Introduction
Head and neck carcinomas comprise a group of malignant tumours originating from the upper aerodigestive tract, including the oral cavity, pharynx and larynx.Reference Vokes, Weichselbaum, Lippman and Hong1 More than 90 per cent of head and neck carcinomas arise from the epithelial tissue of these regions, and are termed head and neck squamous cell carcinomas (SCCs).Reference Marur and Forastiere2
Half a million new cases of head and neck SCC are diagnosed annually worldwide. However, the incidence varies greatly by region, being higher in developing countries.Reference Marur and Forastiere2 Although tobacco and alcohol consumption are recognised as the most common aetiological factors for head and neck SCC, the disease occurs only in a small number of smokers. Non-users of tobacco and alcohol and young adults also develop head and neck SCC.Reference Vokes, Weichselbaum, Lippman and Hong1–Reference Lund and Howard3 Head and neck SCC patients have variable prognoses, even those at the same clinical stage and receiving similar treatments.Reference Hopkins, Cescon, Tse, Bradbury, Xu and Ma4 These differences in head and neck SCC susceptibility and prognosis may be due to heterogeneity of study populations, specifically regarding genetic polymorphisms.
In recent decades, an increasing number of studies have assessed genetic polymorphism within head and neck SCC, aiming to identify new prognostic markers and therapeutic targets. Several studies have investigated polymorphisms in genes coding for enzymes involved in tumour suppression, growth factor pathways, tobacco-related carcinogen metabolism and the cell cycle, and have found associations between these polymorphisms and head and neck SCC susceptibility and survival.Reference Marur and Forastiere2, Reference Hopkins, Cescon, Tse, Bradbury, Xu and Ma4 Other studies have found an association between head and neck SCC and polymorphisms of pro-inflammatory cytokine genes.Reference Vairaktaris, Yiannopoulos, Vylliotis, Yapijakis, Derka and Vassiliou5
Interleukin (IL)-18 is a novel, pro-inflammatory cytokine which appears to play a key role in innate and acquired immunity. This cytokine stimulates interferon-γ production synergistically with IL-12, promotes differentiation of T cells to a Th1 phenotype, and enhances the cytotoxic activities of natural killer cells and T cells. Administration of IL-18 results in significant suppression of tumour growth in animal models, suggesting a role for this cytokine in host defence against cancer.Reference Nakanishi, Yoshimoto, Tsutsui and Okamura6 However, interleukin-18 has also been found: to stimulate IL-4 production in the absence of IL-12; to inhibit the recognition of cancer cells by immune cells; to increase adherence of cancer cells to microvascular walls; to induce the production of angiogenic and growth factors; and to promote a prometastatic microenvironment.Reference Vidal-Vanaclocha, Mendoza, Telleria, Salado, Valcárcel and Gallot7
Interleukin-18 gene expression seems to be regulated by two single nucleotide polymorphisms at positions –607 and –137 in the promoter region of the gene. A change from cytosine (C) to adenine (A) at position –607 disrupts a potential cyclic adenosine monophosphate (cAMP)-responsive element-binding protein binding site. A change at position –137 from guanine (G) to C changes the H4TF-1 nuclear factor binding site to a binding site for an unknown factor found in the granulocyte macrophage colony-stimulating factor promoter.Reference Giedraitis, He, Huang and Hillert8 These two single nucleotide polymorphisms have been associated with a variety of inflammatory conditions, such as autoimmune disease,Reference Mojtahedi, Naeimi, Farjadian, Omrani and Ghaderi9 hepatitis C,Reference Bouzgarrou, Hassen, Schvoerer, Stoll-Keller, Bahri and Gabbouj10 and several types of cancers, e.g. ovarian,Reference Bushley, Ferrell, McDuffie, Terada, Carney and Thompson11 breast (Khalili et al. unpublished data) and prostate.Reference Liu, Lin, Huang, Xu and Pang12
The present study aimed to investigate whether polymorphisms of the IL-18 promoter in the regulatory regions of –607 (C/A) and –137 (G/C) confer a genetic risk for head and neck SCC, and to evaluate the possible correlation of these single nucleotide polymorphisms with clinical characteristics.
