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Breast cancer progression and host polymorphisms in the chemokine system: role of the macrophage chemoattractant protein-1 (MCP-1) -2518 G Allele.

Interaction between tumor cells and stroma is essential for tumor growth. Tumor cells stimulate the formation of stromal tissue, which excretes a variety of growth factors, cytokines, and proteases. Tumor-associated macrophages (TAMs) are one of the major components of tumor stromal tissue and are capable of eliciting diverse aspects of tumor growth as either a positive or negative regulator (1). In breast carcinoma, large numbers of infiltrating T cells and TAMs are often observed. The leukocyte infiltrate is found within the tumor stromal areas as well as in the epithelial areas that constitute the tumor mass (2-13).

Recent reports suggest that the inflammatory reaction at the breast tumor site affects tumor growth and progression. Whereas lymphocytes have been shown to have divergent effects on development of breast cancer (2,10-13), it is widely accepted that high concentrations of TAMs are correlated with poor prognosis (2-10). Macrophage infiltration into tumors is regulated by several cytokines and chemokines, in particular macrophage chemoattractant protein-1 (MCP-1). MCP-1 is a member of the C-C chemokine family and possesses chemotactic activity for monocytes and T lymphocytes (14-17). MCP-1 is produced not only by tumor cells, but also by stromal cells such fibroblasts, endothelial cells, and monocytes. MCP gene transfer enhances the metastatic potential of cancer cells with increased neovascularization, whereas MCP-1 itself activates monocyte cytostatic function against tumor cells (18,19).

Recently, several studies have focused on other chemokines and chemokine receptors in the susceptibility and progression of cancer (20), in particular "regulated on activation normal T cell expressed and secreted" (RANTES), a molecule that attracts T cells and monocytes, and its receptor CCR5 (21, 22).

A third chemokine, stromal cell-derived factor-1 (SDF1), seems to be important in breast cancer progression and was demonstrated to be overproduced in breast cancer tissue (23-27).

Genetic variations commonly occur in the regulatory regions of chemokine genes, and such polymorphisms affect chemokine gene transcription in response to inflammatory stimuli. Consistent with this, polymorphisms have been described in the genes encoding for MCP-1, RANTES, CCR5, and SDF-1 (28-31).

The aim of this study was to investigate possible correlations between polymorphisms in the genes encoding for MCP-1, RANTES, SDF-1, and CCR5 and breast cancer clinical phenotypes, specifically the ability of genetic analysis to identify a subgroup of breast cancer patients with a disease that appears more aggressive or prone to metastasize.

We determined the MCP-1 -2518, RANTES -403, CCR5 delta32, and SDF-1 -801 genotypes in DNA isolated from peripheral blood samples of a consecutive unselected series of 83 white women from northern Italy with breast cancer of different stages who underwent surgery, and 141 age-matched healthy white women (control group). The median age of the breast cancer patients was 62 years (range, 24-91 years), and the median age of the controls was 63 years (range, 29-83 years).

We followed all patients in this study for 6-30 months (median follow-up, 21 months). Our Institutional Ethical Committee approved this study, and consent was obtained from patients and controls.

The presence (M+) or absence (M-) of metastasis at the time of operation and during the follow-up, in addition to axillary lymph node invasion at the time of pathologic staging, were considered as well and matched with the distribution of allelic variants (Table 1 in the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem.org/content/ vo151 / issue2 /).

Vascular invasion was defined as the presence of cancer cells within endothelium-lined spaces in the hematoxylin/eosin-stained specimens. Whole blood (3 mL) from patients and controls was collected into potassium EDTA. DNA was prepared with Istagene Matrix extraction reagents (Bio-Rad Laboratories). The PCR reactions for MCP-1, RANTES, CCR5, and SDF-1 were carried out in a total volume of 25 [micro]L with 5 [micro]L of extracted genomic DNA; 100 [micro]M each of dATP, dGTP, dTTP, and dCTP; 1.5 mM Mg[Cl.sub.2; 1 U of Taq polymerase; and the two primers, forward and reverse, each at a concentration of 80 nM. The CCR5 delta32 deletion was identified by electrophoresis on a 2% agarose gel; the other three genotypes were determined with the PCR-restriction fragment length polymorphism assay described by Szalai et al. (32).

We used the Fisher exact test to check for differences in allele distributions among the groups. Odds ratios (ORs; approximate relative risk) were calculated as an index of the association of chemokine genotypes with each phenotype. For each OR, two-tailed probability values and 95% confidence intervals (CIs) were calculated. All statistical analyses were two-sided and were performed with Stata Statistical Software (Stata Corporation). We used P <0.05 as the cutoff point for statistical significance.

Allele frequencies in both control and patient populations were within Hardy-Weinberg equilibrium for the four genotypes. In breast cancer patients, we found no differences in the variant distributions for MCP-1 -2518A/G, RANTES -403G/A, SDF-1 -801G/A, and CCR5 delta32 compared with controls. The relevant values are summarized in Table 2 of the online Data Supplement. SDF-1, RANTES, and CCR5 variant distributions showed no statistically significant differences between subgroup M+ (presence of metastases) vs controls or between subgroups M+ vs M- (absence of metastases) or M- vs controls. We observed no differences in relation to vascular and lymph node invasion.

As for the MCP-1 -2518A/G promoter polymorphism, we observed a strong correlation between the presence of at least one G allele and the M+ subgroup at the end of the follow-up period: for A/A vs A/G + G/G, the OR was 2.83 (95% CI, 1.06-7.64; P = 0.020) for M+ vs M-patients; and for M+ patients vs controls, the OR was 2.09 (95% CI, 1.15-7.52; P = 0.012; Table 1).

