Lack of Association Between Human Leukocyte Antigen-E Alleles and Nasopharyngeal Carcinoma in Tunisians

Abstract

Nasopharyngeal carcinoma (NPC), a cancer with a remarkable geographical and worldwide ethnic distribution, has been strongly associated with human leukocyte antigen (HLA) class I genes. The presence of additional HLA risk factors has been suggested by several reports. In the present study, we analyzed the implication of HLA-E gene polymorphisms in NPC susceptibility in Tunisians, a population characterized by an intermediate incidence of NPC with specific clinical features. Peripheral blood DNA was obtained from 185 patients with NPC and 177 matched controls. Genotyping for three single-nucleotide polymorphisms, codon 83Gly/Arg, codon 157Arg/Gly, and codon 107Arg/Gly, was performed using the polymerase chain reaction method. The HLA-E*01:01 and HLA-E*01:03 were the only alleles found among Tunisians. The HLA-E*01:03 allele had a slight increase in patients with NPC (43%) compared with controls (37%), but the difference did not reach a statistical significance. Our results show the lack of association between HLA-E alleles and NPC in the Tunisian population. This is not in agreement with the previous studies, suggesting a potential implication of HLA-E gene polymorphisms in the susceptibility to NPC among populations with high-risk incidence. Our study further supports the dissimilarity of NPC between populations with different NPC incidence.

Introduction

N asopharyngeal carcinoma (NPC) is a form of head and neck cancer with a remarkable geographical and worldwide ethnic distribution. Its incidence is <1 per 100,000 inhabitants in most parts of the world. However, NPC is a major public health problem in high-risk regions such as Southern China, particularly in people of Cantonese origin where the incidence exceeds 20 per 100,000 inhabitants (Ferlay et al., 2001). Moreover, intermediate incidence values, which vary from 3 to 8 per 100,000 inhabitants, were observed in regions such as Southeast Asia (Thailand and Malaysia) and Northern Africa (Tunisia, Algeria and Morocco) (Parkin et al., 2002; Devi et al., 2004).

Epidemiological studies agree that NPC is a multifactorial disease. The NPC atypical incidence pattern is explained by its complex etiology, which stems from the combined action of multiple etiological factors such as environmental, viral, and genetic ones (Chang and Adami, 2006). Though frequently observed among Asian and North African populations, a number of epidemiological and clinical dissimilarities of NPC between these populations were reported. Since the incidence is restricted to some ethnic groups or some geographical regions, numerous etiological environmental factors associated with lifestyle were initially suggested to be responsible for this cancer. Lifestyle risk factors mainly include frequent consumption of salt-preserved food containing carcinogenic nitrosamines and/or their precursors and professional exposures to wood dust (Yu and Henderson, 1987; Vaughan et al., 2000; Ward et al., 2000; Yuan et al., 2000). Recently, through a multivariate analysis model on a large-scale population, Guo et al. (2009) showed that associated risk factors in high-risk regions of NPC were a first, second, or third degree relative with NPC, consumption of salted fish, exposure to domestic wood-cooking fires, and exposure to occupational solvents. However, consumption of preserved meats or a history of tobacco smoking was not associated with NPC (Guo et al., 2009). However, among the North African patients with NPC, a large-scale case–control study shows evidence that associated risk factors for NPC in this area were tobacco consumption, marijuana smoking, and domestic cooking fume inhalation (Feng et al., 2009). Distinction between high and intermediate NPC risk areas also relates to their age distribution, which is unimodal in China, with one single incidence peak seen at the age of around 50, but bimodal in the Mediterranean area, with a main peak at around 50 years (80% of patients) associated with a secondary peak in the 10–25 age groups (20% of patients) (Lombardi et al., 1982; Khabir et al., 2000). The juvenile form of NPC is characterized by an early metastatic diffusion, and lymph node metastasis involvement is constantly present (69%) on diagnosis (Frikha et al., 2010). Moreover, unlike the adult form, the juvenile form of NPC is also characterized by weak expression of bcl-2 and p53 and strong expression of LMP1 and c-kit (Khabir et al., 2000). Regardless of the patient's geographical origin, Epstein-Barr virus (EBV) infection has been consistently identified as an important NPC risk factor (Raab-Traub, 2002). However, most North African NPCs contain EBV strains with genetic polymorphisms that are distinct from those described in the Southeast Asian studies (Ayadi et al., 2007). For instance, in North Africans, the most frequently detected EBV strains were the F, D, H1-H2, and XhoI+ variants, whereas those detected among high-risk areas are f, C, H, and XhoI−(Ayadi et al., 2007). In addition, according to incidence area, several specific human leukocyte antigen (HLA) haplotypes and genes within the HLA complex were commonly associated with NPC occurrence (Hassen et al., 2010). However, inconsistent associations were found in the different NPC incidence areas. The most important difference was related to the HLA-A2 allele, which is found to be positively associated among the Chinese, negatively associated among Caucasians, and not associated among North African NPCs. Moreover, direct sequencing of the HLA-class I genes showed positive associations for HLA-B*18, -B*51, and -B*57 with NPC risk in Tunisians and allowed the identification of a rare haplotype (HLA-B*14:02/Cw*08:02) in patients with NPC (Li et al., 2007). However, Tang et al. (2010) have recently shown that among a Southern Chinese population, the HLA-A*02:06 and HLA-B*38:02 alleles and the A*02:07/B*46:01 and the A*33:03/B*58:01 haplotypes were associated with high NPC risk. The exact role of HLA-class I alleles in NPC pathogenesis is still unknown. One explanation of the HLA/NPC relationship is that the associated alleles are in linkage disequilibrium with a gene or genes whose functions favor NPC occurrence and/or progression.

