International Journal of

Radiation Oncology biology

physics

www.redjournal.org

Clinical Investigation: Head and Neck Cancer

Genetic Susceptibility to Head and Neck Squamous Cell Carcinoma Martin Lacko, MD, PhD,* Boudewijn J.M. Braakhuis, PhD,y Erich M. Sturgis, MD, MPH,z Carsten C. Boedeker, MD,x Carlos Sua´rez, MD, PhD,k,{ Alessandra Rinaldo, MD, FACS, FRCSEd ad hominem,** Alfio Ferlito, MD, FACS, FRCSEd ad hominem,** and Robert P. Takes, MD, PhDyy *Department of OtorhinolaryngologydHead and Neck Surgery, Maastricht University Medical Center, Maastricht, The Netherlands; yDepartment of OtolaryngologydHead and Neck Surgery, VU University Medical Center, Amsterdam, The Netherlands; zDepartment of Head and Neck Surgery and Department of Epidemiology, The University of Texas MD Anderson Cancer Center, Houston, Texas; xDepartment of OtorhinolaryngologydHead and Neck Surgery, Albert-LudwigsUniversity, Freiburg, Germany and Department of Otorhinolaryngology - Head and Neck Surgery, HELIOS Hanseklinikum Stralsund, Stralsund, Germany; kDepartment of Otolaryngology, Hospital Universitario Central de Asturias, Oviedo, Spain; {Instituto Universitario de Oncologı´a del Principado de Asturias, Oviedo, Spain; **ENT Clinic, University of Udine, Udine, Italy; and yyDepartment of OtolaryngologydHead and Neck Surgery, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands Received Jul 5, 2013, and in revised form Sep 16, 2013. Accepted for publication Sep 17, 2013.

Head-and-neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, and its incidence is growing. Although environmental carcinogens and carcinogenic viruses are the main etiologic factors, genetic predisposition obviously plays a risk-modulating role, given that not all individuals exposed to these carcinogens experience the disease. This review highlights some aspects of genetic susceptibility to HNSCC: among others, genetic polymorphisms in biotransformation enzymes, DNA repair pathway, apoptotic pathway, human papillomavirus-related pathways, mitochondrial polymorphisms, and polymorphism related to the bilirubin-metabolized pathway. Furthermore, epigenetic variations, familial forms of HNSCC, functional assays for HNSCC risk assessment, and the implications and perspectives of research on genetic susceptibility in HNSCC are discussed. Ó 2014 Elsevier Inc.

Head-and-neck squamous cell carcinoma (HNSCC) accounts for about 640,000 new cases of cancer worldwide and approximately 355,000 deaths annually (1). Exposure to tobacco and its smoke and more than moderate alcohol consumption are the most important etiologic factors in HNSCC carcinogenesis (2-6). However, past infections with high-risk serotypes of human papillomavirus (HPV) are responsible for a significant proportion of oropharyngeal cancer cases, whereas infection with Epstein-

Barr virus accounts for the majority of nasopharyngeal carcinomas (NPCs) (7, 8). Other factorsdnotably, chewing betel nut or shamma, poor oral hygiene, exposure to carcinogenic chemicals, and possibly infection with human immunodeficiency virusdare also associated with an increased risk of HNSCC at specific sites (9-14). Exposure to (pro)carcinogens, especially those in tobacco and alcohol, plays a key role in the initiation of head-and-neck

Reprint requests to: Robert P. Takes, MD, PhD, Department of OtolaryngologydHead and Neck Surgery, Radboud University Nijmegen Medical Center, PO Box 9101, 6500 HB Nijmegen, The Netherlands. Tel þ31-24-3613508; E-mail: [email protected]

This article was written by members and invitees of the International Head and Neck Scientific Group (www.IHNSG.com). Conflict of interest: none.