At the end of the follow-up, 26 patients with stage I to II disease developed metastases, whereas 40 remained metastasis free. The genotype distribution was as follows: A/A, 8 M+ and 25 M- patients; A/G + G/G;18 M+ and 15 M- patients (P = 0.023).

This study is the first to implicate host chemokine gene variants in the progression of breast cancer. Analysis of polymorphisms in the chemokine system indicated that breast cancer patients carrying at least one G allele for the MCP-1 gene regulatory region were at increased risk of developing metastases independently of the initial stage. We observed no correlation with RANTES, SDF-1, and CCR5 polymorphisms.

Macrophage infiltration is a cornerstone of inflammation and neoangiogenesis, which negatively affect prognosis of invasive breast cancer (8). Therefore, any genetic variation accelerating transcriptional activity in genes encoding for proteins involved in macrophage infiltration could be suspected to enhance tumor progression and metastases.

Although several studies showed the importance of signaling factors, i.e., RANTES, CCR5, and SDF-1, in the susceptibility and progression of breast cancer, our data failed to demonstrate a possible relationship to the genetic patterns of the patients (21-27).

MCP-1 has been shown to inhibit the generation of T lymphocytes in response to tumor aggression (33), and therefore it is likely to be involved in immune response to breast cancer (34).

The presence of a common functional single-nucleotide polymorphism (SNP) in the MCP-1 gene was identified by Rovin et al. (28), who found that a biallelic G/A polymorphism at position -2518 of the MCP-1 gene 5'-flanking region influenced the transcriptional activity of the putative distal regulatory segment of the gene. Furthermore, this polymorphism correlated with individual differences in monocyte MCP-1 production. Monocytes from individuals carrying a G allele at -2518 produced more MCP-1 after treatment with interleukin-1[beta] than monocytes from A/A homozygous individuals. It was suggested that MCP-1 plays key roles in macrophage recruitment, expression of angiogenic factors, and activation of matrix metalloproteinases in patients with breast cancer (35). These conclusions are substantially confirmed by our study. In the present series, patients classified at stages I and II at the time of the diagnosis developed distant metastases significantly more frequently when carrying at least one G allele compared with A/A homozygotes.

Our study moves upward to a higher level of control of MCP-1 production. The presence of at least one G allele in the MCP-1 gene promoter of patients with stage I or II disease at the time of diagnosis enhances their risk of metastasis by a factor of 2.67 compared with patients diagnosed in the same stage but homozygous for the allele A. This finding coincides with the known over-expression of MCP-1 in breast cancer tissue (35).

In apparent contrast to the findings of Saji et al. (35), who found a significant association between MCP-1 expression in tumor stromal cells and vascular invasion (lymphatic as well as venous vessels) and a tendency of lymph node involvement, we could not confirm any associations between the presence of a MCP-1 G allele in the host and vascular or lymph node invasion.

In a previous study, we reported a correlation between a functional matrix metalloproteinase-3 (MMP-3) gene polymorphism, the more active 5A variant, and breast cancer susceptibility and found that 5A homozygosity is an independent factor of poorer prognosis (36). The results reported here seem to run in the same direction because protease production (including MMP-3) is one of the protumor biological functions of TAMs (20) that are recruited and activated by MCP-1.

Although suggestive and consistent with our hypothesis, the present results must be considered cautiously. Further studies are needed to confirm the role of a functional MCP-1 gene SNP regarding the relationships between breast cancer and host. This is a very complex matter involving an incredibly high number of variables, each of which may influence in some degree this relationship. Functional MCP-1 gene SNPs represent only one of many factors involved in determining the prognosis of breast cancer. Should our data be confirmed, in the future MCP-1 could be a reliable candidate for inclusion in a panel of genetic risk factors conditioning the course of the disease.

DOI : 10.1373/clinchem.2004.041657

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Ghilardi, [1] * Maria Luisa Biondi, [2] Anna La Torre, [2] Lodovica Battaglioli, [2] and Roberto Scorzal ([1] Dipartimento MCO, Clinica Chirurgica Generale, Universita degli Studi di Milano-Polo S. Paolo, Milan, Italy; [2] Laboratorio di Chimica Clinica e Microbiologia, Ospedale S. Paolo-Polo Universitario, Milan, Italy; * address correspondence to this author at: Dipartimento MCO, Clinica Chirurgica Generale, Universita degli Studi di Milano-Polo S. Paolo, Via A. Di Rudim, 8, I-20142 Milan, Italy; e-mail giorgio.ghilardi@unimidt)
Table 1. MCP-1 genotypes in breast cancer patients with (M+) or
without (M-) metastases at the end of follow-up. (a)

 M + patients M - patients
 (n = 39) (n = 44)

 n % n %

MCP-1 genotype
 A/A 14 36 27 61
 A/G 22 56 16 36
 G/G 3 8 1 3

G allele frequency 28 0.36 18 0.21

 OR (95% CI) P

MCP-1 genotype
 A/A
 A/G
 G/G 2.83 (1.06-7.64) 0.020

G allele frequency 2.17 (1.03-4.64) 0.026

(a) Genotype data are expressed as the genotype frequency. The OR for
each genotype was calculated as A/A vs A/G + G/G.
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Title Annotation:Technical Briefs
Author:Ghilardi, Giorgio; Biondi, Maria Luisa; La Torre, Anna; Battaglioli, Lodovica; Scorza, Roberto
Publication:Clinical Chemistry
Date:Feb 1, 2005
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