The HLA-E gene is a nonclassical class I gene localized on the short arm of chromosome 6 between the HLA-C and the HLA-A loci (Koller et al., 1987). Similar to class I molecules, HLA-E is a heterodimer consisting of heavy and β-2 microglobulin chains. HLA-E expression has been reported in most tissues at low levels and requires conserved nanomer peptides derived from the signal sequence of most HLA-A, -B, -C, and -G molecules (Lee et al., 1998). However, the Ulbrecht et al. (1998) in vitro studies showed that HLA-E can also bind synthetic viral peptides from EBV and influenza A virus and that human CMV open reading frame UL40 encodes a ligand for HLA-E, identical with the HLA-Cw03 signal sequence-derived peptide (Ulbrecht et al., 2000). HLA-E acts as a modulating ligand of both the innate and the adaptive immune systems and this is through interaction with CD94/NKG2 receptors present on natural killer (NK) cells and cytotoxic T lymphocytes (CTLs) (Sullivan et al., 2008). HLA-E gene contains eight exons with a relatively limited number of polymorphisms. Exon one encodes the leader peptide, exons 2 and 3 encode the α-1 and α-2 domains, both of which bind the peptide, exon 4 encodes the α-3 domain, exon 5 encodes the transmembrane region, and exons 6 and 7 encode the cytoplasmic tail. So far, only nine official alleles encoding three different proteins have been reported (The Anthony Nolan Research Institute, London, United Kingdom, at www.anthonynolan.org.uk/HIG/data.html, July 2010). Three nonsynonymous polymorphisms at codon 83 in exon 2, codons 107 and 157 both in exon 3 defined the E*01:02, E*01:03, and E*01:04 allelic variants, respectively. Some functional differences between HLA-E allelic variants E*01:01 and E*01:03 have been reported to be related to binding affinity for peptides and to NK cell functions (Maier et al., 2000; Strong et al., 2003). The literature concerning HLA-E polymorphisms and susceptibility to cancer is very poor. However, evidence on HLA-E expression and function on cancer biology is growing. Indeed, HLA-E overexpression has been found in colorectal cancer (Levy et al., 2008), glioblastomas (Mittelbronn et al., 2007), ovarian carcinomas (Malmberg et al., 2002), and melanoma cells (Derre et al., 2006). In the tumor context, HLA-E overexpression might inhibit the lytic process induced by NK and CTLs through its interaction with the CD94/NKG2A receptor, which may provide a mechanism of tumor escape from immune surveillance. Up to now, no data are available on HLA-E expression in NPC cells; nevertheless, in patients with NPC, several studies have indicated impaired peripheral NK cell immunity (Tsukuda et al., 1993; Zheng et al., 2006). Moreover, a positive association between HLA-E and NPC risk was found in a Thai population. In the present study, we analyzed the implication of the HLA-E gene polymorphisms in NPC susceptibility in Tunisians, a population characterized by an intermediate NPC incidence and a juvenile form with specific clinical features.

Subjects and Methods

Patients and controls

This case–control study included 185 patients with NPC and 177 healthy individuals selected from the same population living along the middle coast of Tunisia. Both patients and controls included in this study were unrelated subjects. The control group was comparable to patients with NPC with regard to age and sex. Written informed consent was obtained from all subjects, and approval for the study was given by the National Ethics Committee.

