor profiles, including higher socioeconomic status and lower comorbidity which may increase survival .
Many cancers, including the HNSCC are caused by oncogenic strains of viruses like Human papillomavirus (HPV) and Epstein – Barr virus (EBV). The HPV has two oncogenes, E6 and E7, the expression of which inactivates p53 and retinoblastoma (RB), respectively, causing perturbation of cell cycle regulation in the infected cells and is considered to be the onset of HPV-related carcinogenesis . The detection of viral E6 and E7 transcripts is an acceptable assay for the detection of an oncogenic HPV infection in HNSCC. EB virus is a γ (gamma)-Herpes virus and a member of the Herpesviridae family. Herpes viruses consist of large, complex DNA viruses, able to encode about 100 different proteins, and are one of the largest virus groups . This virus affects more than 90% of the adult population worldwide in some form or other. EB virus is also associated with a variety of malignant disorders. In the head and neck region, the establishment of latent transforming EB virus infection and the potential viral genetic alteration that occurs in epithelial cells may contribute to the cancer development, its growth and invasive capabilities, especially in the subclass of nasopharyngeal carcinoma . Plasma EBV DNA analysis has proven useful in diagnosing early nasopharyngeal carcinoma in the absence of any clinical clue .
Chronic mechanical irritation (CMI) is an important risk factor for oral cancer and is considered a potentially malignant disorder [18, 19]. CMI of the oral mucosa is due to repeated injury by the intraoral injuring agent. Defective teeth (sharp or rough surfaces because of decay or fractures), ill-fitting dentures (sharp or rough surfaces, overextended flanges or lack of retention) and/or habitual acts (e.g. oral mucosa biting or sucking, tongue interposition or thrusting), acting individually or together, may all be responsible. CMI could initiate changes in the healthy mucosa or intensify existing oral diseases and also produces several alterations related to its duration and intensity. Effects usually range from a hyperproliferative epithelial response if the stimulus is mild (frictional keratosis), to several levels of tissue injury (atrophy, erosion, ulcer) if it is intense or is of longer duration (chronic traumatic ulcer), often with fibrous connective tissue growth (reactive hyperplasia, e.g. Denture-induced fibrous hyperplasia). Some authors have suggested that the relationship between oral cancer and CMI could be the result of the tumor’s growth; the larger the tumor, the more probability of being injured. Many cases of oral cancer have been described on the site of CMI because of a broken tooth or a defective denture. Oral cancer occurs mainly in locations that could be exposed to prosthetic or dental CMI, particularly in non-smokers without other risk factors [20, 21].
Clinical and epidemiologic studies support an association between chronic inflammation and cancer in several organs [22–25]. Chronic cervicitis and cervical cancer, ulcerative colitis and colorectal cancer, reflux esophagitis and esophageal cancer, and hepatitis and liver cancer are few examples. In the oral cavity, periodontitis is a chronic inflammatory disease of the structures around teeth . The ensuing chronic inflammation induces local pathologic and anatomic changes, namely, periodontal pocket formation, clinical attachment loss, and alveolar bone loss . If not treated, periodontitis ultimately leads to tooth loss. The pocket epithelium is characterized by continuous proliferation, the formation of rete- ridges, and ulcerations. In the connective tissue, there is more angiogenesis, chronic inflammatory infiltrate, fibrosis, and loss of tissue. The average prevalence of periodontitis among the general population is around 30% . Periodontitis causes the continuous release of inflammatory cytokines, enzymes, and toxins into saliva. The levels of these inflammatory markers in the saliva are directly related to the extent and severity of periodontitis [29, 30]. Periodontal pathogens and inflammatory cytokines can travel with saliva and the blood, causing inflammation and tissue injury at distant sites also [31–38].
