Genetic Susceptibility to Squamous Cell Carcinoma of the Head and Neck
Genetic Susceptibility to Squamous Cell Carcinoma of the Head and Neck
By Erich M. Sturgis, MD, and Qingyi Wei, MD, PhD
The head and neck surgeon treats a variety of neoplasms, including melanomas and other cutaneous cancers, sarcomas of the head and neck, and tumors of the major salivary glands, sinonasal tract, orbit, nasopharynx, and thyroid/parathyroid glands. However, squamous cell carcinoma of the head and neck (SCCHN) is the most common malignancy treated by the specialty, and SCCsHN are the most common malignancies of the upper aerodigestive tract. In the United States, SCCHN accounts for approximately 3% of all newly diagnosed noncutaneous malignancies or 40,000 cases annually.1 However, these represent the fifth most common cancer worldwide, and in South Central Asia (where 20% of the world’s population resides) SCCHN is the most common cancer in men.2
The predominant risk factor for SCCHN is a history of exposure to tobacco and alcohol use. However, since only a fraction of exposed individuals develop cancer, variations in genetic susceptibility may be equally important in the disease etiology. Certain subgroups of head and neck cancer patients suggest that genetic susceptibility contributes to the etiology of SCCHN. For instance, the nonsmoker/nondrinker who develops such a malignancy may be more genetically susceptible to carcinogens, even in trace amounts. The patient who develops a head and neck cancer before age 40 and after a relatively short period of exposure may be more sensitive to mutagens. Each year after diagnosis, between 3% and 5% (15-25% at 5 years) of head and neck cancer patients develop a second cancer, and likewise, these patients may be more genetically susceptible to carcinogens.3
A genetic component to this disease is also supported by large family studies demonstrating a 3- to 8-fold increased risk of SCCHN in first-degree relatives of patients with SCCHN.4-7 Furthermore, as we will present here, there is molecular epidemiologic evidence supporting the concept of genetic susceptibility in head and neck cancer patients in general and in these subgroups in particular. Emerging data from case-control studies of several phenotypic and genotyping assays support the hypothesis that genetic susceptibility plays an important role in the etiology of SCCHN. Identifying such at-risk individuals in the general population by use of these biomarker assays would have a profound affect on primary prevention, early detection, and secondary prevention strategies.
Phenotypic Studies
Mutagen Sensitivity. Laboratory evidence that SCCHN patients are genetically predisposed to the disease chiefly has come from cytogenetic assays quantifying induced chromosome breakage in peripheral blood lymphocytes after in vitro exposure to the mutagen, bleomycin. In several case-control studies, patients with SCCHN were found to have a higher number of breaks per cell than the healthy control group.8-13 This assay has been confirmed in other case-control studies as a risk for lung cancer as well as other malignancies.9,10,14,15 Furthermore, the rare tobacco-free head and neck cancer patient and those younger than 30 years are particularly mutagen sensitive, displaying very high chromosome breaks/cell values.16 One study has evaluated the mutagen sensitivity to bleomycin of SCCHN patients with a family history of cancer. The researchers reported a higher percentage of mutagen sensitive patients in the group with a positive family history.17 Furthermore, when the SCCHN patients were followed longitudinally, those patients who later developed a second primary tumor (SPT) were found to be the most mutagen sensitive.18,19 This finding was confirmed in SCCHN patients with newly diagnosed SPT as compared to cancer-free controls and SCCHN patients without a SPT.20 While this assay seems to be a good marker for risk of SCCHN and predictor of SPT occurrence, it has been criticized because the mutagenic agent, bleomycin, is not a component of tobacco smoke.
Benzo[a]pyrene is a polycyclic aromatic hydrocarbon and is a classic tobacco carcinogen. While it is relatively nontoxic, it is bioactivated by phase I enzymes to form benzo[a]pyrene diol epoxide (BPDE). BPDE induces DNA damage chiefly through the formation of DNA adducts, and a direct link exists between the most frequent locations of BPDE adduct formation and the observed mutation hotspots in lung cancer.21 Consequently, BPDE may represent an appropriate agent to use in mutagen sensitivity and other assays for tobacco-induced cancer risk. In fact, our lab has recently demonstrated that mutagen sensitivity to BPDE is an independent risk factor for SCCHN.22 However, other data suggest that bleomycin-induced mutagen sensitivity and BPDE-induced mutagen sensitivity may be independent markers of susceptibility to SCCHN, and consequently may offer a better risk assessment model when used in conjunction with one another.23
While these mutagen sensitivity assays appear to be phenotypic markers of a patient’s genetic susceptibility, the exact pathways underlying these phenotypes remain to be elucidated. It has been suggested that a patient’s genetically determined DNA repair ability accounts for his or her level of mutagen sensitivity. More direct measures of DNA repair ability have been used to quantify susceptibility in case-control studies.