Int J Radiation Oncol Biol Phys, Vol. 89, No. 1, pp. 38e48, 2014 0360-3016/$ - see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ijrobp.2013.09.034

Volume 89  Number 1  2014 carcinogenesis. Yet, even among highly exposed persons, HNSCC will develop on only a small proportion. This means that an individual’s genetically coded capability to cope with the carcinogens probably determines who experiences cancer and who does not. Processes such as biotransformation, detoxification, and elimination of (pro)carcinogens, together with DNA repair mechanisms and apoptotic pathways, are probably the most important factors in the body’s defense against tobacco- and alcohol-induced cancers like HNSCC (Fig. 1) (15, 16). It should be stressed that structural polymorphic variants exist in the genes that code for the enzymes catalyzing the reactions in the above-mentioned defense mechanisms. A genetic polymorphism may alter the activity of the enzyme encoded by a polymorphic gene, thereby determining the differences in individuals’ responses to carcinogens and in their susceptibility to cancer (17-19). However, one’s genetic predisposition could influence not only the carcinogenesis related to tobacco and alcohol but also the susceptibility to HNSCC related to HPV and Epstein-Barr virus (20-22). Certain types of cancer have a strong genetic predisposition (eg, some breast, ovarian, and colorectal cancers). They are caused by a rare mutation in a single gene with a high penetrance susceptibility (eg, BRCA1, BRCA2, APC) (23). However, except in some rare familial cases, HNSCC is a multifactorial disease. It is caused by interactions between exposure to carcinogens and a particular risk-bearing genotype. In the event of HNSCC, an individual’s genetic susceptibility most likely comes from a combination of several unfavorable but rather common genetic polymorphisms. The most usual are variations of a single nucleotide base in the DNA molecule, a single nucleotide polymorphism (SNP) (23, 24). However, other genetic polymorphisms and structural DNA variations might also modify an individual’s risk for the development of a variety of diseases, including HNSCC (25). Besides the above-mentioned structural variations in the DNA chain, some modifying effects of epigenetic variations in carcinogenesis have recently been recognized (26). The genetic and epigenetic variations probably explain most of the difference in interindividual susceptibility to many types of disease, including cancer (27-29). This article gives an overview of genetic polymorphisms and epigenetic variations in the pathways potentially involved in headand-neck carcinogenesis and their association with the susceptibility to HNSCC. It also discusses the uncommon familial form of HNSCC and the functional assays for HNSCC risk assessment.

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Finally, it presents some implications and perspectives on research into genetic susceptibility to HNSCC. Key elements of the epidemiologic studies mentioned in this review are summarized in Table 1.

Polymorphisms in Genes Coding for Biotransformation and Detoxification Enzymes As DNA analysis technology improves, an increasing number of studies are performed on genetic polymorphisms and their impact on susceptibility to head-and-neck cancer. Given the biological plausibility of an theory that individual differences in biotransformation efficiency and detoxification of (pro)carcinogens might be important HNSCC risk factors, most HNSCC susceptibility studies deal with the polymorphisms in genes encoding the enzymes involved in these reactions. Activated (pro)carcinogens present in tobacco (and its smoke) and alcohol, particularly polycyclic aromatic hydrocarbons and acetaldehyde, respectively, may react with the exposed mucosa of the upper aerodigestive tract and form DNA adducts. The latter can cause mutations in crucial genes involved in carcinogenesis, such as tumor suppressor genes or oncogenes, ultimately leading to cancer (30-32). Biotransformation and detoxification of the main (pro)carcinogens in tobacco (smoke) and alcohol occur in 2 phases. First, mostly lipophilic compounds are transformed into more polar compounds. The intermediate metabolites created in this phase are highly mutagenic and possibly carcinogenic (33, 34). After conjugation, in the second phase of biotransformation, these compounds are converted into a more water-soluble and less biologically active form, which facilitates their excretion from the body (Fig. 1). Existing polymorphisms in the genes coding for the enzymes involved in the biotransformation of (pro)carcinogens can affect enzyme activity. That, in turn, may influence an individual’s susceptibility to HNSCC. The literature contains over 200 publications on polymorphisms addressing their impact on HNSCC risk. These studies cover the most important phase 1 enzymesdeg, the cytochrome P-450 (CYP) family and human microsomal epoxide hydrolase (mEH)dand phase 2 enzymes such as glutathione S-transferases (GSTs), UDP-glucuronosyltransferase (UGTs), aldehyde dehydrogenase (ALDH), and alcohol dehydrogenase (ADH). The findings and conclusions in the literature are often inconsistent. Actually, many studies are too small to yield

Fig. 1. Simplified and modified scheme of tobacco-related and alcohol-related carcinogenesis (black arrows) and related anticarcinogenic defense mechanisms (gray arrows) (16).

