Introduction
Head and neck squamous cell carcinoma (SCC) is the sixth commonest cancer, with a relatively high rate of local regional recurrence. Despite improvements in the diagnosis and treatment of this malignancy, the survival rate has not improved significantly over the last 30 years.Reference Chin, Boyle, Porceddu, Theile, Parsons and Coman1 Many prognostic indicators have been and are currently being investigated in an attempt to define outcome at the time of diagnosis; however, as is the case for most cancers, sufficiently specific markers are hard to find.
The occurrence of nodal metastasis at presentation remains the single most important prognostic factor, being significantly correlated with poor survival rates.Reference Layland, Sessions and Lenox2–Reference Cerezo, Millan, Torre, Aragon and Otero4 Considerable increases in our knowledge of genetics over the past decade have raised hopes that deoxyribonucleic acid (DNA) based technologies may soon play a role in the early diagnosis and management of head and neck SCC. For instance, genetic testing can be performed to complement histopathological analysis at the time of initial biopsy. Rosin et al. have shown that such genetic testing can be used to predict the progression of oral pre-malignant lesions to invasive cancer; they have demonstrated significant differences in loss of heterozygosity patterns involving multiple genes, comparing progressive and non-progressive cases.Reference Rosin, Cheng, Poh, Lam, Huang and Lovas5
Various studies have attempted to characterise the genetic profiles of head and neck SCC, using a variety of techniques, most commonly detection of loss of heterozygosity.Reference El-Naggar, Hurr, Huff, Clayman, Luna and Batsakis6–Reference Califano, Westra, Meininger, Corio, Koch and Sidransky11 Major events in the pathogenesis of head and neck SCC have been associated with various aberrations at the genetic level which underpin key cellular activities. Despite this knowledge, the controlling mechanisms of head and neck SCC carcinogenesis and progression are still not fully understood. Califano et al. proposed a genetic progression model for head and neck cancer, after using microsatellite analysis which revealed that a number of genetic aberrations correlated with the various histopathological steps (i.e. benign hyperplasia to dysplasia, dysplasia to carcinoma in situ, and finally invasive cancer).Reference Califano, van der Riet, Westra, Nawroz, Clayman and Piantadosi12 An updated version of this model, based on numerous study findings, is given in Figure 1.
Fig. 1 Progression of normal mucosa to metastatic head and neck squamous cell carcinoma (HNSCC), with associated genetic changes. Based on the work by Califano et al. Reference Califano, van der Riet, Westra, Nawroz, Clayman and Piantadosi12 and El-Nagger et al. Reference el-Naggar, Hurr, Batsakis, Luna, Goepfert and Huff52, recently updated by Stafford et al. Reference Stafford, Gleeson, Jones, Clarke, Luxon, Hibbert and Watkinson53 EGF = epidermal growth factor; EGFR = EGF receptor; MMP = matrix metalloproteinase
This seminal work by Califano and colleagues has been corroborated by many other studies over the past decade, using both microsatellite analysis and, more recently, array-based comparative genomic hybridisation, in an attempt to map systematically the gene pathway for head and neck SCC.Reference Pershouse, El-Naggar, Hurr, Lin, Yung and Steck13–Reference el-Naggar, Hurr, Luna, Goepfert, Hong and Batsakis15 Array-based comparative genomic hybridisation is a more recent approach used for genome-wide determination of DNA copy number alterations. Its resolution level is higher than conventional chromosome-based comparative genomic hybridisation (CGH) methodology, 1–2 Mb vs 10–20 Mb level respectively.Reference el-Naggar, Hurr, Luna, Goepfert, Hong and Batsakis15
One of the major difficulties in head and neck SCC research is the fact that the various sub-sites behave differently at both the biological and the clinical level. An interesting study by Huang et al. has shown that different chromosomal aberrations apparently play significant roles in the initiation and/or progression of SCC at different sites.Reference Huang, Yu, McCormick, Mo, Datta and Mahimkar16 This study, using comparative genomic hybridisation, studied 75 head and neck SCC patients (18 pharyngeal SCCs, 23 laryngeal SCCs and 34 oral SCCs). They concluded that ‘the most important chromosomal events for progression of head and neck cancer were: +3q, +5p, +8q and −3p for all subgroups of [head and neck] SCC; additionally, +7q, +17q, −9p and −13q were important for [pharyngeal] SCC; +7p, +9q, +11q12–13, +14q and +17q for [laryngeal] SCC; and +1p and +11q12–13 for oral SCC’, where ‘ + ’ means gain and ‘ − ’ means loss of the chromosome arm or sub-region. Others have undertaken similar analyses. The aim of this paper is to discuss briefly the potential for applying our current knowledge of head and neck SCC genetic profiles to clinical practice, either through DNA-based analysis or the translation of the genetic data into knowledge about potentially detectable proteins.