DNA Repair Capacity. As previously discussed, BPDE can irreversibly damage DNA by covalent binding or oxidation. The nucleotide excision repair pathway is responsible for removal of such BPDE-DNA adducts and for the restoration of normal DNA structure. Unrepaired BPDE adducts probably also cause chromosomal aberrations, as evidenced in the BPDE mutagen sensitivity assay described earlier. While there are many assays that measure the efficiency of the different steps of excision repair individually, the ability to test the whole pathway is needed for population studies, in which time, cost, and repeatability of measurements are major concerns. Therefore, measuring expression level of damaged reporter genes in the host cells (their DNA repair capacity) is the assay of choice. This host-cell reactivation assay uses undamaged cells, is relatively fast, and is an objective way of measuring repair. In the assay, a damaged, nonreplicating recombinant plasmid (pCMVcat) harboring a chloramphenicol acetyltransferase reporter gene is introduced by transfection into lymphocytes. Reactivated chloramphenicol acetyltransferase enzyme activity is measured as a function of excision repair of the damaged bacterial gene in the host cells. We have demonstrated that the cells from individuals with xeroderma pigmentosum (a genetic syndrome of nucleotide excision repair deficiency) have extremely poor repair capacity.24 We also demonstrated that both lymphocytes and skin fibroblasts from patients who have basal cell carcinoma and lung cancer have lower excision-repair rates than individuals without cancer.25-27 Consequently, the repair capacity of lymphocytes has been considered a reflection of an individual’s overall repair capacity.
We have assessed DNA repair capacity in SCCHN patients using a BPDE-damaged reporter gene assay.28 In this pilot case-control study, the DNA repair capacity of the cases was significantly lower than that of the controls. On multivariate analysis using logistic regression models controlling for age, sex, ethnicity, alcohol status, and smoking status, DNA repair capacity was an independent risk factor for the disease. A dose-response effect between DNA repair capacity and risk of SCCHN was observed and confirmed in a logistic regression model including age, gender, ethnicity, smoking status, and alcohol status. As we expect with a phenotypic marker of genetic susceptibility, there was no significant difference among cases based on stage or site of disease, but the younger patients tended to have the lowest DNA repair capacity.
Genotypic Studies
While the phenotypic assays previously discussed suggest that individuals possess underlying genetic differences in their response to carcinogens and ultimate risk of cancer, these assays are only indirect measures of such. However, work is under way to determine whether actual germline genetic differences exist between SCCHN patients and cancer-free controls.
Several syndromes exist that include inherited defects in DNA repair enzymes or tumor suppressor proteins; however, these are exceedingly rare and contribute little to the overall risk of SCCHN in the general population. In most of these syndromes, the genetic alteration profoundly impacts the protein’s function, thus putting the affected individual at high risk of certain cancers. However, it is likely that some genetic alterations exist at higher frequencies (polymorphisms) in the general population but only subtly affect protein function and consequently result in relative, mild increases in cancer risk. Such genetic polymorphisms have been identified for several genes involved in the metabolism of tobacco carcinogens and alcohol, and more recently in DNA repair and tumor suppressor genes.
DNA Repair Gene Polymorphisms. In an attempt to uncover the genotypes responsible for the phenotypic variations noted in DNA repair ability and by inference mutagen sensitivity, a third potential marker of susceptibility to SCCHN has been used in our laboratory (DNA repair gene restriction fragment length polymorphisms). Recently, Shen and colleagues identified polymorphisms in five DNA repair genes (XPD, XRCC1, XPF, ERCC1, and XRCC3).29 While the majority of these variants were either silent or occurred in introns, nine were identified that resulted in an amino acid change, and six of these were in highly conserved regions (3 each for XPD and XRCC1). Because these genes are involved in the repair of DNA damage induced by tobacco carcinogens, their polymorphic variants are potential markers for susceptibility to SCCHN.
The XRCC1 gene is involved in the recombination repair pathway. This pathway is responsible for the repair of DNA strand breaks as measured in the mutagen sensitivity assay. In a case-control study of 203 SCCHN patients and 424 controls without cancer, we have recently demonstrated frequency differences in the XRCC1 genotype at two polymorphic sites.30 These genotypes were each associated with borderline increased risk of SCCHN. However, when these risk genotypes were combined, the risk of SCCHN was 50% higher for those possessing either one of the risk genotypes and doubled for those having both risk genotypes. Furthermore, the patients who developed SCCHN at a young age had the highest frequency of these risk genotypes. In fact, one-quarter of these young patients had both of the risk genotypes, as opposed to only 13% of the middle-age and older patients.
The XPD gene functions as a 5’-3’ helicase required for the nucleotide excision repair pathway. This pathway is responsible for the repair of DNA adducts as induced by BPDE and measured in the DNA repair capacity assay discussed above. While germline mutations in XPD are well described in patients with xeroderma pigmentosum, germline variants in the general population have only recently been described.29 We have genotyped four of the polymorphisms in a case-control study (in revision). Once again, the risks associated with only a single polymorphism were borderline, but the combination of these XPD genotypes appears to offer utility in identifying those at risk are even more pronounced when the XPD and XRCC1 risk genotypes were combined (unpublished data).
