Diversity of Head and Neck Squamous Cell Carcinoma (HNSCC)
There is a remarkably diverse array of anatomy and tumor morphologies, with at least ten anatomic subsites of the head and neck, challenging all members of the multidisciplinary team to precisely define the extent of a patient’s disease. (Figure 1) While the majority of the histopathology consists of squamous cell carcinoma (SCC), there are dozens of other pathologic diagnoses. Accordingly, a broad spectrum of treatment modalities are offered, frequently in combination, including chemotherapy, radiation including Intensity Modulated Radiation Therapy (IMRT), and surgery with and without reconstruction. Historically, the treating teams have labored to join the anatomic and morphologic considerations (as well as patient preference and comorbidities) of a patient’s disease to select the appropriate range of treatment options. Increasingly clinicians are also required to consider a new set of issues, the so-called molecular determinants of head and neck cancer. In the sections below we will attempt to highlight the spectrum of these targets most likely to impact clinicians and patients in the coming years. We will touch briefly on inherited and somatic aberrations that predispose to tumorigenesis, both genetic and epigenetic, as well as a number of specific cancer pathways as targets of tumorigenesis and therapeutics. Finally, we will consider the role of new and developing molecular diagnostics in the management of patient care.
Molecular Basis of Risk Factors for Development of Head and Neck Cancer
The most well known risk factor for developing head and neck cancer is the deleterious effects of tobacco. Indeed, HNSCC was one of the first carcinomas to be linked with p53 mutations caused by tobacco usage . Alcohol use is synergistic with tobacco in causing HNSCC. There are other cultural habit-forming risk factors that have an association with HNSCC. Betel nut, a fruit that is the basic ingredient of a stimulant chew, is used by an estimated 200 to 400 million throughout Southeast Asia . Betel nut is incorporated into Asian medicines to treat a variety of complaints from headaches to rheumatism . The odds ratio of developing leukoplakia and submucous fibrosis from using betel nut is five compared to one in non-chewers . The addition of tobacco raises the risk 3-fold . The duration and frequency of betel nut use increase the risk of developing cancer, suggesting a dose-response relation .
Tobacco smoke is associated with structural changes in DNA, particularly those induced by oxidative damage. Benzo[α]pyrene diol epoxide (BPDE), a known tobacco carcinogen, induces genetic damage by forming covalently bound DNA adducts throughout the genome, including p53 . Damage induced by BDPE and other such carcinogens can be repaired through the nucleotide excision repair (NER) system. Along with the NER the base excision repair (BER) system is another set of multi-step enzymatic complexes involved in the repair of nonspecific DNA damage, including gamma and ultraviolet radiation, cross linking, and chemical intra-/interstrand adduct formation. The BER handles the largest number of cytotoxic and mutagenic base lesions by specifically removing alterations of a single base pair that has been methylated, oxidated, or reduced and corrects single strand interruptions in DNA . Therefore, individual variations in NER/BER are one of the factors that may influence tobacco smoking related cancer risks like HNSCC.
Interestingly, several studies have demonstrated that sequence variations in NER/BER genes contribute to HNSCC susceptibility [8–11]. The ERCC1 gene product is a key enzyme in the NER system, and one particular polymorphism at the ERCC1 gene (C8092A) may affect its mRNA stability, resulting in impaired DNA repair capacity . Two single nucleotide polymorphisms (SNPs) in the XPD gene (Asp312Asn and Lys751Gln), also part of the NER cascade have been associated with suboptimal DNA repair capacity . There are conflicting data regarding SNPs in the BER system and the predilection for developing HNSCC. Li et al, in one of the largest case-control studies of 830 patients with HNSCC and 854 cancer-free controls, evaluated the progression to HNSCC based on polymorphisms in 3 BER non-synonymous SNPs . The BER system enzyme XRCC1 (Arg399Gln), actually inconsistently increases the risk of HNSCC in Caucasians [13–16]. On the other hand, Li et al conclude that polymorphisms in the ADPRT enzyme of the BER system are associated with HNSCC and they demonstrate that individuals with the ADPRT 762Ala/Ala and Ala/Ala1Val/Ala genotypes were at lower risk of developing HNSCC compared with individuals who had the Val/Val genotypes . Further studies to elucidate the genetic predisposition of developing HNSCC in the face of total tobacco burden may provide preventative health benefits to individuals with susceptible polymorphisms in the future.