Patients with NPC were recruited from the Department of Radiation Oncology and Medical Oncology of Sousse Hospital, Tunisia. The histological type was undifferentiated NPC (Type III, WHO classification) for all the included patients, and the clinical stages ranged from I to IV (TNM classification, 1997). The sex ratio was 2.2 (127 men and 58 women), and the mean age was 42.4 ± 16.1 years (range, 10–85). The controls had a mean age of 42.1 ± 17.2 years (range, 11–92) and a sex ratio of 2.5 (127 men and 50 women). They were healthy blood donors recruited from the same area and having no evidence of any personal or family history of cancer or other illnesses.

HLA-E genotyping

Genomic DNA was extracted from peripheral blood leukocytes by a salting-out procedure (Olerup and Zetterquist, 1992). Each DNA sample was analyzed for variation in their DNA sequence for three nonsynonymous single-nucleotide polymorphisms. The 83Gly/Arg codon (exon 2) and the 157Arg/Gly codon (exon 3) were genotyped using the restriction fragment length polymorphism–polymerase chain reaction (PCR). PCR amplification of HLA-E exons 2 and 3 was performed as described by Matte et al. (2000). Polymorphism at codon 107Arg/Gly in exon 3 was analyzed by the amplification refractory mutation system–PCR. The procedure was performed as described by Matte et al. (2000). Each completed reaction was run in 2% agarose gel, stained with ethidium bromide, and visualized with ultraviolet light. Restriction fragment length polymorphism–PCR and amplification refractory mutation system–PCR results were confirmed by sequencing analysis performed on 20 samples for each analyzed exon. The PCR products were purified and sequenced on an ABI-PRISM 310 Genetic Analyzer (Applied Biosystems).

Data analysis

Genotype frequencies were tested for the Hardy–Weinberg equilibrium for both patients and controls using the Chi-square analysis (χ ²). The same test was applied to compare the allele and genotype frequencies between patients with NPC and healthy controls, except for cases where Fisher's exact test was appropriate. Odds ratios with 95% confidence intervals were calculated to estimate the relative risk. p < 0.05 was required for statistical significance. The data were analyzed using the Epi-Info statistical program (version 5.01a-1991; Centers for disease Control and Epidemiology Program office, Atlanta Georgia).

With regard to the bimodal age onset distribution in intermediate risk regions, young patients were defined as those aged 30 years or younger, whereas adult patients were those aged over 30. The clinicopathological parameters were dichotomized as follows: the primary tumor extension T1-T2 versus T3-T4; the regional lymph node status N-negative versus N-positive; the metastatic status M-negative versus M-positive; the clinical stages I-II versus III-IV; and complete remission versus partial or disease progression.

Results

Table 1 summarizes the main features of patients and controls enrolled in the study. The mean age and the sex ratio were statistically similar in both patients and controls.

Table 1.

Characteristics of Study Populations

Characteristics	Patients n = 185	Controls n = 177
Mean age (mean ± SD)	42.4 ± 16.1	42.1 ± 17.2
Age (years)
≤30	47	48
>30	138	129
Gender (male/female)	127/58	127/50
Tumor size
T1-T2	64	—
T3-T4	118	—
Lymph node status^a
N0	47	—
N+	135	—
Metastasis^a
M0	145	—
M+	7	—
Mx	30	—
Overall stage^a
I-II	12	—
III-IV	170	—
Treatment responses^a
Complete remission	33	—
Partial remission	69	—
No response	8	—

The sum does not equal the total due to unavailable data.

SD, standard deviation.

Among both controls and patients, 107Arg/Gly codon shows allelic variations, whereas allelic variations were not detected in 83Gly/Arg and 157Arg/Gly codons. The genotypic distributions of the codon 107Arg/Gly polymorphism were consistent with Hardy–Weinberg equilibrium in both controls and patients. Since the 107Arg/Gly codon is the only analyzed polymorphic codon, only the E*01:01 and the E*01:03 alleles were detected in Tunisians. The HLA-E allele and genotype frequencies observed among controls did not differ from those observed among patients with NPC (Table 2). Based on the particular age distribution of NPC incidence in Tunisia (Fig. 1), we stratified patients and controls in two age subgroups, 10–30 years old and over 30. The comparison of the frequency distribution of HLA-E alleles and genotypes in the two age subgroups for patients versus controls or in the two age subgroups of the patients did not show any genotypic association either (Table 2). Then, we looked whether there was any link between the HLA-E allele or genotype frequencies and the clinical parameters of the patients with NPC (TNM, disease stages, and treatment responses). The differences were again not statistically significant (data not shown).

