Interleukin-1 receptor antagonist gene polymorphism is associated with increased risk of epithelial ovarian cancer

J. Sehouli, A. Mustea+, D. Koensgen, F. C.-K. Chen and W. Lichtenegger

Department of Obstetrics and Gynecology, Charité, Humboldt University, Berlin, Germany

Received 18 February 2003; revised 26 May 2003; accepted 17 June 2003


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background:

Different studies indicate that immunological components play a key role in the development of cancer. Interleukin-1 (IL-1) is known to be critically involved in ovarian carcinogenesis and in other solid tumors. Therefore, we investigated the possible influence of the polymorphism of the IL-1 receptor antagonist (IL-1 RA) genes on the development of ovarian cancer.

Patients and methods:

In a prospective study we analyzed the polymorphism of the IL-1 RA gene in 108 women with ovarian cancer compared with 112 patients with benign gynecological diseases. Genomic DNA fragments were amplified by PCR.

Results:

The distribution of genotype frequencies was significantly different between the study and control group with respect to allele 1/2 heterozygotes (32.4% versus 15.2%; P = 0.004). Patients who were heterozygous at allele 2 for IL-1 RA (IL-RA 1/2) had a significantly higher risk of ovarian cancer with a calculated odds ratio of 2.7 (95% confidence interval 1.4–5.2). There were no differences between IL-1 RA 1/2 polymorphism and all other alleles in tumor stage (International Federation of Gynecology and Obstetrics), histological type, grading, postoperative tumor volume, volume of ascites, recurrence status or age.

Conclusions:

The allele 2 polymorphism of the IL-1 RA gene seems to play a role in the occurrence of ovarian cancer and should be investigated for screening and risk evaluation.

Key words: interleukin-1 receptor antagonist, ovarian cancer, polymorphism


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Ovarian cancer is the leading cause of death of all the gynecological malignancies [1]. The 5-year survival rate remains poor despite significant improvements in surgical treatment and chemotherapy [2, 3].

More than 75% of all patients are classified as being International Federation of Gynecology and Obstetrics (FIGO) stage III and IV at the time of diagnosis [4].

Over the last decade, ovarian cancer screening using the serum marker CA-125 and transvaginal ultrasound examination has been investigated, but neither of these methods fulfills the necessary criteria to be considered as an effective screening test [5]. Therefore, new predictive and prognostic factors for ovarian cancer are needed to influence clinical management.

Unlike gene mutations, polymorphisms in specific genes are common genetic events. It is estimated that there are ~200 000 single-nucleotide polymorphisms within the coding regions of the 80 000 human genes [6, 7]. These could be either a single base pair (bp) substitution of one nucleotide for another or a variable repeated number of a short, repetitive DNA sequence [8]. These variations may affect the rate of gene transcription, the stability of the mRNA or the quantity and activity of the resulting protein.

The prevalence and severity of a number of disorders will be influenced by certain polymorphic genes possessing specific alleles [7]. The polymorphic gene that encodes the interleukin-1 (IL-1) receptor antagonist (IL-1 RA) seems to play an important role in the development of various diseases [8].

IL-1 RA belongs to the IL-1 family, which consists of three linked genes mapping within a 430-kb region of the long arm of chromosome 2 in humans, encoding the secreted glycoproteins IL-1{alpha}, IL-1ß and IL-1 RA. All three molecules bind to IL-1 receptors [911]. IL-1{alpha} and IL-1ß are potent proinflammatory cytokines, while IL-1 RA as an anti-inflammatory cytokine competes with IL-1{alpha} and IL-1ß in binding to IL-1 receptors without intrinsic effects [12, 13]. Polymorphisms have been reported in all three genes [12]. The polymorphism of IL-1{alpha}, IL-1ß and IL-1 RA produce alterations of the IL-1{alpha}, IL-1ß and IL-1 RA protein expression [1419] and it may have crucial effects on oncogenic processes [20].

The role of IL-1 RA in ovarian carcinogenesis is currently being investigated. IL-1 increases the growth of ovarian carcinoma cell lines [21] and tumor proliferation [22]. Furthermore, different studies have reported an association of IL-1 gene polymorphisms with gastric, pancreatic and cervical cancers [2326]. Based on these data, we analyzed the influence of IL-1RA polymorphism on the prevalence of ovarian cancer and its correlation with established clinical prognostic factors.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This prospective, mono-institution study was reviewed and approved by the Clinical Review Board and Ethics Committee of the University Hospital, Charité, Berlin.