Marijuana is the most commonly used illegal drug in the United States and the second most commonly smoked substance after tobacco . Habitual marijuana smoking manifests with similar signs and symptoms associated with chronic tobacco use [18, 19]. Furthermore the carcinogenic properties of marijuana smoke are similar to those of tobacco and numerous studies parallel the use of cannabinoids to cancer development [20–22]. Marijuana has been shown to induce cytogenic changes consisting of chromosomal breaks, deletions, and translocations in mammalian cells in vivo . Until recently, there was not enough evidence to suggest a causative relation with oropharyngeal HNSCC, especially those caused by tobacco use . However, HNSCC caused by human papilloma virus (HPV) may be associated with marijuana (see below).
Clearly, normal variation in patient genotype for genes in DNA repair pathways appears to modify baseline risk for cancer development, especially when impacted by environmental toxins such as smoking. In parallel there are a range of germline variants that are much more rare than SNP’s, and accordingly called mutations. These rare heritable events can be sporadic or conserved in families and are frequently recognized due to the high penetrance of one of a number of recognized familial cancer syndromes. Fanconi’s anemia, known for the risk of developing lympho-reticular malignancies due to germline mutations in the care-taker genes FAA, FAD, and FCC, carries a risk for developing second primary cancers in the tongue, pyriform sinus and post cricoid region . Patients with Bloom syndrome are characterized to have mutations in the helicase genes and are predisposed to developing solid tumors in a number of anatomical sites, 6–8% of which arise from the tongue and larynx, respectively . Homozygotes with ataxia telangiectasia who survive into their 20s and 30s are at increased risk of developing chronic T-cell leukemia and solid malignancies of the oral cavity as well as breast, stomach, pancreas, ovary and bladder . Xeroderma pigmentosum, an autosomal recessive disorder of one or more of the XP genes in the NER system, manifests second primaries within the oral cavity in addition to the known risks of skin malignancies [26, 28]. Other such syndromes (affected gene indicated in parentheses) with primary manifestations in the head and neck include Cowden Syndrome (PTEN), Multiple endocrine neoplasia Type I (MEN I), Multiple endocrine neoplasia Type II (MEN II), Neurofibromatosis Type II (NF-2), Retinoblastoma (Rb).
Genetic cancer syndromes are generally recognized by the early age of onset of malignancies in impacted individuals as well as specific or unusual patterns of tumors. Cancer syndrome tumors are of scientific importance out of proportion to their incidence as they point clearly at specific pathways and targets that are key to the development of malignancy, in contrast to sporadic tumors where the causative lesion may be difficult to identify. The importance of this is highlighted in the case of the RET where a number of drugs such as axitinib and vandetanib have been developed, aimed at the mutation’s effect [29, 30].
Viral Associations and New Epidemic of HNSCC caused by HPV
Recently, human papillomavirus (HPV) infection has been identified as an etiologic agent for oropharyngeal carcinoma, a subset of squamous cell carcinomas, which comprises the tongue base and tonsil. Patients with oropharyngeal squamous cell carcinomas that have the HPV genes incorporated in their tumor genome are younger in age (by 3–5 years) and are less likely to have a history of tobacco and alcohol use .
What is most disconcerting is that while the overall incidence of HNSCC (1973–2004) has steadily declined according to the Surveillance Epidemiology and End Results (SEER) data base, the incidence of oropharyngeal cancer is increasing among younger age groups [32–35]. The unsettling implication is that the incidence of HPV-related HNSCC of the oropharynx could overtake HPV-unrelated HNSCC, thought to be associated more with traditional risk factors.
There is substantial evidence that infection with high-risk HPV subtypes, in particular HPV-16, is a risk factor for the development of oropharyngeal cancers [36–47]. In fact, Gillison et al. purport that HPV-positive and HPV-negative HNSCC of the oropharynx should be classified as two distinct cancers based on the clinical and molecular risk factors and etiology . According to their case-controlled study, patients at risk factors for HPV-16 positive oropharyngeal squamous cell carcinoma were more likely to be white (p = 0.06), married (p < 0.001), college educated (p = 0.03) and have an annual income over $50,000 (p < 0.01); while neither intensity or duration of tobacco smoking or alcohol consumption did not increase the odds ratio of HPV-16 positive HNSCC . Although case control studies have not linked marijuana use to HPV-negative HNSCC, Gillison et al. demonstrate a strong association of marijuana use and HPV-16 positive HNSCC and further theorize plausible mechanisms of cannibinoid modulation of the immune system .
