Significant Reduction in Breast Cancer Risk for Japanese Women with Interleukin 1B 31 CT/TT Relative to CC Genotype

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1 Jpn J Clin Oncol 2002;32(10) Significant Reduction in Breast Cancer Risk for Japanese Women with Interleukin 1B 31 CT/TT Relative to CC Genotype Lucy Sayuri Ito 1, Hiroji Iwata 2, Nobuyuki Hamajima 3, Toshiko Saito 3, Keitaro Matsuo 3, Mitsuhiro Mizutani 2, Takuji Iwase 2, Shigeto Miura 2, Katashi Okuma 4, Manami Inoue 3, Kaoru Hirose 3 and Kazuo Tajima 3 1 Department of Neurology, School of Medicine, São Paulo University, São Paulo, Brazil and 2 Department of Breast Surgery, 3 Division of Epidemiology and Prevention and 4 Department of Clinical Laboratories, Aichi Cancer Center, Nagoya, Japan Received May 7, 2002; accepted June 12, 2002 Objective: The present case-control study aimed to examine the associations between breast cancer risk and three functional polymorphisms (Interleukin (IL) 1A C-889T, IL-1B C-31T and IL-1RN 86-bp variable number tandem repeat) related to expression of IL-1, which combines estrogen receptor. Methods: Cases were 231 patients with breast cancer who had been diagnosed 1 month to 6 years before their enrollment in at Aichi Cancer Center Hospital. Controls were 186 non-cancer outpatients recruited during the same period at the digestive tract, breast surgery and gynecology clinics. Results: There were no differences in the genotype distributions of the IL-1A and IL-1RN polymorphisms, but individuals harboring a IL-1B C-31T T allele (high expression allele) were less frequent among cases (74.3%) than among controls (84.9%). The age-adjusted odds ratio (OR) relative to CC genotype was 0.52 (95% confidence interval, ) for CT genotype, 0.58 ( ) for TT genotype and 0.54 ( ) for CT/TT genotype. Subgroup analysis showed that the preventive effect was significantly stronger for postmenopausal women than for premenopausal women (interaction 0.30, ). Conclusions: Although this is the first report on the association between breast cancer risk and IL-1B C-31T, the observed association seems plausible in a biological sense. Key words: breast cancer interleukin 1B polymorphisms PCR-CTPP INTRODUCTION The interleukin (IL)-1 family includes three members, IL-1, IL-1 and IL-1 receptor antagonist (IL-1Ra), which play important roles in inflammatory and immunological responses (1). IL-1 is a membrane-bound IL-1, whose precursor, proil- 1, is fully active in the cytosol. Some investigators consider that the intracellular form regulates normal cellular differentiation, particularly in epithelial cells (1). It is encoded by IL-1A, which is mapped to chromosome 2q13, along with the other two member genes. IL-1 is a molecule that triggers inflammation, by binding to IL-1 receptor type I. It is encoded by IL-1B with several promoter elements, including a TATA box, a typical motif of inducible genes. IL-1Ra is an antagonist, For reprints and all correspondence: Nobuyuki Hamajima, Division of Epidemiology and Prevention, Aichi Cancer Center Research Institute, 1 1 Kanokoden, Chikusa-ku, Nagoya , Japan. nhamajim@aichi-cc.jp which combines with IL-1 receptors (IL-1RI and IL-1RII) competitively. Its encoding gene is called IL-1RN. Recently, IL-1 was reported to contribute to metastasis of breast cancer cells by enhancing their expression of IL-6 and IL-8 and to induce in fibroblasts these two interleukins and matrix metalloproteinase 3 (2). In breast cancer cells, IL-1 was found to combine with the estrogen receptor (ER), resulting in transcriptional activation (3). The concentration of IL-1 is reported to be higher in invasive breast cancer tissue than in benign lesions (4). In breast cancer tissue homogenates, IL-1 concentration correlates inversely with ER, a wellknown prognostic factor for breast cancer, while the IL-1Ra concentration correlates with both ER and IL-1. However, there does not appear to be any difference in IL-1 and IL-1 levels between ER-negative and ER-positive breast cancer tissues (5). Some of the above findings suggest that interindividual variations in IL-1 production may impact on the prognosis with breast cancers. Although prognostic factors are not necessarily risk factors, the functional polymorphisms of IL-1s may influence breast cancer risk Foundation for Promotion of Cancer Research