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International Journal of Radiation Oncology  Biology  Physics

Table 1 Epidemiologic studies on genetic polymorphisms, epigenetic variations, and germline mutations in relation to HNSCC risk, as highlighted in this review Object of performed epidemiologic study (and racial characteristics of study population) Genetic polymorphisms Genetic polymorphisms related to biotransformation and detoxification enzymes GSTM1 (null), pooled analysis (C, AA, As) (37) GSTT1 (null), pooled analysis (C, AA, As) (37) GSTM1 nullþGSTT1 nullþ GSTP1 Val105, pooled analysis (C, AA, As) (37) rs476364 (variant in multiple genes cluster including ALDH2), GWAS, (C,AA) (39) rs1573496-ADH7 (ADH gene cluster variant), GWAS, (C,AA) (39) rs1229984-ADH1B (ADH gene cluster variant), GWAS, (C,AA) (39) rs698-ADH1C (ADH gene cluster variant), GWAS, (C,AA) (39) Genetic polymorphisms related to the DNA repair pathway XRCC1 exon 6, codon 194 (C/T), meta-analysis, (significant only for Asians) (18) XPD exon 6, C22541A codon 156 (A/A), meta-analysis, (significant only for Caucasians) (18) XPD Asp312Asn (G/A), meta-analysis, (significant for both Asians and Caucasians) (18) rs1494961 (variant near DNA repair genes HEL308 and FAM175A), GWAS, (C,AA) (39) rs927220-RAD51L1 DNA repair gene, (As) (43) rs11158728-RAD51L1 DNA repair gene, (As) (43) Genetic polymorphisms in the apoptotic pathway rs3810294 (C/T) in PUMA gene, (C) (46) rs9904341 (C/G) in BIRC5 gene, (As) (47) rs9904341 (C/G) in BIRC5 gene, (As) (19) rs2071214 (A/G) in BIRC5 gene, (As) (19) rs1042489 (C/T) in BIRC5 gene, (As) (19) Genetic polymorphisms in HPV-related HNSCC pathways TGF-beta1 (T869C; C509T; G915C), HPV16þ versus HPV16- oropharyngeal carcinoma (53) (without racial restriction, >90% Caucasians) rs11801299 (G/A) in MDM4 gene, (C) (22) rs10900598 (G/T) in MDM4 gene, (C) (22) rs1380576 (C/G) in MDM4 gene, (C) (22) Polymorphisms in mitochondrial DNA (mtDNA) np16362 (T/C) mtDNA; np16519(T/C) mtDNA; (mtMSI) at D310(63), (As) (63) np12308 (A/G) mtDNA; np11467 (A/G) mtDNA; (As) (64) np12308 (A) in combination with GSTP1 Ile/Ile; (As) (64) Genetic polymorphisms in bilirubin-related pathway rs8175347, TATA-box repeat polymorphisms in UGT1A1, (C) (25) Genetic polymorphisms in E2F of transcription factors E2F1 and E2F2 Combination of 9-10 risk genotypes: rs3213182AA, rs3213183GG, rs3213180 GG, rs321318121

Cases/controls (and tumor localizations)

Strength of association with HNSCC

2224/2517 (mostly O,P,L) 1929/1830 (mostly O,P,L) -

OR 1.32 (95% CI 1.07-1.62) OR 1.25 (95% CI 1.00-1.57) OR 2.06 (95% CI 1.11-3.81)

8232/11064 (O,P,L,E)

OR 1.12 (95% CI 1.07-1.17)

8545/11657 (O,P,L,E)

OR 0.74 (95%CI 0.69-0.80)

