Department of Obstetrics and Gynecology, Charité, Humboldt University, Berlin, Germany
Received 18 February 2003; revised 26 May 2003; accepted 17 June 2003
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 1.45.2). 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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, IL-1ß and IL-1 RA. All three molecules bind to IL-1 receptors [911]. IL-1
and IL-1ß are potent proinflammatory cytokines, while IL-1 RA as an anti-inflammatory cytokine competes with IL-1
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
, IL-1ß and IL-1 RA produce alterations of the IL-1
, 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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).
|
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 2681). From 108 patients, only 15.7% were diagnosed in FIGO stage I, 77.7% were in FIGO stage IIIIV. The most common histological type was serous-papillary (66.7 %).
|
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 1.45.2). The allele frequencies of IL-1 RA wild-type and polymorphic alleles in the study and control groups are given in Table 2.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Polymorphisms exist in all three genes of the IL-1 family (IL-1, 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 1.54.9) and 3.1 (95% CI 1.56.5), 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 1.45.2). 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 casecontrol 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 IIIIV, 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 |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Chang J, Bridgewater J, Gore M et al. Non-surgical aspects of ovarian cancer. Lancet 1994; 343: 335341.[ISI][Medline]
3. Dhar K, Branigan K, Howells R et al. Prognostic significance of cyclin D1 gene (CCND1) polymorphism in epithelial ovarian cancer. Int J Gynecol Cancer 1999; 9: 342347.[CrossRef][ISI][Medline]
4. Lichtenegger W, Sehouli J, Buchmann E et al. Operative results after primary and secondary debulking-operations in advanced ovarian cancer (AOC). J Obstet Gynaecol Res 1998; 24: 447451.[Medline]
5. Schrecengost A. Ovarian massbenign or malignant? AORN J 2002; 76: 792802.[Medline]
6. Jeremias J, Ledger WJ, Witkin S. Interleukin 1 receptor antagonist gene polymorphism in women with vulvar vestibulitis. Am J Obstet Gynecol 2000; 182: 283285.[ISI][Medline]
7. Strauss JF. Unravelling the genetics of complex disorders of reproduction. In Countinuto EM, Spinola P (eds): Reproductive Medicine: a Millennium Review. New York, NY: Parthenon Publishing Group 1999; 284287.
8. Witkin SS, Gerber S, Ledger WJ. Influence of interleukin-1 receptor antagonist gene polymorphism on disease. Clin Infect Dis 2002; 34: 204209.[CrossRef][ISI][Medline]
9. Dinarello CA. Biologic basis for interleukin-1 in disease. Blood 1996; 87: 20952147.
10. Iizuka N, Hazama S, Hirose K. Interleukin 1 receptor antagonist mRNA expression and the progression of gastric carcinoma. Cancer Lett 1999; 142: 179184.[CrossRef][ISI][Medline]
11. Eisenberg SP, Brewer MT, Verderber E et al. Interleukin-1 receptor antagonist is a member of the interleukin 1 gene family. Evolution of a cytokine control mechanism. Proc Natl Acad Sci USA 1991; 88: 52325236.[Abstract]
12. Dinarello C. Interleukin-1, interleukin-1 receptors and interleukin-1 receptor antagonist. Int Rev Immunol 1998; 16: 457499.[Medline]
13. Arend WP. Interleukin 1 receptor antagonist: discovery, structure and properties. Prog Growth Factor Res 1990; 2: 193205.[Medline]
14. Danis VA, Millington M, Hyland VJ, Grennan D. Cytokine production by normal human monocytes: inter-subject variation and relationship to an IL-1 receptor antagonist (IL-1Ra) gene polymorphism. Clin Exp Immunol 1995; 99: 303310.[ISI][Medline]
15. Hurme M, Santtila S. IL-1 receptor antagonist (IL-1Ra) plasma levels are co-ordinately regulated by both IL-1Ra and IL-1ß genes. Eur J Immunol 1998; 28: 25982602.[CrossRef][ISI][Medline]
16. Santtila S, Savinainen K, Hurme M. Presence of the IL-1RA allele 2 (IL1RN*2) is associated with enhanced IL-1ß production in vitro. Scand J Immunol 1998; 47: 195198.[CrossRef][ISI][Medline]
17. Pociot F, Molvig J, Wogensen L et al. A TaqI polymorphism in the human interleukin-1 ß (IL-1ß) gene correlates with IL-1ß secretion in vivo. Eur J Clin Invest 1992; 22: 396402.[ISI][Medline]