Materials and methods
Patients
A total of 111 non-related patients (86 men and 25 women; mean age 56.7 ± 13.7 years) and 212 controls (165 men and 47 women; mean age 53.3 ± 12.2 years) were enrolled in this study. The patients were admitted at Khalili Hospital, Shiraz, Iran. The diagnosis of SCC was confirmed histopathologically. The mean age at the onset of the disease was 55.7 ± 13.3 years, ranging from 19 to 83 years. Information on clinicopathological parameters at diagnosis was collected, including tumour site, tumour size, lymph node involvement, distant metastasis and stage; see Table I.
Clinicopathological characteristics of 111 patients with head and neck SCC
SCC = squamous cell carcinoma
Control subjects comprised 212 regional volunteers referred to Motahari Clinic, Shiraz, Iran, for routine check-ups, or cancer-free individuals presenting with otolaryngological disease.
All subjects were informed that blood samples would be used for genotyping, and their consent was obtained. The study was approved by the ethics committee of the Shiraz University of Medical Sciences.
Deoxyribonucleic acid preparation
Peripheral blood samples were obtained from patients and control subjects, in 5-ml volumes, and genomic deoxyribonucleic acid (DNA) was extracted from leukocytes by the salting-out method.Reference Miller, Dykes and Polesky13
Interleukin-18 gene amplification
Polymorphisms were detected by allele-specific polymerase chain reaction. For each blood sample, two separate reactions were conducted. For the −137 single nucleotide polymorphism, polymerase chain reaction was performed using a common reverse primer, 5′-AGG AGG GCA AAA TGC ACT GG-3′ (where T = thymine) and two sequence-specific forward primers, 5′-CCC CAA CTT TTA CGG AAG AAA AC-3′ and 5′-CCC CAA CTT TTA CGG AAG AAA AAG-3′. A control forward primer, 5′-CCA ATA GGA CTG ATT ATT CCG CA-3′, was used to amplify a 446-bp fragment covering the polymorphic site to serve as an internal positive amplification control. Polymerase chain reaction for the polymorphism at −607 was performed using a common reverse primer, 5′-TAA CCT CAT TCA GCA CTT CC-3′, and two sequence-specific forward primers, 5′-GTT GCA GAA AGT GTA AAA ATT ATT AC-3′ and 5′-GTT GCA GAA AGT GTA AAA ATT ATT AC-3′. A control forward primer, 5′-CTT TGC TAT CAT TCC ACG AA-3′, was used to amplify a 301-bp fragment covering the polymorphic site as an internal positive amplification control.
All polymerase chain reactions were performed in a mixture containing 0.3 µg of genomic DNA, 0.8 pM of common reverse primer, 0.8 pM of sequence-specific forward primer, 0.3 pM of control forward primer (primers from Tib Molbio, Berlin, Germany), 0.3 mM of deoxyribonucleotide triphosphate (dNTP) (Boehringer, Ingelheim, Germany), 0.3 mM of MgCl2, 2 units of Taq DNA polymerase (CinnaGen, Tehran, Iran), 2.5 µl polymerase chain reaction buffer (CinnaGen, Tehran, Iran) and double distilled H2O added to make up a final volume of 25 µl. The cycling conditions for the −137 single nucleotide polymorphism were 2 minutes at 94º C followed by five cycles of 20 seconds at 94º C, 40 seconds at 64º C, 70 seconds at 72º C, and then 25 cycles of 20 seconds at 94º C, 40 seconds at 57º C and 40 seconds at 72º C. The cycling conditions for the −607 single nucleotide polymorphism were 2 minutes at 94º C followed by seven cycles of 20 seconds at 94º C, 30 seconds at 64º C, 80 seconds at 72º C, and then 25 cycles of 20 seconds at 94º C, 40 seconds at 57º C and 50 seconds at 72º C. All polymerase chain reaction products were separated in 2 per cent agarose gels and stained with ethidium bromide. Amplification products of 196 and 261 bp were detected for the −607 and −137 single nucleotide polymorphisms, respectively.