8527/11653 (O,P,L,E)

OR 0.62 (95% CI 0.56-0.68)

7890/10938 (O,P,L,E)

OR 1.10 (95%CI 1.05-1.15)

844/963 (O,P,L)

OR 1.78 (95% CI 1.13-2.82)

923/1731 (O,P,L)

OR 0.74 (95% CI 0.57-0.95)

2103/3719 (O,P,L)

OR 1.14 (95% CI 1.01-1.29)

8136/11032 (O,P,L,E)

OR 1.13 (95% CI 1.08-1.17)

2323/2052 (N) 2323/2052 (N)

OR 1.20 (95% CI 1.10-1.30) OR 1.17 (95% CI 1.08-1.27)

380/333 (O,P,L) 855/1036 (N) 439/424 (O) 439/424 (O) 439/424 (O)

OR 1.5 (95% CI 1.0-2.1) OR 1.40 (95% CI 1.13-1.73) OR 1.7 (95% CI 1.0-3.1) OR 4.5 (95% CI 1.1-17.5) OR 2.8 (95% CI 1.5-4.9)

HPV16þ/HPV16-: 147/53 (P)

OR 2.28 (95% CI 1.16-4.50)

86 HPV16þ vs 100 HPV16-/321 (O,P, L) 86 HPV16þ vs 100 HPV16-/321 (O,P, L) 86 HPV16þ vs 100 HPV16-/321 (O,P, L)

OR 6.1 (95%CI 2.9-12.9) OR 11.4 (95%CI 4.9-26.4) OR 6.1 (95% CI 3.4-11.1)

7 FNPCþ 20 SNPC/142 (N)

Significant, but small study size

Cases/controls: 310/389 (O)

OR 1.7(95% CI 1.1-2.6)

Cases/controls 307/381 (O)

OR 2.6 (95% CI 1.4-4.9)

421/417 (O,P,L)

OR 1.37 (95% CI 1.02-1.83)

1096/1090 (O,P,L)

OR 1.62 (95% CI 1.14-2.30) (continued on next page)

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Table 1 (continued ) Object of performed epidemiologic study (and racial characteristics of study population) GG, rs2742976 GTþTT, rs6667575 GAþAA, rs3218203 CC, rs3218148 AA, rs3218211 CC, and rs3218123 GTþTT (C) (76) Epigenetic variations rs1057035, DICER, miRNA pathway, (As) (84) rs8126 (T/C), TNFAIP2, (C) (85) rs3746444 (A/G) hsa-mir-499 (C) (86) rs11614913 (C/T) MIR196A2 (92% C 8% non-C) (87) Hereditary form of HNSCC (Germline mutation, Familial clustering) Germline mutations in FA gene family (94) Risk of HNSCC in the smoke- and alcohol-exposed firstdegree relatives of patients with HNSCC, pooled analysis (without racial restriction) (95) 3p21.31-21.2, tumor suppressor cluster, linkage analysis in high-risk families, (As) (20)

Cases/controls (and tumor localizations)

Strength of association with HNSCC

397/900 (98% O,P,L)* 1077/1073 (O,P,L) 1109/1130 (O,P,L) 484/555 (O,P,L)

OR 0.65 (95% CI 0.46-0.92) OR 1.48 (95% CI 1.06-2.05) OR 0.83 (95% CI 0.69-0.99) OR 0.8 (95%CI 0.56-0.99)

754 FA patients 8967/13627

SIR 500 (95% CI 300-781) OR 7.2 (95% CI 5.5-9.5)

46/96 (N)