Current and future clinical applications
Diagnosis
The current methods for diagnosing head and neck SCC have major limitations. Usually, diagnosis involves a combination of clinical history-taking, endoscopy and, possibly, tissue biopsy. Despite arriving at a diagnosis, at this stage it is impossible to predict accurately an individual patient's outcome, as head and neck SCC tumours vary significantly in their growth behaviour and response to treatment (be it radio- or chemotherapy). It is anticipated that identification of a tumour's genetic profile, and comprehension of the implications of this, will both aid diagnosis and inform prognosis and treatment. The ability to make an early, personalised diagnosis without undergoing a significantly invasive procedure would be of great advantage to both patient and clinician.
Patmore et al. have shown that the most common aberrations in head and neck SCC are 3q (90 per cent), 8q (65 per cent), 1q (50 per cent), 5p (43 per cent), 2q (41 per cent) and 11q (41 per cent), and that the most common deletions are 3p (57 per cent), 1p (54 per cent), 4p (48 per cent), 13q (48 per cent), 11q (41 per cent) and 10q (37 per cent).Reference Patmore, Ashman, Cawkwell, MacDonald, Stafford and Greenman17 These authors assessed DNA from 23 paired specimens of primary tumour from various sub-sites and their matched lymph node metastases. In a similar study, Sparano et al. used array-based comparative genomic hybridisation to map the genetic profile of oral SCC.Reference Sparano, Quesnelle, Kumar, Wang, Sylvester and Feldman18 They reported results as follows:
[The] genomic regions most frequently amplified (>35 per cent) were located on 3q, 5p, 8q, 9q and 20q, although [the] regions most frequently deleted (>40 per cent) involved chromosomes 3p, 8p, 13q and 18q. Cancer-related genes altered in greater than 25 per cent of [oral] SCC samples were identified (22 amplified, 17 deleted)… .
Other studies have shown similar findings of distinct chromosomal aberrations being associated with different types of head and neck SCC, based on the site of origin.Reference Noutomi, Oga, Uchida, Okafuji, Ita and Kawauchi19, Reference Oga, Kong, Tae, Lee and Sasaki20
Prognosis (Table 1)
Table I Studies analysing chromosomal loss of heterozygosity and association with prognosis: 1994–2008
Data obtained by a comprehensive search of PubMed, Ovid and Google, using the key words ‘genetic profile’, ‘loss of heterozygosity’, ‘prognosis’, ‘head and neck’, ‘oral’, ‘hypo-pharyngeal’, ‘laryngeal’, ‘squamous cell carcinoma’ and ‘head and neck squamous cell carcinoma’. LOH = loss of heterozygosity
The prognostic markers currently used to predict the survival and likely disease course of head and neck SCC are based on the histological tumour–node–metastasis (TNM) staging system (i.e. T stage, presence and extent of nodal metastasis, tumour site, tumour volume and thickness).Reference Le Tourneau, Velten, Jung, Bronner, Flesch and Borel21 The drawback of using T stage and tumour site is that these are average parameters which alone are not significant predictors of survival.Reference Baatenburg de Jong, Hermans, Molenaar, Briaire and le Cessie22, Reference Verschuur, Irish, O'Sullivan, Goh, Gullane and Pintilie23 Attempts at using histological characteristics (e.g. lymphovascular and perineural invasion as well as regional extracapsular spread), identified in the tumours and/or nodes as determinants of prognosis, have been helpful; however, these are also relatively non-specific and have not been shown to be independent predictors of survival.Reference Soo, Carter, O'Brien, Barr, Bliss and Shaw24, Reference Unal, Ayhan and Hosal25 Various studies have shown that nodal status is a bad prognostic marker and that spread to the nodes is associated with a significantly reduced time to recurrence and death.Reference Zatterstrom, Wennerberg, Ewers, Willen and Attewell26–Reference Shingaki, Suzuki, Kobayashi and Nakajima28 However, all the conventional parameters are general markers which lack the ability to predict individual prognosis or response.