FIG. 1.

Bimodal age distribution of the study populations: controls, (A) and patients with nasopharyngeal carcinoma (NPC) (B).

Table 2.

Distribution of HLA-E Allele and Genotype Frequencies in Patients with Nasopharyngeal Carcinoma and Healthy Controls

	All subjects		Age ≤30 years		Age >30 years
	NPC	Controls	NPC	Controls	NPC	Controls
	n = 185	n = 177	n = 47	n = 48	n = 138	n = 129
HLA-E	n (%)	n (%)	n (%)	n (%)	n (%)	n (%)
Alleles
E01:01* ^r	212 (57.3)	223 (63.0)	52 (55.3)	63 (65.6)	160 (58.0)	160 (62.0)
E01:02*	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)
E01:03*	158 (42.7)	131 (37.0)	42 (44.7)	33 (34.4)	116 (42.0)	98 (38.0)
E01:04*	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)
Genotypes
E0101/E0101 ^r	64 (0.35)	74 (0.42)	15 (0.32)	20 (0.42)	49 (0.35)	54 (0.42)
E0101/E0103	84 (0.45)	75 (0.43)	22 (0.47)	23 (0.47)	62 (0.45)	52 (0.40)
E0103/E0103	37 (0.20)	28 (0.15)	10 (0.21)	10 (0.10)	27 (0.20)	23 (0.18)

reference group.

HLA, human leukocyte antigen; NPC, nasopharyngeal carcinoma.

The geographical distribution of NPC incidence and the genetic divergence between ethnicity prompted us to compare HLA-E allele frequencies in our control population with those of low NPC incidence populations such as European (Antoun et al., 2009) and Danish (Steffensen et al., 1998), and with high NPC incidence populations such as Chinese (Kimkong et al., 2003) and Thai (Hirankarn et al., 2004) (Table 3). We noted the lack of both E*01:02 and E*01:04 alleles in all the populations above. The E*01:01 allele was the most frequent one among Tunisians, Europeans, and Danes, whereas E*01:03 was the most frequent allele among Thai and Chinese populations. Compared with Chinese, E*01:03 allele frequency highly differs from the frequency observed in Tunisians (p < 10⁻⁷). Indeed, in Thai and Chinese populations, there were increased frequencies of the E*01:03 allele and a decrease in the wild-type E*01:01 allele. A high significant difference was also observed when HLA-E allele frequencies of Tunisian controls and patients with NPC were compared with those from Thailand (p < 310⁻⁶ and p < 10⁻⁷, respectively). The huge allele frequency differences observed among patients might have resulted from the difference observed among controls. A less important, though significant, difference was noted when Tunisian controls were compared with European individuals (p = 0.04).

Table 3.

Comparison of HLA-E Allele Frequencies Between Tunisians and Populations with Low or High Nasopharyngeal Carcinoma Incidence

	Tunisians		Thai Hirankarn et al. (2004)		Danish Steffensen et al. (1998)	Europeanss Antoun et al. (2009)	Chinese Kimkong et al. (2003)
	NPC n = 185 (f)	Controls n = 177 (f)	NPC n = 100 (f)	Controls n = 100 (f)	Controls n = 150 (f)	Controls n = 223 (f)	Controls n = 50 (f)
HLA-E allele
E01:01* ^r	212 (0.57)	223 (0.63)	56 (0.28)	85 (0.42)	170 (0.57)	249 (0.56)	32 (0.32)
E01:02*	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)
E01:03*	158 (0.43)	131 (0.37)	144 (0.72)^a	115 (0.58)^a	130 (0.43)	197 (0.44)^a	68 (0.68)^a
E01:04*	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)

The chi-squared test was used to determine whether significant differences (p-value) were observed when Tunisian NPC controls or patients with NPC were compared with other populations.

p-value < 0.05.

n, number; f, frequencies.

Discussion

In this study, HLA-E allele frequencies were investigated in Tunisian healthy blood donors and patients with NPC. To our knowledge, this is the first study conducted on a North African population sample. Consistent with the majority of previous reports, the E*01:01 and E*01:03 alleles were found, but not the E*01:02 and E*01:04, which were missing in both healthy and NPC individuals. Conflicting data were reported on the existence of both the E*01:02 and E*01:04 alleles; indeed, their detection among Japanese and Spanish populations was not confirmed. Thus, as previously suggested, our findings confirm the fact that their existence needs to be further investigated (Grimsley et al., 2002). A strong disparity in distribution of HLA-E alleles among human populations was observed. This may influence susceptibility to diseases and may partly explain the peculiar ethnic distribution of diseases such as NPC. Compared with populations from low and intermediate NPC risk areas, the HLA-E*01:03 allele, which was found to increase NPC risk among Thai population, shows an extremely elevated frequency in high NPC risk areas.