Logarithm of odds for linkage 4.18

Abbreviations: A Z adenine; AA Z African-American; As Z Asian; C Z Caucasian; C Z cytosine; CI Z confidence interval; E Z esophagus; FA Z Fanconi anemia; FNCP Z familial nasopharyngeal carcinoma; G Z guanine; HNSCC Z head-and-neck squamous cell carcinoma; L Z larynx; N Z nasopharynx; np (mtDNA) Z nucleotide pair number (in mitochondrial DNA); O Z oral cavity; OR Z odds ratio; P Z pharynx (without nasopharynx); rs (number) Z reference number for a particular genetic polymorphism (reference SNP cluster ID); SIR Z standardized incidence ratio; SNPC Z sporadic nasopharyngeal carcinoma; T Z thymine. * 98% cases with HNSCC of oral cavity, pharynx, and larynx; 2% cases with carcinomas localized in nasal sinuses and salivary glands. Names of listed gene polymorphisms were taken from original publications; where mentioned, rs-numbers were used. Explanation of the enzymes names is given in the text. If not otherwise specified, the data are based on patient populations with HNSCC localized in more than 1 particular cancer subsite.

a significant correlation between genetic polymorphisms and disease risk. On the whole, though, the body of genetic epidemiologic research performed to date suggests that genetic polymorphisms in some biotransformation enzymes have a modest influence on the susceptibility to HNSCC (31, 35-40). Considering only studies with sufficient statistical power, the most consistent significant associations were obtained for genetic polymorphism in GST (GSTM1null ), where the variant with absent enzyme function increases HNSCC risk (36, 37). In a meta-analysis and pooled analysis, Hashibe et al (37) found a borderline association with a modest increased risk of HNSCC for the GSTT1 null, the GSTP1 polymorphism (ILE105VAL), and the CYP1A1 polymorphism (CYP1A1 Val462) with an absent or decreased enzyme function associated with an increased risk of HNSSC. Having all 3 of the above-mentioned GST polymorphisms led to a higher HNSCC risk in comparison with having only 1. A large genome-wide association (GWA) study was conducted recently by the International Head and Neck Cancer Epidemiology (INHANCE) consortium. It found that 3 common genetic variants in alcohol metabolizing enzyme ADH were associated with an increased risk of HNSCC (rs1573496-ADH7, rs1229984-ADH1B, rs698-ADH1C). In the same study, the 12q24 variant (rs4767364) located in the region containing multiple genesdincluding 1 encoding for another enzyme involved in alcohol metabolism, ALDH2dwas associated with an increased risk of HNSCC (39). The small sample size in many studies impedes a proper analysis of the gene-environment interaction and thus poses restrictions on the interpretation of the results. The reason is that a study’s power to detect a given odds ratio (ie, risk/association) is inversely related to the prevalence of the investigated genetic polymorphism and the size of the study population (41). For instance, to detect an odds ratio of 1.25 with a risk allele

frequency of 20%, about 1800 study subjects (cases and controls) are required, assuming a power of 80% and P60 years), heavy smokers, and heavy drinkers.

Genetic polymorphisms in E2F transcription factors E2F is a family of genes coding for the transcription factor proteins involved in cell cycle regulation (74, 75). Dysregulated expression of most members of the E2F family has been detected in many human cancers. A large study that included 1096 patients with HNSCC and 1090 healthy control individuals investigated the association of 10 common SNPs of the E2F gene for transcription factors 1 and 2 (E2F1 and E2F2) and the risk of HNSCC (76). It reported a statistically significant increase in the risk of HNSCC associated with the combined risk genotypes. A significantly increased risk was observed for those who carried 9 to 10 risk genotypes compared with those carrying 5 to 8 or 0 to 4 genotypes. The joint effect was more pronounced among patients with oropharyngeal cancer, younger adults (57 years), men, nonsmokers, nondrinkers, and individuals with a family history of cancer in first-degree relatives.

Epigenetic Variations and Susceptibility to Head-and-Neck Cancer Epigenetic information can be characterized as stable, heritable, and transmissible during cell division but not encoded by the DNA sequence (26). This information is important in the activation or silencing of specific genes involved in physiologic processes, but also in diseases like cancer. Therefore, pre-existing interindividual epigenetic variation might partly determine one’s susceptibility to cancer, including HNSCC.

