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Technical Briefs |
Genes
1 Department of Biomedicine Evolutive Age, University of Bari, 70124 Bari, Italy
2 Department of Clinical Epidemiology, National Cancer Research Institute, 16148 Geneva, Italy
3 Department of Pediatrics, University of Foggia, CEINGE-Advanced Biotechnology, 80131 Naples, Italy
Departments of
4
Experimental Oncology and
5
Female Cancer, IRCCS-Oncologic Hospital, 70100 Bari, Italy
6 Department of Biochemistry and Biophysics, "F. Cedrangolo", II University of Naples, 80138 Naples, Italy
aaddress correspondence to this author at: Pediatric Department, University of Foggia, CEINGE-Advanced Biotechnology, Via Pansini 5, 80131 Naples, Italy; e-mail iolascon{at}dbbm.unina.it
We investigated the potential relationship between breast cancer and polymorphisms of the interleukin 10 (IL-10) and tumor necrosis factor-
(TNF-) genes.
High plasma TNF- concentrations predict poor cancer outcome (1), weight loss, cachexia, poor immune response, and anemia (2)(3). We studied the -308(G/A) polymorphism because it is the most frequently investigated, it is included in an AP-2 transcription-factor binding site of the TNF- gene (4)(5)(6), and its importance in TNF- gene expression has been demonstrated by reporter gene assays (7). The TNF- -308 polymorphism affects TNF- gene transcription in both a cell-type and stimulus-specific manner, with a remarkable effect in macrophage-like cells (8).
IL-10, an important immunoregulatory cytokine, induces T-cell anergy (9) and prevents tumor antigen presentation to CD8+ cytotoxic T lymphocytes by suppressing expression of MHC class I and II antigens (10). This effect may contribute to the lack of immune response toward transformed cells (11)(12)(13). Three polymorphisms [-1082(G/A), -819(C/T), and -592(C/A)] have been described in the IL-10 promoter region, but data are unclear regarding their influence, alone or in reciprocal linkage, on transcription (14). Higher IL-10 production seems to be associated with -1082 G/G. The positive effect on IL-10 gene expression was substantiated by reporter gene assays (15)(16), although, in some cases, differing results have been described in cells undergoing distinct stimuli (17).
There appears to be conflicting evidence on the interplay between IL-10 and cancer. Indeed, it has been proposed that IL-10 might contribute to tumor escape from the immune response, but it may also exert an antitumor effect. In one study, IL-10-deficient mice developed colitis and colorectal cancer, mirroring the increased colorectal cancer incidence observed in patients with inflammatory bowel disease (18). In another study, the results indicated that the G/G genotype, associated with high IL-10 expression, protects against cutaneous malignant melanoma (CMM), whereas a low-expression genotype is a risk factor for more advanced disease and may confer CMM susceptibility. This finding also suggests an IL-10 antitumor effect in CMM, possibly via inhibition of angiogenesis (19). Finally, several anticancer protocols include the use of IL-10 treatment. These reported observations led us to investigate a possible link between breast cancer and TNF- or IL-10 gene polymorphisms.
We studied 125 consecutive breast cancer patients (age range, 25–86 years) seen for surgery between February and September 2001 at the Female Cancer Department of the Cancer Research Institute in Bari, Italy. The diagnosis was confirmed by histologic examination. The stage of the disease, according to the TNM system, histologic type, and tumor grade, was recorded for each patient. The majority (106 of 125; 85%) of cases were invasive breast cancers. Six (5%) were ductal breast carcinomas in situ, whereas 13 (10%) were metastatic infiltrative breast cancers; 93% (116 of 125) of the cancers were of the ductal histotype.
We selected 100 controls from unrelated female volunteers attending the same clinic, who were free from breast cancer and without a history of tumor (or other serious illness). Written informed consent was obtained from all breast cancer patients and controls.
Genomic DNA was extracted from peripheral blood leukocytes (20). A PCR–restriction fragment length polymorphism assay was performed to detect the -1082(G/A) IL-10 and -308(G/A) TNF- polymorphisms by introducing a single base change (C
T and GC) in the primer sequence, according to the amplification-created restriction site method (21)(22). PCR amplification of IL-10 and TNF- promoter sequences was performed with primer pairs 5'-GTCAGTGTTCCTCCCAGT-3' and 5'-TTACCTATCCCTACTTCCTC-3' and 5'-AATAGGTTTTGAGGGCCATG-3' and 5'-TCATCTGGAGGAAGCGGTAG-3', respectively (the underlined bases indicate the CT and GC changes). The PCR mixture contained 100 ng of genomic DNA, 0.2 mM each of the deoxynucleotide triphosphates, 0.2 mM each of the primers, and 1 U of Taq polymerase in a 25-µL final volume. The samples were heated at 95 °C for 3 min followed by 35 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s, with a final extension at 72 °C for 10 min. PCR products were visualized on a 2% agarose gel stained with ethidium bromide.