Patmore et al. have shown that dysplasia correlates with loss of heterozygosity at 3p21, 5q21, 9p21 and 17p13 in early laryngeal carcinogenesis.Reference Patmore, Ashman, Cawkwell, MacDonald, Stafford and Greenman17 They concluded that genomic changes in pre-malignant laryngeal lesions could be of potential use as markers of progression to invasive carcinoma. Others have investigated these associations and shown similar changes associated with dysplastic tissue. A comprehensive study by Bockmühl et al., using comparative genomic hybridisation analysis on 113 primary head and neck SCC patients, demonstrated that gains at 3q21–29 and 11q13 and loss at 8p21–22 acted as independent prognostic markers which carried a higher statistical significance than nodal status.Reference Bockmühl, Schlüns, Küchler, Petersen and Petersen29 Genetic aberrations were independent markers, and this allowed for molecular dissection of patients at low clinical risk (i.e. pN0 and pT2 tumours). Therefore, a sub-group of patients of N0 status, who would not normally be treated, potentially could now be selected for aggressive adjuvant chemotherapy and treated differently due to their specific genetic phenotype. Similarly, head and neck SCC studies have consistently associated chromosome 11q13 gains with a poor prognosis.Reference Akervall, Jin, Wennerberg, Zätterström, Kjellén and Mertens30, Reference Meredith, Levine, Burns, Gaffey, Weiss and Erickson31 A number of groups have attempted to identify a metastatic phenotype.Reference Patmore, Ashman, Cawkwell, MacDonald, Stafford and Greenman17, Reference Wreesmann, Wang, Goberdhan, Prasad, Ngai and Schnaser32 Although there is no clear consensus, largely because the study cohorts have been relatively small, a number of aberrations have been reported, such as gains at 10p11–12 and 11p and deletions at chromosomes 4q22–31, 9p13–24 and 14q in the nodal metastasis, when compared with the corresponding primary tumours.Reference Wreesmann, Wang, Goberdhan, Prasad, Ngai and Schnaser32 These aberrations were seen in two matched tumour–node pairs assessed using a modified comparative genomic hybridisation assay, whereby DNA from the tumour and nodes was differentially labelled and then hybridised to a normal karyotype.
It is hoped in the future that genetic stratification of disease can be performed, in order to allow clinicians to determine the most appropriate treatment for patients, particularly those with early stage disease. In the same way, this information could inform decisions for patients with advanced stage disease, helping to predict whether patients will benefit from resection and reconstructive surgery or from a more conservative approach. If this is to become common clinical practice in head and neck cancer management, appropriately powered, large scale studies need to be undertaken.
Treatment
The current treatment modalities for head and neck SCC largely comprise surgery, radiotherapy and, to a lesser extent, chemotherapy. However, a number of studies (albeit early-stage) have investigated the correlation of genetic profile with response to certain treatments.