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The most frequently studied and elucidated epigenetic mechanisms regulating the expression of genes and their role in carcinogenesis are DNA methylation and, more recently, the posttranscriptional regulation of gene expression by microRNAs (miRNAs) (77-80). MiRNAs are single-stranded, 21- to 23nucleotide-long RNA molecules encoded in the genome. Although their function is not entirely clear, they seem to play an important role in cellular processes like differentiation, proliferation, cell cycle progression, and apoptosis (81). To date, over 1000 miRNAs have been identified. Genetic variations in miRNA processing genes and miRNA binding sites could promote carcinogenesis by affecting interactions between miRNA and miRNAmessenger RNA (mRNA), the degradation of target mRNAs, or the repression of their translation. Elevated or decreased expression of miRNAs has been found in cancers of various types, including HNSCC (82). The study of genetic variants of miRNAs in cancer susceptibility is interesting for several reasons. The sequence variations in miRNA binding sites and target genes may influence miRNA expression and thereby affect the expression of mRNA levels of the specific target genes. There is also some evidence that miRNA is closely involved at the genetic level in the susceptibility to progression and to the prognosis of, and response to, therapy for many complex diseases, including cancer (83). A few recent studies have investigated the association between genetic variants and levels of expression of miRNAs and target genes as a risk factor for HNSCC. In a case-control study involving 397 HNSCC patients and 900 healthy individuals, it was suggested that potentially functional polymorphisms of DICER, an enzyme responsible for the cleavage of miRNA precursors, may play a role in the development of HNSCC in general and oral cavity cancer in particular (84). Another case-control study, including 1077 patients with HNSCC and 1073 cancer-free control individuals, investigated the associations between the risk of HNSCC and 4 common polymorphisms at the miRNA binding sites of the tumor necrosis factor-aeinduced protein TNFAIP2, an important gene in apoptosis. The rs8126 variant CC and CC/CT genotypes were found to be associated with a significantly increased risk of HNSCC as compared with the TT genotype (85). The same group also studied SNPs in 4 miRNA polymorphisms (pre-miRNAhsa-mir-146a, hsa-mir-149, hsa-mir196a2, and hsa-mir-499). These 4 had previously been associated with a risk of lung cancer (86). Inasmuch as smoking is a risk factor for both types of cancer, it is plausible that these variants might play a role in the susceptibility to HNSCC as well. Of the 4 polymorphisms, only the hsa-mir-499 was associated significantly with the risk of HNSCC. Nonetheless, the results also suggested that the 4 pre-miRNA polymorphisms may have a joint effect on the risk of HNSCC, especially among men who were never smokers, and on the risk of oropharyngeal cancer. However, the exact mechanism by which these variants may influence the risk of HNSCC remained unclear. The risk-modifying effect of the hsa-mir-196a2 for HNSCC was also studied by another group in a population-based casecontrol study of 1039 individuals (87). In this study, carriers of a variant allele in MIR196A2 were shown to confer a significantly decreased risk for the development of HNSCC compared with individuals carrying the wild type. Although all of these studies have shown promising results, their conclude that their findings need to be confirmed by independent studies.

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Hereditary Form of HNSCC Germline mutation The existence of families with multiple cases of HNSCC and the occasional occurrence of HNSCC in young patients suggest a heritable gene defect with a high relative risk of cancer. Certain germline mutations have been discovered in association with HNSCC and explain a limited number of the cases. Such mutations are always found in connection with certain cancer syndromes in which HNSCC is not the sole type of tumor. At present, 3 genes are known whereby a mutation leads to an unusually high risk of HNSCC. The first of these is CDKN2A, which encodes the p16 protein. This gene plays an important role in cell cycle regulation and is somatically impaired in HNSCC. The syndrome FAMMM (familial atypical multiple mole melanoma) is caused by a specific inactivating mutation in CDKN2A. It confers a high risk for the development of melanoma, pancreatic cancer, and HNSCC (88-92). A common feature among such patients is that the second allele of CDKN2A is deleted in the HNSCC tumor cells (ie, somatic second event). A recent report described the second gene in a family that had multiple members with HNSCC of the oropharynx (93). The germline mutation was identified in an ATR (ataxia telangiectasia and Rad3)-related gene, which is involved in DNA damage response. Other malignancies reported there were skin, breast, and cervical cancer. The germline-mutated ATR is functionally compromised in these patients. A loss of heterozygosity of the ATR locus was noted in the oropharyngeal cancer tissue. The Fanconi anemia (FA) gene is the third gene, or rather gene family, for which a germline mutation is linked to HNSCC. FA is a rare recessively inherited disease. It is characterized by congenital abnormalities, bone marrow failure, and a genetic predisposition to leukemia and squamous cell carcinomas, including HNSCC (44). More than 13 different FA genes have been described. All of them belong to the FA pathway, which is important in DNA damage control. An FA patient has a relative risk more than 500 to 1000 times higher than that in an unaffected individual for the development of HNSCC (94).

