Risk Factor Analysis

Tobacco is associated with HNSCC [4]. More than 70 known carcinogens were found in cigarette smoke [5]. 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 [6]. Seven TSNA have been identified in tobacco products and N′- nitrosonornicotine (NNN), 4(methylnitrosamino)-1- (3-pyridyl)-1-butanol (NNAL) are two of them [6]. 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 [7]. 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 [8].

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 [9]. 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. [10]. 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 [11]. In general, there is a strong association between alcohol and tobacco use and the combined use of these further increases the risk [12].

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 [8], 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 [12]. 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 [13]. 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 [14].

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 [15]. 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 [4]. 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 [16]. Plasma EBV DNA analysis has proven useful in diagnosing early nasopharyngeal carcinoma in the absence of any clinical clue [17].

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 [26]. The ensuing chronic inflammation induces local pathologic and anatomic changes, namely, periodontal pocket formation, clinical attachment loss, and alveolar bone loss [27]. 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% [28]. 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 [51] 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 [54]. 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 [63]. 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 [75]. High consumption of fruit and vegetable and low intake of red meat was associated with reduced risk for HNSCC [76].

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 [82].

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 [86]. 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 [96]. 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 [110]. 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 [111]. Further investigation concentrating on the transcriptional changes that occur during the progression from normal-appearing mucosa to dysplastic tissue and then invasive HNSCC [112].

Cytogenic Features

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 [113].

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 [114]. 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 [115].

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.

Molecular Features

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 [121]. In fact, alterations in p16 are considered the most common genetic alteration in HNSCC [122]. 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 [123].

EGFR/STAT3

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 [126].

Features in Squamous Cell Carcinoma (HNSCC)

Molecular Features

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 [156]. 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 [157]. Overexpression of matrix metalloproteinase-2 (MMP2) and MMP9 are associated with invasion, metastasis, and poor prognosis [158]. Likewise, upregulation of EGFR, proangiogenic genes such as VEGF and IL-8, and chemokine receptor-7 (CCR7) have all been implicated in HNSCC metastasis [159]. 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 [160].

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 [161]. 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

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

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 [167]. Seventy to ninety percent of cases are tobacco consumers, the most consistent causal factor [168]. 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 [171]. 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 [172].

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