Radiotherapy and genetic profile screening for head and neck squamous cell carcinoma
Radiotherapy is a key treatment modality for head and neck SCC, both in the early and advanced stages. Up to 27 per cent of patients with T2 tumours of the larynx are said to demonstrate locally persistent or recurrent disease at the original site, requiring salvage surgery to achieve a definitive cure.Reference Stevens, Castle and O'Brien33 It would therefore be extremely beneficial to patients if clinicians could identify, prior to treatment, whether their tumour was radiotherapy-responsive or -resistant. This would prevent a worthless therapy being given, saving the patient the associated loss in quality of life as well as saving the resources of the health service.
A study by Singh et al., at the Memorial Sloan–Kettering Cancer Center, screened for genetic aberrations associated with radiation response, using comparative genomic hybridisation on five head and neck SCC cell lines after exposing the cells to a single course of radiation (400 cGy).Reference Singh, Kim, Carew, Yu, Shaha and Wolden34 They demonstrated that no recurrent aberrations were unique to the radiation-resistant cell lines. However, the three radiation-sensitive cell lines did have recurrent gains at 7p and 17q and losses at 5q, 7q and 18q. This means that comparative genomic hybridisation analysis may enable prediction of radiation response even before treatment. However, the small number of cell lines analysed means that this result can only be considered a preliminary finding. To address the same question from a clinical perspective, Nix et al. assessed pre-treatment tissue biopsies from 124 patients with early stage (T1–T2, N0) laryngeal SCC.Reference Nix, Cawkwell, Patmore, Greenman and Stafford35 Patients were split into two equal-sized groups (n = 62); one group had failed radiotherapy (and hence were considered radio-resistant) and the other had been successfully treated. Both groups were matched for T stage, laryngeal sub-site and smoking history. Using immunohistochemistry, Nix and colleagues demonstrated that the expression of the apoptotic proteins bcl-2, bcl-XL, bax, bak and survivin was associated with radio-resistance in laryngeal cancer. The radio-resistant group over-expressed bcl-2 and bcl-XL and had a loss of bax expression in pre-treatment biopsies. Nix et al. reported that bcl-2 had an accuracy of 71 per cent in predicting radiotherapy outcome. Predicting radio-resistance or -responsiveness should significantly enhance outcome, as the clinician would be able to recommend conservative laryngeal surgery as an alternative first-line treatment to radiotherapy, or consider other modalities from the outset. This study confirmed the earlier work of the same group using a smaller cohort of laryngeal tumours.Reference Condon, Ashman, Ell, Stafford, Greenman and Cawkwell36
p53 and p53 vaccines
The p53 gene is the most commonly mutated gene in all cancers, including head and neck SCC. This subsequently leads to over-expression of a mutant form of the p53 protein. The normal p53 protein is activated in response to cellular stress, in order to arrest the cell cycle so that there is an opportunity for the damaged DNA to be repaired or, if this cannot happen, to initiate apoptosis. Therefore, when there is a mutation of the p53 gene, as in cancer, there is loss of the function of p53 protein. Numerous studies have investigated the importance of various p53 mutations in the development of head and neck SCC, because of this cancer's high prevalance.Reference Delfino, Casartelli, Garzoglio, Mereu, Bonatti and Margarino37, Reference Shin, Charuruks, Lippman, Lee, Ro and Hong38 Furthermore, it has been shown that the introduction of a wild-type p53 gene results in a promising anti-tumour strategy.Reference Brown and Benchimol39–Reference Hoffmann, Donnenberg, Finkelstein, Donnenberg, Friebe-Hoffmann and Myers41