High-risk HPV strains (16 and 18) associated with oropharyngeal squamous cell carcinoma (as well as cervical cancer) manipulate cellular pathways within affected cells to activate cell growth and suppress apoptosis. Malignant transformation begins with inactivation of the p53 tumor suppressor gene by E6, while a second HPV protein, E7, inactivates the retinoblastoma tumor suppressor protein (Rb). The HPV E6 and E7 proteins, encoded in the HPV-16 genome, functionally disrupt regulatory cell-cycle and DNA-repair pathways that drives genetic or epigenetic changes during molecular progression of HNSCC . E6 targets the cellular ubiquitin-protein ligase E6-AP, which then targets p53 for ubiquitination and degradation, leaving cell growth unregulated. E7 associates with Rb and p21 blocking the interaction of Rb with E2F and initiating uncontrolled cell division .
Not only is the nascent HPV positive tumor subject to inactivation of tumor suppression genes from the viral genome, but several genes involved in transcription and cell cycle regulation are among the most prominent up-regulated in these tumors. One such cell cycle inhibitor is CDKN2A, which encodes the p16INK4A tumor suppressor protein that functions as a cyclin-dependent kinase inhibitor in the Rb tumor suppressor pathway. Increased expression of p16INK4A may potentially reflect loss of a negative feedback loop associated with inactivation of Rb by HPV E7 . Overexpression of p16INK4A is strongly correlated with HPV infection in head and neck carcinomas and has been used as a surrogate marker for HPV .
Detecting high-risk HPV using in situ hybridization (ISH) from ethanol-fixed and Papanicolaou-stained smears after FNA has a 93% correlation to corresponding tissue sections positive for HPV-16 with PCR . Interestingly, HPV-positive tumors are associated with nonkeratinizing cytomorphology . A recent meta-analysis showed that 26% of HNSCCs from all subsites contain HPV genomic DNA , and it is now estimated that over 50% of oropharyngeal HNSCCs are related to HPV infection . Although ISH and PCR techniques are available, there are no standardized clinical tests approved by the FDA for HPV positive HNSCC tumors.
Whereas in the case of HPV, a virus can usurp normal cellular processes, in the case of most patients, the development of carcinoma is the result of a stepwise accumulation of genetic alterations . Three main steps include initiation, promotion, and progression. For this multiple-step process to succeed, numerous cellular processes and derangements must occur. The creation of an initial, critical, early genetic change helps set into motion the carcinogenic process . Exposure to carcinogenic factors may lead to the abnormal expression of tumor suppressor genes and/or proto-oncogenes, which in turn, activate pathways that lead to the malignant transformation of cells. Oftentimes, this abnormal expression may include a sporadic mutation, deletion, loss of heterozygosity, overexpression, or epigenetic modification such as hypermethylation. For example, telomerase, an enzyme involved in immortalization, has been shown to be reactivated in roughly 90% of HNSCCs, while a deletion of 9p21 is found in 70–80% of these cases. Various point mutations in TP53 and the loss of heterozygosity of 17p are shown to exist in over 50% of HNSCC lesions . Once this occurs, secondary genetic changes create greater genetic instability, shifting the cell toward a more malignant phenotype (Figure 2). Specifically, inactivation of tumor-suppressor genes allow for cellular proliferation to continue with unregulated and autonomous, self-sufficient growth. Proto-oncogenes also play a key role in tumorigenesis, helping the cell attain a malignant phenotype.
Six hallmarks of cancer cells, distinguishing them from their normal counterparts, have been described: (i) self-sufficiency in growth signals, (ii) insensitivity to growth-inhibitory signals, (iii) evasion of programmed cell death, (iv) immortality or unlimited replicative potential, (v) sustained angiogenesis, and (vi) tissue invasion and metastasis . In the following sections we will discuss major pathways, receptors, and proteins implicated in the initiation and/or progression of HNSCC as they relate to aspects of all six of these hallmarks. It is the accumulation of specific abnormalities such as those we describe, likely along with other genetic events and alterations that account for the process of carcinogenesis in HNSCC.