The restriction fragment length polymorphism assay was performed in a 15-µL reaction volume containing PCR product and specific restriction enzymes (EarI for the IL-10 polymorphism and NcoI for the TNF- polymorphism). The digestion products were visualized on a 4% agarose gel stained with ethidium bromide. In the presence of the TNF- -308 G allele, NcoI cut the 224-bp PCR product into two bands of 208 and 16 bp, respectively. The IL-10 -1082 G allele gave a product of 295 bp, whereas the A allele gave two fragments, of 275 and 20 bp (Fig. 1
). IL-10 and TNF- typing by the amplification-created restriction site method was successfully confirmed by automated DNA sequencing of 20 PCR products on an ABI Prism 310 with use of the BigDye-Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems).
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The 
2 test was used to assess the statistical significance of the association between breast cancer and TNF- or IL-10 genotypes. Relative risk of breast cancer associated with a particular genotype was estimated by the odds ratio (OR). To obtain age-adjusted estimates of the OR associated with the various genotypes, a multivariate conditional logistic regression model was fitted to the data, with the case/control status as the dependent variable and age (in 10-year classes) and TNF- and IL-10 genotypes as covariates.
The distributions of IL-10 and TNF- genotypes are summarized in Table 1
. We observed a significant association between IL-10 genotype and the risk of breast cancer (Table 1
). Forty-eight percent of breast cancer cases and 33% of controls were AA homozygotes. Compared with AA homozygotes, the ORs of breast cancer for AG heterozygotes and GG homozygotes were 0.58 (95% confidence interval, 0.32–1.07) and 0.38 (95% confidence interval, 0.14–0.99), respectively (P = 0.046). Conversely, we observed no difference between cases and controls in the distribution of TNF- genotypes (Table 1
). These findings were also confirmed when adjusted for age in the multivariate logistic regression analysis.
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The IL-10 -1082 genotype frequency we observed in Italian individuals is slightly at variance with that observed by Lio et al. (23). However, very recent data reported by Uboldi de Capei et al. (24) in a detailed study on genotype frequencies are almost completely in agreement with our findings. This indirectly confirms the statistical soundness of our investigation.
The results of this association study suggest that the IL-10 AA genotype is correlated with a marked increase in breast cancer risk, whereas there is no indication of an association between the TNF- polymorphism and breast cancer risk.
Before we discuss the inferences of our findings, however, some limitations of the study, mainly related to its explorative nature, are worth mentioning. Possible bias, arising from the selection of controls, could lead to underestimation of the association between breast cancer risk and the factors under investigation because of the overrepresentation, in this group, of women at risk of developing breast cancer. Moreover, the limited sample size produces relative risk estimates lacking adequate precision. In view of these limitations, our investigation should be considered a hypothesis-generating study, providing suggestive but preliminary evidence in favor of the association between breast cancer risk and the IL-10 genotype. The protective effect of the IL-10 GG genotype appeared to be independent of tumor histology or stage (not reported), although limited power was available for these subgroup analyses because the majority of the cases were of the ductal type and in stage I-II.
The lack of association between TNF- genotype and breast cancer risk is at variance with recent reports by Mestiri et al. (25), who demonstrated a fourfold increase in breast cancer risk in TNF2 (A) homozygous individuals. However, the different genetic background of Tunisians, as demonstrated by the TNF2 allele frequency [16% in the study by Mestiri et al. (25) and 8% in our study] and the higher proportion of T3 and T4 cases included in the Tunisian study (40% vs 25% in our study) might explain the different results obtained for TNF-. Moreover, and possibly more interesting, another group (26) has very recently investigated the -308(G/A) TNF- polymorphism in 95 breast cancer patients and 190 healthy individuals. This study, in agreement with our findings, concluded that breast cancer risk was not associated with TNF- promoter single-nucleotide polymorphisms (26).
In conclusion, this case-control study reports, for the first time, an association between the IL-10 AA genotype and increased risk of breast cancer. Because this polymorphism is associated with lower production of the cytokine (15) and because IL-10 may play a protective and preventive role, at least in some cancers, it is conceivable that the IL-10 AA genotype might represent a breast cancer risk factor of low penetrance. Future studies appear necessary to confirm and extend our observations, taking into account its association with other known risk factors of breast cancer.
Acknowledgments
We thank Nicola De Marzo for technical support. This work was supported in part by grants from the Fondazione Italiana per la Ricerca sul Cancro (FIRC), the Associazione Italiana per la Ricerca sul Cancro (AIRC), MURST, the Associazione Italiana per la Lotta al Neuroblastoma, and by "progetto finalizzato Oncologia" CNR-MIUR, Italy.
References
B: a pivotal transcription factor in chronic inflammatory diseases. N Engl J Med 1996;336:1066-1071.
(TNF) gene detectable by Nco1 restriction of PCR product. Hum Mol Genet 1993;1:353-359.
A substitution at position -863) in the promoter region of the tumour necrosis factor- (TNF-) gene associated with reduced circulating levels of TNF-. Hum Mol Genet 1999;8:1443-1446. promoter polymorphisms influence the phenotypic expression of hereditary hemochromatosis. Blood 2001;97:3707-3712. promoter region and in the stress protein hsp70–2: susceptibility and prognostic implications in breast carcinoma. Cancer 2001;91:672-678.[CrossRef][ISI][Medline]
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