Patients with histologically confirmed ovarian cancer were allocated to this trial. Written informed consent was provided by each patient. Borderline ovarian tumors are different tumor entities and therefore were excluded from this study [27].

The control group consisted of women of a similar age with no history of cancer. None of these women had previous oophorectomy. Because germline mutations do not fluctuate with age, we did not specify the age and include only women >55 years of age, the median age of the patients with ovarian cancer [4]. To avoid confounding by genetic admixture, only white Caucasian women were enrolled in this study.

Genetic studies
Two milliliters of blood were drawn from the antecubital vein, and DNA was extracted using the Qiagen system (QIAamp DAN Blood Midi kit; Qiagen, Hilden, Germany).

DNA was stored at 4°C until analyzed. A genomic DNA fragment was amplified by PCR to determine IL-1 RA genotypes. Oligonucleotide primers flanking the 86-bp repeat region in intron 2 of IL-1 RA were used. The sequence of the forward primer was 5'-CTCAGCAACACTCCTAT-3'. The reverse primer sequence was 5'-TCCTGGTCTGCAGGTAA-3' [28]. PCR conditions included an initial denaturing step at 94°C for 5 min, followed by 30 cycles of 94°C for 1 min, 60°C for 1 min, and 70°C for 2 min, and a final extension at 70°C for 4 min.

Using this PCR strategy, the common allele (allele 1) generated a 410-bp band (including four copies of an 86-bp repeat). The uncommon alleles generated a 240-bp band (two copies of the same repeat; allele 2) a 500-bp band (five copies of the same repeat; allele 3) and a 325-bp band (allele 4). PCR products were resolved on a 3% agarose gel and stained with SYBR Green I (FMC Bio Products Europe, Vallensbaek Strand, Denmark) (Figure 1).



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Figure 1. Photograph of a 3% agarose gel used to resolve alleles 1, 2 and 3 of interleukin-1 receptor antagonist (IL-1 RA). Lane 1: the heterozygous allele 1/allele 2 pattern (IL-1 RA 1/2). Lane 2: the homozygous allele 1 pattern (IL-1 RA 1/1). Lane 3: the homozygous allele 2 pattern (IL-1 RA 2/2). Lane 4: the heterozygous pattern for allele 1/allele 3 (IL-1 RA 1/3).

 
Statistics
SPSS (10.0; SPSS Inc., Chicago, IL, USA) for Windows statistical software was used for statistical analyses. Differences in the frequencies of IL-1 RA alleles in the groups were analyzed by the two-tailed Fisher’s exact test. All P values were two-tailed. P <0.05 was considered statistically significant. Odds ratios with a 95% confidence interval (CI) were calculated.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
From June 2001 to September 2002, 125 women with ovarian cancer were enrolled in this study. DNA extraction was not possible because of technical reasons in two cases. Fifteen patients were excluded from this study because of different histological diagnosis (borderline tumor of the ovary, colon carcinoma). One hundred and eight women with ovarian cancer and 112 women with no history of cancer were analyzed.

The characteristics of the women with ovarian cancer are summarized in Table 1. The median age of the patients with ovarian cancer was 56 years (range 26–81). From 108 patients, only 15.7% were diagnosed in FIGO stage I, 77.7% were in FIGO stage III–IV. The most common histological type was serous-papillary (66.7 %).


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Table 1. Characteristics of the ovarian cancer group
 
To enhance the relevance of the statistics, the following subgroups were combined for statistical analysis: stage I + II and III + IV; histological grading I + II and III + IV.

No statistically significant differences were observed between the study and control groups with respect to allele 1/3 (0.9% versus 2.7%; P = 0.61), homozygous allele (IL-1 RA 1/1; 73.2% versus 62.0%; P = 0.12). One woman in the study group was found to be homozygous for allele pattern 4 and six women for allele 2. One women of the control group was heterozygous for allele 3 pattern (IL-1 RA 2/3).

With respect to allele 1/2 heterozygotes, the distribution of genotype frequencies was significantly different between the study and control groups (32.4% versus 15.2%; P = 0.004). Patients who were heterozygous at allele 2 for IL-1 RA (IL-RA 1/2) had a significantly higher risk of ovarian cancer with a calculated odds ratio of 2.7 (95% CI 1.4–5.2). The allele frequencies of IL-1 RA wild-type and polymorphic alleles in the study and control groups are given in Table 2.