Statistical analysis
All genotype frequencies were tested for the Hardy–Weinberg equilibrium. The fit to the equilibrium was tested by calculating the chi-square test. Haplotype frequencies were calculated by Arlequin population genetic software (http://anthropologie.unige.ch/arlequin). Data were analysed using the Statistical Package for the Social Sciences software (version 11.5.0; SPSS, Chicago, Illinois, USA). Pearson's chi-square test and Fisher's exact probability test were used, when appropriate, to estimate the differences in the distribution of alleles, genotypes and haplotypes in the groups studied. Findings were considered statistically significant at a p value less than 0.05.
Results
Neither patient nor control genotype frequencies significantly differed from those expected according to the Hardy–Weinberg equilibrium.
As shown in Table II, the frequencies of the interleukin (IL)-18 single nucleotide polymorphisms at positions −607 and −137 did not differ significantly, comparing patients and controls. At position −607, the respective frequencies of CC, CA and AA genotypes were 43 (38.7 per cent), 53 (47.7 per cent) and 15 (13.5 per cent) in patients, versus 82 (38.7 per cent), 101 (47.6 per cent) and 29 (36 per cent) in controls. At position –137, the respective frequencies of GG, GC and CC genotypes were 65 (58.6 per cent), 37 (33.3 per cent) and nine (8.1 per cent) in patients, versus 116 (54.7 per cent), 79 (37.3 per cent) and 17 (8 per cent) in controls. The allele distribution of the single nucleotide polymorphisms at positions −607 and −137 showed no significant difference, comparing patients and controls (Table II). The haplotype frequencies also did not differ, comparing patients and controls (Table III).
Genotype and allele frequencies of IL-18 gene promoter in 111 head and neck SCC patients and 212 controls
No statistically significant difference was found between patient and control groups for any comparison (chi-square test on 2 × 3 or 2 × 2 tables). IL = interleukin; SCC = squamous cell carcinoma
Haplotype frequencies of IL-18 gene promoter in head and neck SCC patients and controls
* 2n = 222; †2n = 424; ‡For differences in frequency of a given haplotype, comparing patients and controls; chi-square test. IL = interleukin; SCC = squamous cell carcinoma; C = cytosine; G = guanine; A = adenine; NS = not significant
The frequency of the IL-18 alleles and genotypes was also compared with subjects’ clinical parameters at diagnosis, including tumour size, tumour stage, lymph node involvement and metastasis. No statistically significant correlation was observed (data not shown).
Discussion
The development of head and neck SCC is a multifactorial process affected by genetic factors and also environmental agents, including tobacco and alcohol consumption, viral infection and chronic inflammation.Reference Vokes, Weichselbaum, Lippman and Hong1, Reference Marur and Forastiere2 The association of head and neck SCC with inflammatory cytokine genes has been the subject of several studies.Reference Vairaktaris, Yiannopoulos, Vylliotis, Yapijakis, Derka and Vassiliou5, Reference Vairaktaris, Serefoglou, Yapijakis, Agapi, Vassiliou and Nkenke14, Reference Khademi, Razmkhah, Erfani, Gharagozloo and Ghaderi15 Single nucleotide polymorphisms at positions –607 and –137 of the interleukin (IL)-18 promoter have been reported to cause differences in transcription factor binding and to have an impact on the genetic expression of this pro-inflammatory cytokine. Upon stimulation, higher promoter activity has been observed for C and G alleles in the −607 (C/A) and −137 (G/C) positions, respectively.Reference Giedraitis, He, Huang and Hillert8 These two single nucleotide polymorphisms of the IL-18 promoter have been found to affect the susceptibility to and prognosis of several types of malignant tumours, including ovarianReference Bushley, Ferrell, McDuffie, Terada, Carney and Thompson11 and breast (Khalili et al. unpublished data).