Carcinogen Metabolizing Gene Polymorphisms. At least 50 known carcinogens are components of tobacco smoke, including the important groups of polycyclic aromatic hydrocarbons, aromatic amines, and nitroso compounds. Interindividual differences in carcinogen absorption, distribution, or accumulation in target tissue should also affect cancer susceptibility. The dose of tobacco carcinogens to which upper aerodigestive tract mucosa are exposed is modulated by the enzymes responsible for activation (phase I) and detoxification (phase II) of these carcinogens. Genetic differences or polymorphisms in these pathways potentially are an important source of interindividual differences in susceptibility. The phase I oxidative enzymes, such as the cytochrome P450 (CYP) multigene family of enzymes, may create carcinogenic or mutagenic intermediates that are more reactive than the parent compounds and may covalently bind to DNA, forming carcinogen-macromolecular adducts. Phase II metabolic processes generally inactivate these genotoxic compounds by conjugation (e.g., with glutathiones, glucuronides, and sulphate esters) that promote cellular excretion.
CYP1A1, the gene that codes for aryl hydrocarbon hydroxylase, which initiates a multienzyme pathway that activates polycyclic aromatic hydrocarbons, including benzo[a]pyrene, to highly electrophilic metabolites such as BPDE. CYP2D6 metabolizes a wide range of nitrogen-containing drugs, including neuroleptics, antidepressants, and beta-blockers, as well as the tobacco-specific nitrosoamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, to mutagenic products. CYP2E1 is involved in the oxidation and adduct formation of several components of cigarette smoke, such as N-nitrosamines and benzene, as well as contributing to the metabolism of ethanol and acetone. Various case-control studies have evaluated polymorphisms of CYP1A1, CYP2D6, and CYP2E1 as risk factors for SCCHN.31-37 However, the findings of these studies have been mixed, with most only demonstrating a borderline affect on risk.
The glutathione S-transferase (GST) genes catalyze glutathione conjugation to the carcinogen electrophilic epoxide intermediates to protect against DNA damage and adduct formation. Four different gene classes (a, m, p, and q) collectively make up the GSTs, and each class includes several genes. For instance, the GST mu family (GSTM) includes GSTM1, GSTM2, GSTM3, GSTM4, and GSTM5. Overlap in substrate specificity and variation in tissue expression results in a complex phenotypic picture. However, genotypic differences of a number of GSTs have been studied, particularly, GSTM1 and GST theta 1 (GSTT1). For these two isoenzymes, a common null genotype exists in which there is no enzyme activity. In the Caucasian population, approximately 50% and 15% are homozygous for the GSTM1 and GSTT1 null genotype, respectively, but considerable ethnic differences exist in the polymorphic rates for these two genes.38
A plethora of studies have examined the risk for cancer (particularly lung and bladder) among individuals possessing the GSTM1 or GSTT1 null genotype.38 Several studies have evaluated the risk of SCCHN in individuals possessing the GSTM1 null genotype and the GSTT1 null genotype.34-37,39-44 Most of these case-control studies have found a borderline increased risk in those with the null genotype of either gene, however the combined effect of the two genotypes on risk was examined by some.40,44 In these studies, a dose-response effect was seen, with those lacking both isoenzymes having double the risk of individuals lacking one and almost three-fold the risk of subjects expressing both isoenzymes.
Future Directions
As discussed above, genetic polymorphisms of DNA repair genes and carcinogen metabolizing genes seem to marginally impact the risk of SCCHN when analyzed separately. However, some utility may exist in combining the risk genoypes into genetic risk models based on multiple polymorphic sites. At present, the ability to genotype multiple polymorphic sites simultaneously at a reasonable speed and cost is somewhat limited. The new technology of the DNA microchip will allow for the simultaneous and rapid genotyping of hundreds of polymorphic sites. Large case-control studies will be needed to determine the pattern(s) of genetic polymorphisms predisposing an individual to a significant risk of cancer. Of course, phenotypic studies run in parallel will be equally important to determine the actual effect these polymorphisms and their combinations have on DNA repair and carcinogen metabolizing function.
Conclusions
In multiple case-control studies, the mutagen sensitivity assay has demonstrated that SCCHN patients are more sensitive to chromosomal damage induced by mutagens such as bleomycin and BPDE. It also appears that SCCHN patients have a reduced ability to repair genetic damage induced by tobacco carcinogens as measured by the DNA repair capacity assay. SCCHN patients have somewhat higher frequencies of germline genetic variants of DNA repair and carcinogen metabolizing genes, and a model of genetic susceptibility may represent a combination of these variants. While phenotypic and emerging genotypic evidence supports the hypothesis that genetic susceptibility plays an important role in the etiology of SCCHN, future work is needed with much larger studies to confirm these findings, to study their interactions with environment (tobacco and alcohol), to identify the complex pattern of genetic variants leading to risk, and to determine the actual functional effects of these genetic variants. If high-risk individuals can be identified, it would have a major affect on primary and secondary prevention strategies. (Dr. Sturgis is a Fellow, Department of Head and Neck Surgery, and Dr. Wei is an Associate Professor of Epidemiology, The University of Texas—M.D. Anderson Cancer Center, Houston, TX.)
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