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Table 2. Allele frequencies (%) of interleukin-1 receptor antagonist (IL-1 RA) among women with ovarian cancer and the control group
 
We compared all relevant clinical data of the patients with ovarian cancer and allele 2 heterozygotes genotype (IL-RA 1/2) with patients of the other genotypes in the cancer group. No statistically significant differences were found in the correlation between IL-1 RA 1/2 polymorphism and FIGO stage (P = 0.53), histological grading (P = 0.67), recurrence status (P = 0.72), ascites volume (<=500 ml versus >500 ml; P = 0.98), residual tumor mass (P = 0.17) and age (P = 0.27).


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
IL-1 RA may have a function in the host immune responses in the local and general environments of gynecological cancers [29]. IL-1 RA decreases tumor growth and tumor angiogenesis [30].

Polymorphisms exist in all three genes of the IL-1 family (IL-1{alpha}, IL-1ß and IL-1 RA) and are located very close to each other on the long arm of the human chromosome 2q [31, 32]. Mutation in one of these genes up-regulates the expression of these genes, e.g. IL-1 RA polymorphism is associated with enhanced IL-1ß production in vitro [16]. Also, IL-1 RA genotype plays a major role as a modulator in IL-1ß release [33].

The allele 2 of IL-1 RA seems to be the critical point in the molecular pathway of different diseases. The two-repeat allele has been associated with different benign diseases: vestibulites, ulcerative colitis, alopecia areata, psoriasis, autoimmune conditions [6, 34] and idiopathic recurrent miscarriage [35].

There are some data that indicate an additional role in cancer. El-Omar et al. [23] and Machado et al. [36] have reported an association of the IL-1 gene cluster polymorphism with enhanced production of IL-1ß and gastric cancer. Carriers of IL-1 1B-511T and allele 2 of IL-1 RA homozygotes had an increased risk of developing gastric cancer with odds ratios of 2.7 (95% CI 1.5–4.9) and 3.1 (95% CI 1.5–6.5), respectively. Their findings complement the most widely accepted multistage model of gastric carcinogenesis and underline the fact that host genetic factors may determine why some people infected with Helicobacter pylori develop gastric cancer while others do not [23, 36].

This hypothesis can be transferred and applied to other solid tumors. Recently we have published that IL-1 RA is also associated with cervical cancer. Allele 2 heterozygotes have a greater risk of developing cervical cancer (P = 0.04) [25, 37].

In our study, we have demonstrated that the allele 2 polymorphism of the IL-1 RA gene is significantly associated with ovarian cancer. Allelic frequencies were different between patients with ovarian cancer and controls. Patients who are heterozygotes at allele 2 for IL-1 RA (IL-RA 1/2) have a significantly higher risk of ovarian cancer with an odds ratio of 2.7 (95% CI 1.4–5.2). But IL-1 RA mutation seems to give no information about the prognosis of the patients. We have not observed any association between specific alleles and clinical features such as FIGO stage, histological type or grading.

Recently, Hefler et al. [38] published a case–control study, where the IL-1 gene cluster was genotyped in 94 ovarian cancer patients and 27 patients with borderline ovarian tumors and 134 healthy women. In contrast to our results they found no differences in the prevalence of polymorphisms in the IL-1 RA gene between the ovarian cancer and control group. The distribution of the tumor stage of the enrolled patients in our study was different: stage I, 15.7% versus 40.8%; stage II, 6.5% versus 12.7%; stage III–IV, 77.7% versus 45.6%. Hefner and co-workers enrolled more patients at early stages than we did. But this cannot be the only explanation for the different results.

Ovarian cancer is known to be a polygenic disease and genetic factors must play an additional role in the induction of this malignancy, whereas only some of the genes were identified in ovarian cancer patients. Maybe some of them can mask potential influences. Polymorphisms in the IL-1 RA gene must be included in a panel of polymorphic genes which are associated with ovarian cancer, such as the androgen receptor [39], MTHFR [40] or cyclin D1 genes [3]. Therefore, these specific genes should also be analyzed in further studies.


    Footnotes
 
+ Correspondence to: Dr A. Mustea, Charité, Humboldt University, Augustenburger Platz 1, 13353 Berlin, Germany. Tel: +49-30-450-564052; Fax: +49-30-450-564952; E-mail: amustea{at}yahoo.com Back


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