2 Jpn J Clin Oncol 2002;32(10) 399 Table 1. Characteristics of cases and controls Characteristic Cases Controls n = 231 % n = 186 % Age at diagnosis for cases or at interview for controls Age at menarche (years)* < Age at first birth (years) < No birth Menopause Premenopausal Postmenopausal BMI (kg/m 2 ) < Breast cancer family history No Yes *Unknown for one case. BMI, body mass index. Breast cancer history of the mother and/or sister (s). Several polymorphisms have been reported for the three IL- 1 members (6,7). Among those, IL-1A C-889T, IL-1B C-31T and IL-1RN 86-bp variable number tandem repeat (VNTR) at intron 2 are reported to be functional (8,9). The IL-1A polymorphism has been reported to have an association with Alzheimer s disease (10). Associations with IL-1B polymorphisms have been reported for stomach cancer risk (7), Helicobacter pylori infection (11), periodontitis (12) and inflammatory bowel diseases (13). The inflammatory bowel diseases were also associated with the IL-1RN polymorphism (13 15). The present case-control study was conducted to investigate possible associations with the IL-1A C-889T, IL-1B C-31T and IL-1RN 86-bp VNTR polymorphisms among Japanese women, using the same approach as in previous reports (16 18). PATIENTS AND METHODS STUDY SUBJECTS Cases were female breast cancer patients aged years histologically diagnosed at Aichi Cancer Center Hospital. Controls were female patients without cancer who visited outpatient clinics at the same hospital. This study was openly announced to female outpatients at the reception desk for firstvisit patients and in waiting rooms of breast surgery clinic. In addition, doctors at the breast surgery clinic invited eligible patients to participate in this study. Between March 1999 and April 2000, 243 breast cancer patients were enrolled. Twelve cases diagnosed 6 years before were excluded and the remaining 231 cases were used for analysis. Controls were 186 women without a history of cancer; 123 outpatients mainly of the gastroenterology clinic and 63 outpatients mainly of the breast surgery or gynecology clinics, aged years. It was not rare that outpatients visited two or more clinics, because of anxiety about whether they might be suffering from cancers. In our hospital, approximately 70% of first-visit non-cancer patients were found to be disease-free, presenting for the purpose of an annual checkup or further examination after a positive result at a cancer screening facility (19). Those who signed an informed consent form were asked to complete a self-administered questionnaire and to provide a 7 ml peripheral blood sample. This study was approved by the Ethical Committee at Aichi Cancer Center in 1999 (Ethical Committee Approval Numbers and 12-23). GENOTYPING DNA was extracted from 200 l of buffy coat preserved at 40 C with a QIAamp DNA Blood Mini Kit (Qiagen, Valencia, CA). PCR-CTPP (polymerase chain reaction with confronting two-pair primers) was applied for IL-1A C-889T and IL-1B C-31T polymorphisms as described in our previous papers (11,20) and IL-1RN genotyping was conducted by the same method as described by Mansfield et al. (14).

3 400 Breast cancer and IL-1B polymorphism Table 2. Genotype frequencies for IL-1A, IL-1B and IL-1RN polymorphisms Genotype Cases Controls 2 test IL-1A C-889T STATISTICAL ANALYSIS An unconditional logistic model was applied for estimating odds ratios (ORs) and 95% confidence intervals (CIs), as well as interaction terms, with the computer program STATA Version 7 (STATA Corp., College Station, TX). All the ORs were adjusted for age by the logistic model including a continuous variable of age at diagnosis for cases and age at enrollment for controls. Women less than 50 years of age with hysterectomy before menopause were classified as being premenopausal. Those younger than 50 years whose menstruation stopped after chemotherapy were also classified into the premenopausal group. RESULTS n = 231 % n = 186 % CC = 1.70 CT d.f. = 2 TT P = Not genotyped 3 1 IL-1B C-31T CC = 6.85 CT d.f. = 2 TT P = Not genotyped 4 1 IL-1RN 86-bp VNTR = d.f. = P = Not genotyped 4 1 Table 1 summarizes data for 231 cases and 186 controls. The controls were slightly older, with a mean age of 52.9 as compared with 50.4 years for the cases. There were no significant differences in age at menarche, age at first birth and body mass index (BMI, kg/m 2 ) between cases and controls. The number of postmenopausal subjects was greater in controls than in cases, which reflected the age distribution of non-cancer patients of our hospital. The cases had a higher percentage for mother