Evidence strongly suggests that diet can influence the risk for HNSCC, and data shows a probable causal relationship for decreased HNSCC risk with non-starchy vegetables, fruits, and food containing carotenoids. A relation between poor oral hygiene and HNSCC has been suggested [39–50], but the underlying mechanism was not clear. Chronic periodontitis, an outcome of poor oral hygiene, is associated with oral premalignant lesions  and HNSCC [52, 53]. The potential association between chronic inflammation and HNSCC is further supported by two case-control studies suggesting a beneficial effect of nonsteroidal anti inflammatory drugs (NSAID) against HNSCC [54, 55]. Aspirin administration in a hospital-based case-control study between patients with HNSCC and control subjects similar by age, sex, and smoking status, revealed a 25% decrease in the risk of HNSCC. Risk reduction was observed in all primary subsites, with cancers of the oral cavity and oropharynx . The mechanism of the biological relation between chronic inflammation and cancer, although described extensively but it is evolving continuously since both are complex processes under the control of several driving factors. Bacteria and their products, including endotoxins, enzymes, and metabolic by-products, may directly induce genetic and epigenetic changes in surrounding epithelial cells [55–57]. They also increase the production of carcinogenic acetaldehydes [58–60] and nitrosamines [61, 62]. However, the available evidence supports an indirect association through stimulation of inflammation. Host cells, including neutrophils, macrophages, lymphocytes, monocytes, fibroblasts, and epithelial cells, respond to bacteria by generating cytokines, chemokines, prostaglandins, growth factors, and other signals that provide an environment for cell survival, proliferation, migration, angiogenesis, and inhibition of apoptosis . This environment facilitates epithelial cells for mutations and drives these mutant epithelial cells to proliferate and migrate and gives them a growth advantage. Numerous studies have confirmed the associations of several genes and proteins involved in different stages of inflammation with carcinogenesis [64–74]. Chronic inflammation may also act synergistically with other carcinogens to increase the risk of HNSCC. For example, breaks in the mucosal barrier due to chronic inflammation may favor enhanced penetration of other carcinogens e.g. tobacco, alcohol, and dietary metabolites . High consumption of fruit and vegetable and low intake of red meat was associated with reduced risk for HNSCC .
Exposure to the carcinogen, oral hygiene, chronic irritation to the oral mucosa, dental plaque formation, positive family history, and exposure to ultraviolet rays all play a role individually or in combination in the HNSCC development because they can modulate toxin and carcinogenic metabolism [77–79]. The contribution of family factors in HNSCC development maybe because of familial aggregations of inheritable genetic factors. Several genetic polymorphisms in genes involved in the carcinogen metabolism, DNA repair, and several other processes may also have contributed to this.
Inflammation, A Hallmark of HNSCC
Inflammation in and around cancer is considered as a “hallmark of cancer”. Many studies demonstrate that tumors may develop and progress within inflammatory diseases. The main steps in the development of cancer are genetic changes that make these cancer cells potential with many of the hallmarks of cancer, such as self-sufficient growth and resistance to anti-growth and pro-death signals. While the genetic changes that occur within cancer cells themselves, such as activated oncogenes or dysfunctional tumor suppressors, are responsible for many aspects of cancer development. Tumor promotion and progression are dependent on ancillary processes involving cells of the tumor environment that are not necessarily cancerous themselves. Infiltration of immune cells helps tumor development by the production of factors that facilitate carcinogenesis by enabling tumors to evade the host immune response. Small molecules including cytokines, chemokines, and growth factors play main roles in both inflammation and cancer by promoting proliferation, angiogenesis, and carcinogenesis and by recruiting immune cells. The extracellular matrix is changed in inflammation and provides structural support to developing tumors. Hypoxia is a common condition in cancers and inflamed tissues that causes DNA damage and induces tumorigenic factors. Finally, tissue vasculature is a vital part of its microenvironment, supplying oxygen, nutrients, and growth factors to rapidly dividing cells and providing a mechanism for metastatic spread [80, 81]. Inflammation often exists in the tumor microenvironment and is induced by inflammatory mediators (cytokines, chemokines, and growth factors) produced by the tumor, stroma, and infiltrating cells. These factors modulate tissue remodeling and angiogenesis and actively help tumor cell survival and chemoresistance through autocrine and paracrine mechanisms. HNSCC has got high inflammatory and aggressive character, and they express a number of cytokines and growth factors involved in inflammation. These cytokines and growth factors activate signal transduction pathways, which regulate the expression of genes controlling growth, survival, and chemosensitivity. This is an important update on recent advances in the understanding of the mechanisms driving cancer-related inflammation in HNSCC and on targeted molecular therapies under preclinical and clinical investigations .