Familial clustering Often the inheritance patterns of HNSCC are not immediately evident. Yet, it has repeatedly been shown that HNSCC clusters within certain families. A family history of HNSCC increases the risk of HNSCC twofold to fourfold (95) The risk is higher when the affected relative is a sibling (95-97). When the analysis is limited to tobacco users, the risk associated with family history appears to be even stronger. This suggests that susceptibility to tobacco-induced DNA damage may play a role in HNSCC heritability (95). Familial clustering is even more pronounced in HNSCC patients with second primary tumors in the upper respiratory and upper digestive tract (98). NPC has a well-documented familial aggregation. Highincidence families have been reported in both high-NPC incidence and low-NPC incidence areas (99). Nonetheless, the proportion of NPC patients with a family history in high-incidence areas is greater (100, 101). In NPC families, the incidence in firstdegree relatives of NPC patients is 410 times that in the control

International Journal of Radiation Oncology  Biology  Physics group (100, 101). A large Chinese case-control study reported a significant 12-fold elevated risk of NPC associated with a positive family history. The association was strongest for the less common form, keratinizing NPC (102). Epidemiologic studies suggest that most of the familial aggregation of NPC derives from inherited susceptibility. However, segregation analysis in high-incidence families in southern China found no evidence to support the implication of a major single gene in this inherited pathogenesis. Other studies have provided evidence for the linkage of NPC to chromosome 3p and fine mapping of an NPC susceptibility locus to a 13.6-cM region on 3p21.31-21.2 (20). These results are in agreement with several previous reports suggesting that deletion of chromosome 3p is a common genetic event in NPC (103). Many tumor suppressor candidate genes, notably CACNA2D2, DLC1, FUS1, H37, HYAL1, RASSF1A, SEMA3B, and SEMA3F, and tumor susceptibility genes such as hMLH1, are located in this region (20, 91). Additionally, haplotypes that have been associated with NPC include certain human leukocyte antigens (HLA), particularly HLA-A2, HLA-B46, and HLA-B58 (91). GWA studies have yielded the strongest evidence for NPC association within the major histocompatibility complex region of chromosome 6p21, where the HLA genes are located (21). These findings are further supported by case-control studies reporting an association between specific alleles, haplotypes of HLA genes, or both and an increased or decreased risk of NPC. Recently, it has been demonstrated that in addition to the known HLA-A gene, there are multiple chromosome 6p susceptibility loci that contribute to the risk of NPC, possibly through GABBR1 and NEDD9 loss of function (104). Furthermore, Hsu et al (105) evaluated SNPs within the major histocompatibility complex region of chromosome 6p21 that were recently reported to be significantly associated with NPC (21). Of the 12 SNPs considered, all but 1 appeared to reflect previously reported associations between HLA risk and protective alleles and NPC. Only rs29232, an SNP located within the GABBR1 gene, remained significant after adjustment for HLA-A effects, suggesting that it may represent an independent genetic determinant.

Functional Assays It is known that only a fraction of structural gene variations such as SNPs will result in a functional variation of the protein encoded by such a polymorphic gene. Moreover, external environmental factors might also influence gene expression and the final efficiency of the encoded enzyme. This has already been demonstrated for several biotransformation enzymes (106-109). Therefore, as suggested by Ahsan and Rundle (110), it may be more relevant to measure the interested phenotype (enzyme activity) instead of genotyping each gene in a particular involved pathway. A mutagen sensitivity assay, which is used to measure DNA repair capacity, is an example of this functional approach (110-112). Functional assays, which are based on the measurement of DNA or chromosomal damage processing, have successfully been used in studies on cancer risk. One such biomarker of genome instability is micronuclei frequency. It has been measured in the peripheral blood lymphocytes of newly diagnosed/untreated cancer patients. A meta-analysis showed a small but significant increase in baseline micronucleus frequency for various cancer types, including HNSCC (113).