The literature concerning HLA-E polymorphisms and susceptibility to cancer is controversial and too poor. The HLA-E*01:03 allele was associated to NPC but not to cutaneous melanoma (Hirankarn et al., 2004; Moya-Quiles et al., 2005). Compared with the HLA-E*01:01 wild-type allele, the E*01:03 allele differs by a nonsynonymous R to G substitution at codon 107 in the α-2 domain of the heavy chain. These two alleles, also called HLA-E^R and HLA-E^G respectively, show some functional disparities that might affect the modulator role of HLA-E on NK and CTL cytolysis. The E*01:03 allele presents a greater affinity for distinct binding peptides and a higher cell surface expression than the wild-type allele (Strong et al., 2003). In the tumor context, one possible hypothesis is that tumor escape might be the result of the increased expression of cell surface HLA-E^G that increases the inhibitory effect on NK cell lysis through interaction with the NKG2A receptor. In the presence of anti-NKG2A monoclonal antibody, a recent report conducted on colorectal cancer cells where HLA-E expression is significantly increased has shown that the NK cytolytic activity is restored through the HLA-E/NKG2A pathway (Levy et al., 2009). Further, the consequence of the HLA-E/NKG2A interaction depends on both the bonded HLA class I signal sequence derived peptides and on the presenting HLA-E (Maier et al., 2000). For instance, the same leader peptide derived from HLA-A2 has a different effect, as it is bound to the HLA-E^R or HLA-E^G. When bound to HLA-E^G, a more efficient inhibition effect on NK cell mediated lysis was observed. Interestingly, one of the main differences between the several incidence risk areas is that the HLA-A2 allele is strongly associated with NPC only in high-risk incidence areas. Unlike North African countries, both discrepancies in HLA-A2 and HLA-E*01:03 associations with NPC in high-risk areas may contribute to the increased NPC incidence and further confirm the peculiar incidence pattern of NPC worldwide.

In contrast to Hirankarn et al. (2004), our study showed that, among Tunisian NPCs, none of the HLA-E alleles is likely to have major effects on NPC susceptibility or progression. The Hirankarn et al. (2004) study is not the first to suggest the HLA-E and NPC relationship among populations having high NPC incidence. Indeed, through a microsatellite marker analysis, a marker localized near the HLA-E gene, the D6S1624 locus was found to be associated with NPC in Singaporean Chinese (Ooi et al., 1997). Moreover, using high-resolution microsatellite mapping, Lu et al. (2005) suggested that, in the Taiwanese population, NPC susceptibility genes might be located between the D6S510 and D6S211 markers, which cover the HLA-A, HLA-B, HLA-C, and HLA-E loci. These previous findings suggest that the HLA-E locus is probably involved with NPC susceptibility. However, this implication is not exclusive. It is not ruled out that other genes in linkage disequilibrium within or near this region may be implicated in NPC susceptibility. So far, regardless of the study population's ethnic origin, the HLA-NPC association is clear. However, HLA-NPC association results seem to be conflicting among different ethnic populations. Earlier studies showed that allele and haplotype frequencies are characteristic features of particular populations and certain alleles are exclusively found in some ethnic groups (Arnaiz-Villena et al., 1999; Barquera et al., 2008). Discrepancies may be related to ethnic differences in haplotype diversity, distribution, and, consequently, in linkage disequilibrium with HLA alleles.

Bearing in mind that according to its origin, NPC does not result from the same combination of genetic factors, this study shows a new dissimilarity in NPC risk factors between high and intermediate incidence risk areas. These dissimilarities also suggest that NPC tumor cells might use different pathways or strategies to escape the immune system response. In conclusion, the present study does not confirm the HLA-E and NPC association. These data, however, do not exclude a possible physiopathological role of the HLA-E molecules in the NPC disease. HLA-E expression in soluble and/or membrane-bound forms in NPC tumor cells needs to be analyzed, and the link between HLA-E expression and clinical parameters should be evaluated.

Footnotes

Acknowledgments

This work was supported by le Ministère de l'Enseignement Supérieur, de la Recherche scientifique et de la Technologie, and le Ministère de la Santé Publique de la République Tunisienne. We thank Mr. Adel Rdissi for the English revision.

Disclosure Statement

No competing financial interests exist.

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