There are many different pathways through which a proinflammatory diet can influence the risk of HNSCC. Diet directly contributes to the excessive production of proinflammatory biomarkers such as CRP, IL-6, white blood cell count, and homocysteine [83–85]. The inflammation contributes to the “hallmarks of cancer” by supplying bioactive molecules to the tumor microenvironment . Additionally, inflammatory transcription factors can be activated by inflammatory cytokines and other inflammatory biomarkers, which play a key role in both cancer initiation and promotion. Inflammatory cytokines can change the oral microbiota, which in turn can cause an increased risk of periodontitis and cancer [56, 87].
Bacteria and their products, including endotoxins, enzymes, and metabolic by-products, may directly induce genetic and epigenetic changes in surrounding epithelial cells [88–90]. They also intensify the production of carcinogenic acetaldehydes [91–93] and nitrosamines [94, 95]. However, the available evidence supports an indirect association through stimulation of inflammation. Host cells, including neutrophils, macrophages, monocytes, lymphocytes, fibroblasts, and epithelial cells, respond to bacteria by producing cytokines, chemokines, prostaglandins, growth factors, and other signals that provide an environment for cell survival, proliferation, migration, angiogenesis, and inhibition of apoptosis . This environment favors epithelial cells to accumulate mutations and drives these mutant epithelial cells to proliferate and migrate and gives them a growth advantage. Numerous in vivo and in vitro studies have confirmed the associations of several genes and proteins responsible for different stages of inflammation with carcinogenesis [97–107]. In addition to its independent association with HNSCC, chronic inflammation may also act synergistically with other carcinogens to increase the risk of HNSCC. For example, breaks in the mucosal barrier due to chronic inflammation may enhance the penetration of other carcinogens such as tobacco, alcohol, and dietary metabolites [108, 109].
Pathogenesis of HNSCC Development
HNSCC represents around 90% of all head and neck cancers and arises from the mucosa of the upper aerodigestive tract, including the nose, nasopharyngeal, paranasal sinuses, oral cavity, oropharynx, hypopharynx, larynx, and also the cervical esophagus. Proper understanding of the evolving molecular pathophysiology of head & neck tumorigenesis is essential for the development of feasible as well as effective diagnostic, therapeutic, and preventative strategies. It is established that HNSCC develops by a multistage pathogenesis process as reported in the genetic progression model for colon cancer . Molecular alterations in epithelial cells generally precede phenotypic histologic changes and accumulate along the malignant transformation process from the benign to premalignant and also invasive states. Increased understanding of the cellular and molecular features of solid tumors has further improved the pathologist’s capability for diagnosis and prognosis assessment of HNSCC. Identification of known and potential tumor markers including chromosomal changes, oncogenes, and tumor suppressor genes has far-reaching implications for predicting the biological behavior of a tumor and its response to a particular therapy.
Features in Premalignant Oral Lesions
Multistage pathogenesis of cancer formation was first suggested by Vogelstein et al. specifically for the colon. This genetic model for tumor formation included the activation of oncogenes and inactivation of tumor suppressor genes. He coined that a minimum of four mutations was necessary for malignant transformation. Steps have been described during advancement including initiation, promotion, and progression. This pattern has been observed in many cancers including those of the brain, bladder, and also in the head and neck. Califano et al. reported a “Vogelgram” for HNSCC, which linked histologic features in the progression of HNSCC to specific molecular changes . Further investigation concentrating on the transcriptional changes that occur during the progression from normal-appearing mucosa to dysplastic tissue and then invasive HNSCC .