Volume 89  Number 1  2014 More studies have assessed chromosomal damage in metaphase cells after challenging cultured peripheral blood lymphocytes with DNA-damaging agents. Such DNA damage induced during the G2 phase of the cell cycle appears to be related to cancer development. In this type of assay, the response to DNA damage is measured by scoring the number of persistent chromatid breaks in metaphase cells. This score is considered a measure of chromosomal instability, which is often referred to as mutagen sensitivity (114). Some of the DNA damage inducers that have been used in this setting are benzo(a)pyrenediol epoxide, gamma radiation, and bleomycin. Patients with HNSCC consistently prove to be more sensitive to chromosomal damage than a group of control persons, as found after in-vitro exposure of peripheral blood lymphocytes to gamma radiation (115, 116), benzo(a)pyrenediol epoxide (117), or bleomycin. This assay, with bleomycin as the mutagen of choice, has been studied extensively with peripheral blood lymphocytes from HNSCC patients. The results obtained from multiple groups have been convincing. In the normal population, considerable variation exists in bleomycin sensitivity, reflecting a normal distribution. The results also indicated that bleomycin sensitivity can reproducibly be measured at multiple sampling times. It does not appear to be influenced by removal of the tumor, smoking, alcohol abuse, or the age or sex of the individual (118). Details on the characteristics of this type of mutagen sensitivity in relation to HNSCC susceptibility are presented in Table 2.

Limitations, Implications, and Perspectives of the Research on Genetic Susceptibility to HNSCC There is evidence that several genetic polymorphisms and epigenetic variations might be associated with a modest modification of head-and-neck cancer susceptibility. However, it now appears that the possibilities for using genetic polymorphisms as a (single) biomarker of genetic susceptibility for HNSSC are more limited than has been originally thought or hoped for. However, if such a genetic biomarker, or a combination of these biomarkers, proves to be useful for genetic screening in a multifactorial disease like HNSCC, it is likely to give rise to new ethical questions and dilemmas. Would the information provided by the genetic profile of high-risk individuals help them take preventive measures and change their lifestyle to reduce the risk of cancer? Could they cope psychologically with the knowledge that they have a high-risk genetic profile? Would the result of such genetic susceptibility testing for HNSCC affect the medical

Table 2 Highlights of mutagen (bleomycin) sensitivity studies in head-and-neck squamous cell cancer A high mutagen sensitivity phenotype is a profound risk factor for tobacco users (111, 125) A very high mutagen sensitivity is related to the development of second primary tumors in the upper aerodigestive tract (126, 127) Family and twin studies show a high heritability estimate (117, 128) Expression array study: a group of low-penetrance genes most likely explains mutagen sensitivity (129)

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insurance coverage for high-risk persons? Or conversely, might individuals who do not have a high-risk genetic profile be more inclined to engage in high-risk behavior such as smoking and drinking because they assume a protective effect, but in reality increasing their risk for the development of HNSCC? These and other issues, like the reliability and correct interpretation of the rapidly increasing availability of commercial genetic screening tests, will be a challenge for both physicians and patients. On the other hand, research in the field of genetic polymorphisms and susceptibility to HNSCC might detect and elucidate the important pathophysiologic pathways involved in head-and-neck carcinogenesis. Moreover, there is some evidence that genetic polymorphisms and interindividual epigenetic variations not only would modify the risk of cancer but also may play a role in prognostication and possibly serve as a predictive tool in treatment decisions (119-124). It can be hoped that further integration and application of this knowledge in the therapeutic interventions and secondary prevention programs for HNSCC will contribute to the survival of patients with this disease.

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