There is evidence that human leukocyte antigen (HLA) A2 restricted cytotoxic T lymphocytes specific for human wild-type sequence p53 epitopes lyse tumour cells expressing mutant p53.Reference Umano, Tsunoda, Tanaka, Matsuda, Yamaue and Tanimura42–Reference McArdle, Rees, Mulcahy, Saba, McIntyre and Murray44 Therefore, treatment modalities that would target tumours over-expressing mutant p53 are being studied. Hoffmann et al., using cytotoxic T lymphocytes from circulating precursor T cells from 30 HLA-A2.1 positive head and neck SCC patients and 31 controls, have demonstrated the potential of using HLA-A2 restricted cytotoxic T lymphocytes specific for human wild-type p53 epitopes which lyse tumour cells expressing mutant forms.Reference Hoffmann, Bier, Donnenberg, Whiteside and De Leo45 However, Hoffman and colleagues warn that ‘…in vivo p53-specific cytotoxic T lymphocytes might play a role in the elimination of tumour cells expressing the p53 264–272 epitope (‘immunoselection’), leading to the outgrowth of ‘epitope loss’ tumour cells'. They also suggest that more immunogenic variant peptides of the 264–272 epitope could be used in those patients whose cytotoxic T lymphocytes do not respond against the original wild-type form. Other studies have demonstrated similar outcomes with different combinations of HLA molecules and peptides.Reference Eura, Chikamatsu, Katsura, Obata, Sobao and Takiguchi46, Reference Asai, Storkus, Mueller-Berghaus, Knapp, DeLeo and Chikamatsu47 This remains a promising modality of treatment, although large scale clinical trials involving multiple peptides are required.
Intra-operative surgical margin analysis
One of the difficulties in surgical management of head and neck SCC is determination of whether a surgical margin is free of tumour or not. Between 10 and 30 per cent of all surgically treated patients develop local recurrence despite having a histologically free margin at the time of surgery.Reference van Houten, Leemans, Kummer, Dijkstra, Kuik and van den Brekel48 Hence, predicting sub-clinical tumourigenesis could play a role intra-operatively in determining surgical margins and reducing local recurrence.Reference Brennan, Mao, Hruban, Boyle, Eby and Koch49 Chromosome imbalance associated with head and neck SCC malignancy can be detected between tumour margins and clinically normal adjacent cells. Barrera et al. have shown that this is possible using the fluorescence in situ hybridisation technique and DNA probes specific for 14 human chromosomes (1, 2, 3, 6, 7, 8, 9, 10, 11, 12, 15, 17, X and Y).Reference Barrera, Ai, Pan, Meyers and Varella-Garcia50 However, it is important to note that this was a small study involving only 10 patients. In a similar study involving 52 patients undergoing primary resection for head and neck SCC, Nathan et al. assessed the proto-oncogene eIF4E (4E), which they report as being ‘elevated in 100 per cent of head and neck squamous cell carcinoma tumours and […] of prognostic value in predicting recurrence’.Reference Nathan, Amirghahri, Rice, Abreo, Shi and Stucker51 They histologically examined all tissue from tumours classified as ‘tumour-free’ for the presence or absence of 4E, p53 and MMP-9. They found that 4E over-expression in the margins was a more sensitive predictor of recurrence compared with p53. In the future, surgical treatment of head and neck SCC could incorporate an intra-operative genetic test to ascertain tumour-free margins, based on over- or under-expression of specific genes such as 4E. Such a test would hopefully contribute to reducing the significant problem of local and regional recurrence and the associated poor outcome.
Future and conclusion
Head and neck squamous cell carcinoma at distinct sub-sites behaves very differently. Current methods of evaluation and treatment make it extremely difficult to individualise treatment. However, with our growing understanding of the genetic changes that underlie head and neck SCC development, it would seem a realistic aspiration that tumours could be characterised based on a specific genetic profile. Once this is possible, patients could receive individualised prognostic advice, with the next logical step being highly personalised treatment strategies. It is hard to predict when genetics-based diagnosis and treatment will become widespread; however, the rapid pace of advances in the field over the past decade leads the authors to believe that this will become a reality. Screening head and neck SCC prior to radiotherapy could be a common practice within the decade; however, genetics-based targeting therapies still require the development of safe and effective delivery vehicles.