Genomic alterations happening in precancerous and cancerous lesions can express themselves on different levels (chromosomal, DNA, RNA, and protein), in many ways such as point mutations, amplifications, deletions, and chromosomal alterations. Common methods to detect the presence of these changes include conventional cytogenetics, comparative genomic hybridization (CGH), spectral karyotyping, and cDNA microarrays. CGH is a technical cytogenetic process for assessing the gains or losses in DNA content (e.g., chromosomal imbalances) within a tumor’s entire genome. CGH does not show morphological changes between chromosomes .
Weber et al. used this technique to see the average number of chromosomal imbalances in 12 oral premalignant lesions (including dysplasia and carcinomas-in situ) and invasive oral HNSCCs . An average of 3.2 ± 1.2 imbalances was seen in premalignant lesions while invasive HNSCCs had a significantly higher average of 11.9 ± 1.9 (p = 0.003) imbalances. In the premalignant lesions, gains were identified on 8q and 16p, while losses were found on 3p, 5q, 13q, and 4q, 8p, and 9p. In individual biopsies from the same subject that contained both premalignancy and invasive carcinoma, most of the genomic alterations discovered in premalignancy were also found in HNSCC. Brieger et al. used CGH to analyze chromosomal alterations in OPCs and their surrounding benign mucosa. In the morphologically healthy mucosa collected 2 cm from the primary tumor margin, no chromosomal changes could be identified. In normal-looking mucosa located 1 cm from the tumor, the most common amplifications were in 15q and 21q. Almost all of these alterations were found in the primary tumor also .
These cytogenetic changes are consistent with previous reports implicating molecular features appear early in head and neck tumorigenesis before histologic and phenotypic changes and also accumulate through successive stages. CGH is a very important tool, but the other aforesaid techniques are also frequently used.
Microsatellites are repeats of non-coding DNA sequences that normally occur within the human genome. Defects in the DNA healing process may lead to microsatellites that are abnormally short or long; this process has been termed microsatellite instability (MI). MI is indirect evidence of a mismatch repaired (MMR) protein’s function. A suggested mechanism relevant in HNSCC tumorigenesis is through promoter hypermethylation. When MMR promoters are hypermethylated, it provides indirect evidence of the increased possibility that promoters of tumor suppressor genes are also hypermethylated, and therefore not functional . But when a microsatellite repeats replication error, if it goes uncorrected, a germline hereditary mutation could result in inactivation of tumor suppressor genes and uncontrolled cell and tumor growth. This idea of a mutator phenotype provides an alternate option to a multistage accumulation of genetic alterations to explain head & neck tumorigenesis.
Loss of Heterozygosity (LOH)
Mutation can inactivate an allele of a gene, e.g. tumor suppressor gene. When this occurs in a parent’s germline cell, the inactivated allele is passed onto the offspring resulting in heterozygosity. If genomic loss takes place in the somatic cell of the offspring affecting the remaining allele, LOH occurs and tumor-suppressive function in that cell is lost. LOH assays commonly employ microsatellite analysis to assess polymorphic chromosomal regions that map in or around tumor suppressor genes.
In premalignant oral lesions, LOH at 9p21 and/or 3p14 increases the probability of malignant transformation . Other chromosomal losses have been associated with increased risk: 4q, 8p, 11q, 13q, and 17p. Hyperplastic or dysplastic lesions with LOH at 3p and/or 9p plus one of the other above losses were found to have a 33-fold increase in cancer risk . Lesions that are lack significant dysplasia to necessitate estimation of cancer risks, molecular markers may yet to be proved useful. Zhang et al. proposed a staging system incorporating assessments from both histology and LOH criteria .
A well-characterized tumor suppressor gene p53 is located on chromosome 17p. Its role is to control cell growth arrest and apoptosis. The p53 protein has a too short half-life, and thus, is difficult to detect in benign tissues. Overexpression of p53 can happen from mutation, a defect in its degradation, or from binding to other proteins. On the whole, mutations of the p53 tumor suppressor gene have been found in half of HNSCC tumors and are the most common genetic alterations found in the malignancy of human beings. The types of mutations vary from mutations, transversions, transitions, and deletions. Various proteins usually bind with p53, such as SV40 large T, which blocks its DNA binding capability. Binding with adenovirus E1B blocks p53’s transcriptional activity. Finally, binding with HPV E6 targets p53 for accelerated degradation .