It is believed that tumourigenesis is largely influenced by the pleotropic, systemic IL-18 cytokine, either in protective or permissive ways.Reference Vidal-Vanaclocha, Mendoza, Telleria, Salado, Valcárcel and Gallot7 Interleukin-18 was initially identified as a potent inducer of interferon-γ production, a cytokine which in turn strengthens the cellular arm of the immune response. Anticancer effects of IL-18 were demonstrated in animal models treated with this cytokine.Reference Nakanishi, Yoshimoto, Tsutsui and Okamura6 Conversely, serum IL-18 levels were reported to be higher in patients with cancer compared with healthy donors.Reference Vidal-Vanaclocha, Mendoza, Telleria, Salado, Valcárcel and Gallot7 Interleukin-18 levels have also been observed to increase as the pathological stage of cancer progresses, and the serum IL-18 level has been suggested as a non-invasive marker for suspected metastasis in certain types of cancer, e.g. breast cancer.Reference Vidal-Vanaclocha, Mendoza, Telleria, Salado, Valcárcel and Gallot7, Reference Günel, Coşkun, Sancak, Günel, Hasdemir and Bozkurt16 Several mechanisms have been suggested by which IL-18 could promote a prometastatic environment.Reference Vidal-Vanaclocha, Mendoza, Telleria, Salado, Valcárcel and Gallot7
Therefore, we investigated two functional single nucleotide polymorphisms in the promoter region of IL-18 as possible genetic risk factors for head and neck SCC. We found no significant association between polymorphisms of the IL-18 gene at positions –607 and –137 and head and neck SCC susceptibility or clinical parameters at diagnosis.
• Inflammation plays an extremely complex role in cancer and involves interaction of several inflammatory cytokines
• This study aimed to investigate the association between interleukin-18, pro-inflammatory cytokine and head and neck squamous cell carcinoma (SCC)
• Interleukin-18 promoter polymorphisms were not observed to confer susceptibility to head and neck SCC in southern Iranian patients
In agreement with our results, previous investigation of IL-18 polymorphism and oral SCC found no significant association between IL-18 promoter polymorphisms and head and neck SCC in a Greek population.Reference Vairaktaris, Serefoglou, Yapijakis, Agapi, Vassiliou and Nkenke14 Furthermore, serum IL-10, IL-12 and IL-18 levels have been measured in head and neck SCC patients in a UK population; systemic IL-10 and IL-12 concentrations were found to be significantly altered in patients compared with non-tumour controls, but no such differences in IL-18 levels were observed.Reference Jebreel, Mistry, Loke, Dunn, Hough and Oliver17 Head and neck cancers often drain to the lymph nodes of the neck, and cervical lymphadenopathy is often the first manifestation of disease at the time of diagnosis.Reference Wein, Chandra, Weber, Brunicardi, Andersen, Billiar, Dunn, Hunter and Pollock18 Immunoregulatory molecules secreted within the central nervous system and effluxing along with cerebral extracellular fluid into the cervical lymph nodes provide an immunoregulatory microenvironment.Reference Harling-Berg, Park and Knopf19 It is suggested that these immunoregulatory molecules (such as transforming growth factor-beta) modulate antigen-presenting cells in cervical lymph nodes.Reference Harling-Berg, Park and Knopf19 Macrophages are an important group of antigen-presenting cells, and are thought to be the major site of IL-18 production.Reference Nakanishi, Yoshimoto, Tsutsui and Okamura6 One could argue that, since the major source of IL-18 is modulated by the exceptional microenvironment of the cervical lymph nodes, the effect of this cytokine on head and neck SCC may be too small to detect in a small sample.
Conclusion
This study found that interleukin (IL)-18 promoter polymorphism did not contribute to head and neck SCC in an Iranian population. More data from a larger number of patients are required in order to exclude a possible minor effect of IL-18 gene polymorphism on head and neck SCC susceptibility and prognosis.
Acknowledgements
The authors would like to thank Dr Nasrollah Erfani for his assistance in statistical analysis. This study was supported by a grant from the Shiraz Institute for Cancer Research (ICR-82-93).