18. Licastro F, Pedrine S, Ferri C et al. Gene polymorphism affecting 1-antichymotrypsin and interleukin-1 plasma levels increases Alzheimers disease risk. Ann Neurol 2000; 48: 388391.[CrossRef][ISI][Medline]
19. Hulkkonen J, Laippala P, Hurne M. A rare allele combination of interleukin gene complex is associated with high interleukin-1ß plasma levels in healthy individuals. Eur Cytokine Netw 2000; 11: 251255.[ISI][Medline]
20. Apte R, Voronov E. Interleukina major pleiotropic cytokine in tumorhost interactions. Semin Cancer Biol 2002; 12: 277290.[CrossRef][ISI][Medline]
21. Marth C, Zeimet AG, Herold M et al. Different effects of interferons, interleukin-1ß and tumor necrosis factor- in normal (OSE) and malignant human ovarian epithelial cells. Int J Cancer 1996; 67: 826830.[CrossRef][ISI][Medline]
22. Chen Z, Fadiel A, Feng Y et al. Ovarian epithelial carcinoma tyrosine phosphorylation, cell proliferation, and ezrin translocation are stimulated by interleukin 1 and epidermal growth factor. Cancer 2001; 92: 30683075.[CrossRef][ISI][Medline]
23. El-Omar EM, Carrington M, Chow WH et al. Interleukin-1 polymorphisms associated with increased risk of gastric cancer. Nature 2000; 404: 398402.[CrossRef][ISI][Medline]
24. Sehouli J, Mustea A. Interleukin-1 receptor antagonist gene polymorphism and cancer. Clin Infect Dis 2002; 34: 15351536.[CrossRef][ISI][Medline]
25. Sehouli J, Mustea A, Konsgen D et al. Polymorphism of IL-1 receptor antagonist gene: role in cancer. Anticancer Res 2002; 22: 34213424.[ISI][Medline]
26. Barber MD, Powell JJ, Lynch SF et al. A polymorphism of the interleukin-1ß gene influences survival in pancreatic cancer. Br J Cancer 2000; 83: 14431447.[CrossRef][ISI][Medline]
27. Sehouli J, Lichtenegger W, Hauptmann S, Dietel M. Therapie von Borderline-Tumoren des Ovars. Frauenarzt 2002; 43: 12021207.
28. Tarlow JK, Blakemore AIF, Lennard A et al. Polymorphism in human IL-1 receptor antagonist gene intron 2 is caused by variable numbers of an 86-bp tandem repeat. Hum Genet 1993; 91: 403404.[ISI][Medline]
29. Irvine KR, Rao JB, Rosenberg SA, Restifo NP. Cytokine enhancement of DNA immunization leads to effective treatment of established pulmonary metastases. J Immunol 1996; 156: 238245.[Abstract]
30. Corbacioglu S, Pietsch T, Koenig A, Welte K. Interleukin-1 receptor antagonist: a powerful inhibitor of tumor vascularization and tumor growth in vivo. In VI International Symposium on Pediatric Neuro-Oncology, MD Anderson Cancer Center, Houston, TX 1994; 35 (Abstr).
31. Arend WP. Interleukin-1 receptor antagonist. A new member of the interleukin family. J Clin Invest 1991; 88: 14451451.[ISI][Medline]
32. Dinarello CA. The interleukin-1 family: 10 years of discovery. FASEB J 1994; 8: 13141325.
33. Vamvakopoulos J, Green C, Metcalfe S. Genetic control of IL-1ß bioactivity through differential regulation of the IL-1 receptor antagonist. Eur J Immunol 2002; 32: 29882996.[CrossRef][ISI][Medline]
34. Arend WP, Malyak M, Guthridge CJ, Gabay C. Interleukin-1 receptor antagonist: role in biology. Annu Rev Immunol 1998; 16: 2755.[CrossRef][ISI][Medline]
35. Unfried G, Tempfer C, Schneeberger C et al. Interleukin 1 receptor antagonist polymorphism in women with idiopathic recurrent miscarriage. Fertil Steril 2001; 75: 683687.[CrossRef][ISI][Medline]
36. Machado JC, Pharoah P, Sousa S et al. Interleukin 1B and interleukin 1RN polymorphisms are associated with increased risk of gastric carcinoma. Gastroenterology 2001; 121: 823829.[ISI][Medline]
37. Mustea A, Sehouli J, Sofroni D et al. Interleukin 1 receptor antagonist (IL-1RA) polymorphism in women with cervical cancer. Anticancer Res 2002; 1B: 486 (Abstr).
38. Hefler L, Ludwig E, Lebrecht A et al. Polymorphism of the interleukin-1 gene cluster and ovarian cancer. J Soc Gynecol Invest 2002; 9: 386390.[CrossRef][ISI][Medline]
39. Levine DA, Boyd J. The androgen receptor and genetic susceptibility to ovarian cancer: results from a case series. Cancer Res 2001; 61: 908911.
40. Gershoni-Baruch R, Dagan E, Israeli D et al. Association of the C677T polymorphism in the MTHFR gene with breast and/or ovarian cancer risk in Jewish women. Eur J Cancer 2000; 36: 23132316.[CrossRef][ISI][Medline]