Increasing frequencies of p53 alterations and genomic instability have been identified during progressive steps in HNSCC carcinogenesis. Shin et al. analyzed p53 expression and chromosomal polysomy via immunohistochemistry and chromosome in situ hybridization, in epithelial specimens. 19% of adjacent normal-appearing mucosa, 29% of hyper-plastic lesions, 46% of dysplastic lesions, and 58% of HNSCC tumors expressed p53. Normal-appearing mucosa lacked detectable p53 levels as expected . These findings suggest that premalignancy is usually associated with altered p53 expression and increased genomic instability, which are possibly early markers of carcinogenesis.
Retinoic Acid Receptor-β
Retinoids (natural and synthetic derivatives of vitamin A) regulate cell growth and differentiation and have growth-suppressive effects in epithelial cells. Retinoic acid receptorβ (RARβ), a steroid hormone receptor whose expression is suppressed in premalignant tissues and established HNSCC through an unknown mechanism.
In addition to p53, Rb is the other major tumor suppression pathway in human carcinogenesis. Rb normally suppresses cell growth by binding to E2F1 transcription factors and preventing cells from advancing from G1 into the S phase of the cell cycle (G1 arrest). Altered expression of Rb and p16 has been reported in oral carcinomas . In fact, alterations in p16 are considered the most common genetic alteration in HNSCC . The HPV oncoprotein E7 is known to cause inactivation of Rb.
Opposing to changes in human genomic DNA, the alteration can occur in mitochondrial DNA instead. The usual site for polymorphisms and mutations is the poly-cytosine tract (C-tract) of the displacement loop of the mitochondrial genome. Ha et al., tested 137 premalignant lesions for C-tract DNA changes using PCR and polyacrylamide gel electrophoresis .
Overexpression of epidermal growth factor receptor (EGFR) is present in malignant, premalignant, and normal-appearing tissues from HNSCC patients, which is correlated with poor prognosis [124, 125]. EGFR staining increased linearly in the stratum spinosum in oral leukoplakia with increasing degrees of dysplasia .
Features in Squamous Cell Carcinoma (HNSCC)
A good number of molecular features found in HNSCC are due to cytogenetic changes. The conventional cytogenic study includes the preparation of metaphase spreads of cultured tumor biopsies. The frequent cytogenetic alterations in HNSCC include chromosomal gains on 3q, 8q, 9q, 20q, 7p, 11q13, and 5p. Genomic losses are more frequent than gains and being identified on 3p, 9p, 21q, 5q, 13q, 18q, and 8p . Investigation of these alterations in untreated HNSCC specimens enables the identification of genes responsible for disease phenotypes and also improves the idea about the pathophysiology behind the disease. Both classical and molecular cytogenetic mechanisms are shown in HNSCC to have a complex karyotype, with the most common change being tetraploidization. Moreover, an average number of 15 aberrations are found across the genome . These changes usually include deletions, translocations, and isochromo-somes.
Molecular alterations are also seen in non-squamous cancers of the head and neck region. The most specific mucoepidermoid carcinoma-associated genetic alteration is the t (11;19) (q21; p13) translocation initially detected by traditional karyotyping . This translocation generates a mucoepidermoid carcinoma translocated 1 (MECT1)–mastermind-like 2 (MAML2) gene fusion product consisting of exon 1 of MECT1 fused to exons 2–5 of MAML2 . Patients with fusion-positive tumors are generally younger, have a lower grade tumor, with a significantly lower risk of local recurrence, metastasis, or tumor-related death. Only a few translocation-positive aggressive high-grade mucoepidermoid carcinomas have been described .