While it is unclear exactly why HPV appears to target certain subsites in the head and neck, the pattern is clear. In contrast, there appears to be a more general phenomenon seen in smokers where broad regions of tissue appear to be damaged, giving rise to multiple premalignant and frankly invasive tumors. In 1953, Slaughter et al. first hypothesized that primary tumors emerge from a layer of pre-cancerous tissue and coined the term “field cancerization” after demonstrating histopathologic changes consistent with genetic aberration from normal mucosa . Forty years after Slaughter proposed field cancerization, Califano et al. demonstrated the molecular basis for histopathologic changes. Samples of dysplastic mucosa and benign hyperplastic lesions displayed loss of heterozygosity at specific loci (9p21 (20%), 3p21 (16%), 17p13 (11%)) . In particular, loss of 9p21 or 3p21 is one of the earliest detectable events leading to the progression to dysplasia. From dysplasia, further genetic alteration in 11q, 13q, 14q creates carcinoma in situ. (Figure 2)
The high rate of recurrence in the location of the primary tumor is thought to be a result of the fact that 30% of histopathologiclly benign squamous cell epithelium consists of a clonal population with genetic alterations seen in HNSCC . Studies using microsatellite analysis and X chromosome inactivation have verified that metachronous and synchronous lesions from distinct anatomic sites in HNSCC often originate from a common clone . This evidence confirms that genetically altered mucosa is difficult to cure in the HNSCC patient since it is on the path to tumorigenesis. Indeed, second primaries are common in the patients with HNSCC.
There is evidence to suggest fundamental changes to the programming of cells, including stem cells, may also be involved in tumorigenesis. One program that is particularly dangerous is the Epithelial-to-Mesenchymal Transition (EMT), a phenotypic change in cells that provides them with the ability to escape from constraints of surrounding tissue architecture. It has been postulated that EMT is the means by which epithelial tumors invade and metastasize to other tissues. As defined by Hugo et al, EMT is a culmination of protein modifications and transcriptional events in response to extracellular stimuli. These changes lead to long term, yet sometimes reversible, cellular changes . Abnormalities in cadherins, tight junctions and desmosomes lead to a decrease in cell-cell adherence and loss of polarity in the cells, increasing the mobility of these cells. More specifically, epithelial cells disassemble their junctional structures, undergo extracellular matrix remodeling, begin to express proteins of mesenchymal origin, and subsequently become migratory . This process has been postulated to be a part of normal embryogenesis, as well as the inflammatory process and wound healing . When the process of EMT becomes pathological, it lacks the tight coordination and regulatory checkpoints that are normally present. Specifically, in the carcinogenic process, EMT causes changes in tumor cell properties that contribute to tumor invasion and metastasis, enabling cancer cell dissemination and self-renewal capabilities . In HNSCC, EMT has been found to play a role, especially in high-risk tumor subtypes. Chung and colleagues showed that genes involved in EMT and nuclear factor-KB (NF-KB) signaling deregulation are the most prominent molecular characteristics of the high-risk tumors in the subset they examined . While it is clear that EMT plays a role in tumorigenesis in many cancers, the complete clinical significance of this process is yet to be fully defined.
While many programs (such as those discussed in Figure 2) are the result of direct damage to the genome, there are other mechanisms of heritable somatic changes in gene expression that do not require direct alteration of the DNA sequence itself. The DNA molecule can be modified, such as by the addition or subtraction of methyl groups without a change in the base composition. Similarly, histones, the structural proteins found in close association with DNA, can be modified such as by acetylation, methylation and ubiquitylation. These non-DNA encoded modification can result in heritable changes in gene expression that are clinically significant, including in the setting of cancer. Different cancers display varying behaviors, likely due to the multiple epigenetic changes and genetic mutations that occur within a tumor environment. Hypermethylation is one such type of epigenetic modification that is increasingly well-characterized. Recently identified as a probable component in the development of carcinoma, hypermethylation in certain promoter regions of a gene can lead to repression of transcription . Numerous studies have implicated this process of aberrant methylation in many tumor suppressor genes, causing them to become inactive .
Molecular Pathways Involved in HNSCC
Increasingly, model systems and other research techniques have helped to decipher pathways of importance for our patients (Figure 3). Knowledge of these pathways has led investigators to interrogate key pathway components for tumor-specific gene mutations, and many have been reported in head and neck tumors (Table 1). Initial clarity in the activated pathways and mutated genes of head and neck tumors has resulted in clinical trials of host of targeted therapies, such as those documented in table 2.. The most promising pathways and agents from this inventories are discussed below.