and/or sister family history of breast cancer than the controls (P = by Fisher s exact test). Genotype frequencies for IL-1A C-889T, IL-1B C-31T and IL-1RN 86-bp VNTR are shown in Table 2. No significant differences in genotype frequency between the cases and controls were observed for IL-1A and IL-1RN polymorphisms. However, the frequency of CC genotype of IL-1B C-31T was 25.7% for cases and 15.1% for controls; the difference was statistically significant (P < 0.05). Further analysis was focused on the IL-1B C-31T polymorphism. The age-adjusted ORs estimated by a logistic model are shown in Table 3. The estimates relative to the CC genotype were 0.52 (95% CI, ) for the CT genotype and 0.58 ( ) for the TT genotype. When CT and TT were combined, the OR was 0.54 ( ). They did not differ substantially according to the interval between diagnosis and enrollment. No significant differences in the OR were found with reference to age at menarche, age at first childbirth and BMI, although the difference in the point estimate was substantial for the age at menarche and BMI. With regard to menopause status, the difference was significant; the interaction term between menopausal status (0 for premenopausal and 1 for postmenopausal) and genotype (0 for CC and 1 for CT/TT) was 0.31 ( ). The OR among premenopausal women was 0.91 ( ) for CT genotype, 0.74 ( ) for TT genotype and 0.85 ( ) for CT/TT genotype, whereas that for postmenopausal women was 0.24 ( ), 0.37 ( ) and 0.29 ( ), respectively. There was no difference in the OR between the postmenopausal women with a BMI < 22 and those with a BMI 22; OR for CT/TT was 0.27 ( ) and 0.27 ( ), respectively. As shown in Table 3, none of 12 controls with breast cancer history of mother and/or sister(s) had CC genotype, whereas nine out of 29 cases had CC genotype (P = by Fisher s exact test). DISCUSSION The present study showed that women with CT/TT genotype of IL-1B C-31T had a reduced risk of breast cancer. Subgroup analysis revealed that the preventive effect was marked for postmenopausal women. The observed association needs to be discussed in terms of (1) studies with prevalent cases, (2) possible bias for genotype frequency, (3) statistical power and (4) possible biological explanation. Prognostic factors influence ORs derived from prevalent case-control studies seriously for cancers with a poor outcome, such as pancreatic cancer, for which the T allele at 3954 of IL- 1B was reportedly associated with poor prognosis (21). However, the impact may be limited for cancers with a relatively good prognosis, such as breast cancer (22). In this study, the OR for survivors 3 years or longer after diagnosis was not different from that for patients diagnosed in the past 2 years, indicating that no adjustment was required for prognostic effects. Since recall bias is not relevant for genotyping, there seem no logical problems in using case-control studies as long as the OR for the long survivors is the same for the cases enrolled more recently (22). Even in case-control studies with incident cases using a cancer registry, it is not rare that it takes 6 months

4 Jpn J Clin Oncol 2002;32(10) 401 Table 3. Age-adjusted odds ratios (ORs) and 95% confidence intervals (95% CIs) for IL1B C-31T genotype Subjects Cases Controls OR (95% CI) CC CT TT CT/TT All subjects ( ) 0.58 ( ) 0.54 ( ) Interval after diagnosis <3 years ( ) 0.52 ( ) 0.48 ( ) 3 years ( ) 0.66 ( ) 0.60 ( ) Age at menarche (years)* < ( ) 0.85 ( ) 0.83 ( ) ( ) 0.39 ( ) 0.34 ( ) Age at first birth (years) < ( ) 0.63 ( ) 0.49 ( ) 25 or no birth ( ) 0.50 ( ) 0.53 ( ) Menopause Premenopausal ( ) 0.74 ( ) 0.85 ( ) Postmenopausal ( ) 0.37 ( ) 0.29 ( ) Body mass index (kg/m 2 ) < ( ) 1.10 ( ) 0.73 ( ) ( ) 0.28 ( ) 0.35 ( ) Family history of breast cancer (mother or sisters) No ( ) 0.61 ( ) 0.61 ( ) Yes /0 10/9 10/3 20/12 *Unknown for one case. Cases/controls. or so after diagnosis for research staff to contact them, with the result that some patients die before the first contact. The IL-1B C-31T polymorphism was genotyped by the same method for patients with esophageal or colorectal cancer in our laboratory. The CC genotype was found in 17.2% (16/93) and 17.3% (24/139), respectively (unpublished data). The percentages were similar to those in the present controls. Only breast cancer series showed a higher percentage. In addition, the genotype frequency for controls was calculated according to the clinic that they visited; 14.6% for 123 