Early studies have failed to identify a correlation between p53 expression and survival of HNSCC patients. When patients with laryngeal primaries were studied, positive p53 expression was significantly associated with the patient’s poor outcome [132, 133]. In studies focusing on certain types of mutations (e.g., missense, nonsense), correlations were observed with prognosis [134, 135]. Other studies to see the relation between p53 mutations and prognosis have yielded conflicting data [136, 137]. The prevalence of p53 changes in HNSCC underscores its importance in tumorigenesis. Inactivation of this tumor suppressor gene is found to be correlated with resistance to chemotherapy . Restoration of function via intratumoral injection of adenoviral p53 gene therapy (Ad-p53 or INGN-201) has demonstrated favorable results in phase I and phase II trials in patients with advanced and recurrent HNSCC .
Lee et al. measured telomerase activity via TRAP in the peripheral blood mononuclear cells of 120 patients (100 with HNSCC and 20 controls) . Telomerase positivity is highly significant and correlate with higher T stage (p = 0.005); higher N stage (p = 0.002); and higher AJCC stage (p < 0.001). Multivariate analysis reveals telomerase expression is an independent predictor of survival. (p = 0.017) This is very important that the expression of a protein in the peripheral blood cells from HNSCC patients demonstrated prognostic potential. Thurnher et al. used a modified semiquantitative TRAP assay to assess HNSCC tumors. When stratified by the presence or absence of cervical metastases, a statistically significant difference in telomerase activity was found between the two groups . This study shows primary feasibility in using biomarkers, such as telomerase to identify patients at high risk of cervical metastasis.
There is increasing evidence that suggests the association of high-risk HPV strains with a subset of HNSCC as an aetiological factor. Viral DNA is found in Carcinoma in situ (CIS), primary HNSCC, and metastatic lesions by a number of molecular techniques. DNA has been found in around 50% of oropharyngeal cancers, particularly in nonsmokers. Viral oncoproteins E6 and E7 of high-risk HPV strains 16 and 18 disrupt the p53 and Rb tumor suppressor pathways, respectively, thereby providing potential mechanisms for transformation. HPV-positive tumors usually show the following features: usually originate in the oropharynx (tonsil or tongue base), basaloid histology, positive history of tobacco and/or alcohol may or may not be present, and improved survival compared to HPV-negative counterpart . Additionally, there is a rise in Oropharyngeal cancer incidence over the last three decades, detected in younger adults. HPV involvement has implications for the potential transmission of some HNSCC malignancies via Oro-genital contact and the potential application of HPV vaccines targeted at people at risk.
Epidermal growth factor receptor (EGFR) represents an important therapeutic target in HNSCC. Cetuximab is a chimeric monoclonal antibody that binds to the extracellular domain of EGFR. Several trials reported on the efficacy of cetuximab and platinum-based chemotherapy in recurrent and refractory HNSCC cases [143, 144]. Response rates are approximately 10% in patients with progressive disease with combination therapy. The severity of one of the side effects, acneiform rash, is directly related to outcome and survival. The phase III trial conducted by Burtness et al. found increased response rates for recurrent and/or metastatic HNSCC patients treated with cisplatin and cetuximab (26%) vs. cisplatin and placebo (10%) (p = 0.03) . Another class of EGFR-targeted therapy includes the tyrosine kinase inhibitors (TKIs). Gefitinib and erlotonib oral preparations TKIs, have shown clinical activity in a variety of HNSCC treatment settings [146, 147]. Caponigro et al. summarize recent clinical trials for this group of targeted therapies .
Keratinocyte inflammation has been identified as crucial for chemical carcinogenesis in oral HNSCC. Inflammatory mediators such as prostaglandins, interleukin-1, interleukin-6, and tumor necrosis factor α are central to this process. The release of prostaglandins can cause vasodilation, alters vascular permeability, and inflammatory cell infiltration –all potential tumor-promoting effects. COX-2 is overexpressed in much oral dysplasia and HNSCC. Prostaglandin E2 (PGE2) is related to malignant transformation in HNSCC . COX-2 inhibitors exert an anti-proliferative effect and induce apoptosis in oral cancer cell lines. Studies have revealed a decrease in PGE2 levels and dose-dependent reduction of tumor growth .
NF-kB (nuclear factor of kappa light polypeptide in B-cells), upon activation, translocate from the cytosol to the nucleus to induce transcription, leading to cell proliferation and reduced apoptosis. A clinical trial evaluating the proteasome inhibitor bortezomib in combination with cisplatin and radiotherapy for locoregionally advanced HNSCC is ongoing for which standard chemo-radiation regimens are yet to be established .
The contribution of angiogenesis in the progression of solid tumors is crucial. Without adequate blood supply for a relentlessly growing cancer, newly dividing tumor cells are unlikely to survive due to lacking necessary nutrients and oxygen. Vascular endothelial growth factor (VEGF) is a proangiogenic mediator, binds to corresponding receptors on endothelial cells. This results in endothelial migration, proliferation, and increased vascular permeability. High VEGF and VEGF receptor expression in HNSCC patients are associated with increased tumor proliferation rates and worse survival . In HNSCC, angiogenesis represents a potential technique of resistance against anti-EGFR agents. Vokes et al. conducted a phase I-II trial of erlotinib with bevacizumab in patients with metastatic and/or recurrent incurable HNSCC [153, 154]. Favorable results were watched with no dose-limiting toxicities.
Transforming growth factorβ (TGFβ) has played a crucial role in cell differentiation, tissue regeneration, and regulation of the immune system. Ras activation has been associated with malignant transformation. Ras mutations are rare in HNSCC in the western part of the world (< 5%), while Ras overexpression is much more common .
Molecular Features in Metastatic Lesions of HNSCC
Lymph node metastasis in the neck is the single most significant prognostic predictor of HNSCC. This reduces survival by 50% on average. Immunohistochemical profiling of primary and metastatic lesions has revealed various genes implicated in metastasis. Similar gene expression profiles are found in primary tumors and their metastases, which indicates that molecular alterations occur early in the metastatic lesion. E-cadherin is a cell adhesion molecule that regulates adhesion between adjacent epithelial cells. Down-regulation of E-cadherin has been found in HNSCC patients with metastatic tumors . Dissociation of tumor cells is suggested as a probable mechanism. Integrins are transmembrane proteins that take part in cell adhesion in addition to signal transduction. Increased cell motility and growth have been reported in HNSCC via upregulation of integrin α v β 6 . Overexpression of matrix metalloproteinase-2 (MMP2) and MMP9 are associated with invasion, metastasis, and poor prognosis . Likewise, upregulation of EGFR, proangiogenic genes such as VEGF and IL-8, and chemokine receptor-7 (CCR7) have all been implicated in HNSCC metastasis . Detection of tumors, which are at high risk for locoregional and distal metastasis by molecular testing would have a profound impact on patients’ prognosis.
Alcohol-Induced Molecular Changes
A molecular understanding of the pathogenesis of an alcohol-related HNSCC remains elusive and poorly understood. Since ethanol alone is not considered a carcinogen, most studies involving alcohol and cancer focus on its ability to increase penetration of carcinogens, interfere with DNA repair mechanisms, or cause DNA damage through acetaldehyde, the first metabolite of ethanol, which is a known carcinogen. Evidence of an association between pre-treatment alcohol use and HNSCC mortality risk is conflicting. Some studies report an inverse association between alcohol intake and survival whilst others have found little or no evidence of an effect. Consequently, it is unclear whether any association of alcohol consumption with HNSCC cancer mortality is genuine or the result of residual confounding by smoking (or other factors). Recently, it was suggested that the effects of alcohol intake on HNSCC survival may differ by treatment method and primary site but this study only included 427 individuals from a single cancer center in Japan, emphasizing the need for further research in this area .