outpatients of the digestive tract clinic and 16.1% for the remaining 62 outpatients, mainly from breast surgery and gynecology clinics. It seems unlikely that the genotype frequency for the controls was biased. There was no way for patients or research staff to have access to genotype information before genotyping. Most of the breast cancer cases were invited to participate in the study by doctors in charge. Only six patients with breast cancer refused to provide blood because of their anxiety about genotyping. Generally, associations with rare alleles are difficult to examine in case-control studies for a moderately elevated risk. The present study was conducted within a project to detect genotypes related to breast cancer risk, so that the population was not large. However, the statistical power to detect OR = 2 for 231 cases and 186 controls under P < 0.05 (two-sided test) is 0.75 when the high risk genotype is 15% among controls, e.g. for CC genotype of IL-1B C-31T. Among Japanese, the T allele of IL-1A C-889T is present in 8.5% [n = 241 (11)] and alleles other than four repeats of IL- 1RN in 5.4% [n = 241 (1)]. Both alleles are less frequent than in Caucasians; for the IL-1A T allele, 33.1% [n = 400 in Finland (8)] and 28.3% [n = 242 in England (14)] and for the nonfour-repeat alleles of IL-1RN, 27.9% [n = 429 in Poland (7)], 29.9% [n = 400 in Finland (8)], 26.6% [n = 261 in England (14)] and 27% [n = 61 in South Africa (13)]. The T allele of IL- 1B C-31T had a penetration of 55.0% [n = 241 (11)], less frequent than in Caucasians [70.2%, n = 429 in Poland (7)]. These genotype frequencies indicate that the effects of IL-1B C-31T polymorphism can be examined more efficiently among Japanese than among Caucasians. A smaller proportion of the IL- 1A and IL-1RN influential alleles enhances the evaluation of IL-1B C-31T with respect to disease risk among Japanese. As mentioned in the Introduction, the biological findings suggest that high levels of IL-1 predispose to a poor prognosis and possibly to an increase in breast cancer risk. Since the T allele of C-31T is thought to be the higher expression allele, if it is correct, the protective effect observed here is paradoxical. Although biological evidence is scarce, there are two possible interpretations. One is that the T allele is not a high expression allele, but linked to a low expression allele. The T allele of C-31T is linked nearly completely to the C allele of IL-1B C-511T (7,11). It has been reported that individuals with IL- 1A 889T and IL-1B 511T alleles have a higher serum IL-1

5 402 Breast cancer and IL-1B polymorphism level on average (8). This means that IL-1B 31C is a higher expression allele among those with IL-1A 889T, who are rare in Japanese. An alternative interpretation is that the 31T is truly a higher expression allele and plays a role in reducing breast cancer risk. An electrophoretic mobility shift assay indicated that the 31T allele was highly expressed by lipopolysaccharide (7). Since IL-1 is a multifunctional cytokine, the higher expression may induce protective mechanisms against breast cancer cells, as a whole. Nearly every cell type is affected by IL-1, acting in concert with other cytokines or small mediator molecules (1). Even though invasive breast cancer tissues contain higher levels of IL-1 than benign lesions (4) and IL-1 was demonstrated to enhance gene expression with an estrogen responsive element (3), the interleukin might nevertheless be protective against breast cancer carcinogenesis. The observed protective effect is more marked for postmenopause, a condition with a lower estrogen level which could become more sensitive to IL-1, may provide clues to elucidate the biological mechanisms. As noted above, the present study was conducted as part of a project to detect polymorphisms potentially associated with breast cancer risk (16 18). Some significant association may be found by chance, while others could actually be related to breast cancer risk. In this study, the results obtained were opposite to the expectation we had had before the genotyping. Since this is the first report on the association between the IL- 1B polymorphism and breast cancer risk, other studies are required to confirm and extend our findings. Acknowledgments The authors are grateful to Ms Hiroko Fujikura, Ms Keiko Asai, Ms Michiyo Tani, Ms Naomi Takeuchi and Ms Mayumi Kato for their technical assistance. 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