Applications of Altered Molecular Features Of HNSCC
Early Detection of HNSCC
Diagnosing HNSCC at an early stage is a very critical clinical step to improve outcomes. A recently developed oral rinse utilizes ELISA to detect soluble CD44 (an overexpressed protein marker) in HNSCC . This biochemical test revealed elevated CD44 levels in 62% of the 102 HNSCC patients in the study group. This assay also picked up a few false-positives cases. Sensitivity ranged from 62% to 70% while the specificity spectrum is from 75% to 88%.
Biomarkers in Risk Assessment and Molecular Staging
Surgical Margin Assessment for Risk of Local Recurrence
Adequacy of surgical resection of HNSCC is evaluated by frozen sections from margins around the lesion. Local recurrence may occur in up to 50% of cases with histologically negative margins . Detection of pre-malignant molecular changes in histologically cancer-free tissue will further ensure margin safety and also can guide which patient will require postoperative adjuvant therapy. Gene promoter hypermethylation is one of the common mechanisms for loss of tumor suppressor gene function. Rosas et al. showed that at least 56% of HNSCC are characterized by abnormal methylation of p16, MGMT, or death-associated protein kinase . The feasibility of per-operative rapid DNA tissue extraction, rapid bisulfite treatment, and quantitative methylation-specific PCR (QMSP) for p 16 and MGMT was studied by Goldenberg et al . While conventional QMSP requires about 24 hours, the rapid QMSP assay takes 5 hours and requires the simultaneous work of two persons. This approach is useful for cases requiring extensive resection and reconstruction.
Molecular Predictors of Survival
cDNA microarray technology is being utilized for outcome prediction in HNSCC patients. Belbin analyzed gene expression from 17 patients using gene chips containing 9,216 clones . 375 differentially expressed genes, so far identified by this method enabling the investigators to classify patients into two groups. The better prognosis group had a 2-year disease-specific survival of 100%, while the inferior group had a 56% survival. This molecular assay was found to be more efficient in anticipating outcomes than standard clinical and pathological criteria. Genes identified in the unfavorable prognostic group included catalase, cytokine family members, xanthine oxidase, and phosphodiesterase genes.
These are endogenous 21–22 nucleotide long non-protein-coding RNA sequences regulating target mRNA expression by complementary interaction with the 3′ untranslated region of mRNA. The extent of complementarity between microRNA and its target determines the mechanism of translational inhibition: partial complementarity will induce mRNA repression, and perfect complement-arity will cause translational inhibition. MicroRNA biogenesis and its role in carcinogenesis are studied by Gomes et al. .
Histopathological Changes and their Clinical Implications
Premalignant Lesions of the Head and Neck
A precancerous epithelial lesion shows histological changes and also has an increased potential of progressing to an invasive squamous cell carcinoma. These altered situations include hyperplasia, and more ominously, mild to severe dysplasia. Clinically signs of premalignancy include leukoplakia and erythroplakia, which are not the synonyms of histologic diagnoses.
Leukoplakia is a clinical condition where a white patch or plaque cannot be rubbed off and cannot be labeled with a more specific diagnosis. The prevalence of oral leukoplakia in the US is around 2.9%, usually affecting in their fifth to seventh decades irrespective of sexes . Seventy to ninety percent of cases are tobacco consumers, the most consistent causal factor . The majority of white patches are due to chronic irritation (i.e., tobacco or dental trauma) and, histologically, represent an irregularly thickened keratin layer ranging from simple hyper parakeratosis to an early invasive carcinoma. Therefore, leukoplakia does not correlate with the histologic diagnosis of dysplasia.
The malignant transformation potential in oral leukoplakia is well known. Studies performed in the US show an average transformation rate of 15.6% (range 13.6– 17.5%) with varying periods of follow up [169, 170]. Malignancy is considered irreversible, but premalignancy is reversible after withdrawal or removal of the offending agent. A prospective cohort of Indian smokers, who quit smoking showed a marked decrease in the incidence of oral leukoplakia during follow-up). But no decrease could be seen among patients of oral lichen planus . DNA content in the leukoplakia lesions has been found to be a significant predictor of local failure following surgical resection. Lesions harboring aneuploid or tetraploid DNA reflect a higher chance of recurrence compared to their normal diploid counterparts .