Serum IL-8 and IL-12 Levels in Breast Cancer

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1 Medical Oncology, vol. 24, no. 2, , 2007 Copyright 2007 by Humana Press Inc. All rights of any nature whatsoever reserved /(Online) X/07/24: /$30.00 Original Article Serum IL-8 and IL-12 Levels in Breast Cancer Duygu Derin, Hilal Oguz Soydinc, Nese Guney, Faruk Tas, Hakan Camlıca, Derya Duranyıldız, Vildan Yasasever, and Erkan Topuz Institute Of Oncology; Istanbul University, Istanbul, Turkey Abstract Interleukins (ILs) are known to play a fundamental role in cancer. We investigated the serum levels of IL-8 and IL-12, in breast cancer patients, and their relationship with the prognostic parameters and therapy. Fortyeight patients with pathologically verified breast carcinoma and 21 healthy controls were enrolled into the study. Serum samples were obtained at baseline and after two cycles of chemotherapy. Serum IL-8 and IL-12 levels were determined using enzyme-linked immunosorbent assay (ELİSA). There was no significant difference in the baseline serum IL-8 and IL-12 levels between breast cancer patients and healthy controls (p = and p = 0.871, respectively), no significant correlation between the prognostic parameters and the serum IL-8, IL-12 levels. However, in the subgroup consisting of metastatic breast cancer patients, baseline serum IL-8 levels were significantly higher compared with non-metastatic disease (p = 0.047). Anthracycline-based chemotherapy and the addition of taxane did not change the levels of both serum IL-8 and IL-12. Serum IL-8 level may be useful in determining metastatic breast cancer. Larger studies are needed to confirm this finding. Key Words: Breast cancer; chemotherapy; IL-8; IL-12; serum. Introduction The high incidence and the long survival of breast cancer have stimulated the need for new markers for earlier detection of metastases and better prognosis prediction. Cytokines are intercellular short-acting soluble mediators that are involved in the pathogenesis of cancer. Some of them, e.g., IL-8, IL-6, and IL-1, stimulate angiogenesis (1), and some of the others, e.g., IL-12 and IL-10, inhibit angiogenesis (2). Serum concentrations of the cytokines may be utilized as a marker of immunity status and prognosis in cancer (3). Interleukin-12 (IL-12) is known to play a fundamental role in activating antitumor immunity; it Received November 30, 2006; Accepted December 29, 2006 Dr. Duygu Derin, Istanbul Universitesi, Onkoloji Enstitusu, Capa, 34390, Istanbul, Turkey. dygderin@yahoo.com induces T-cell differentiation and activates natural killer cells (4). IL-12 deficiency has been reported in patients with various types of cancer such as colonic, gastric, and breast tumors (5 8). It is also an antiangiogenic agent (9,10) that enhances antitumor activity in preclinical models (2). Interleukin-8 (IL-8) is an angiogenic molecule that induces neovascularization in cancer (11). IL-8 expression is enhanced by vascular endothelial growth factor A, and both of them are modulated by hypoxia (12,13). Further angiogenesis concurs with a higher vessel density, thus an increased propensity for metastasis and enhanced paracrine growth stimulation of the tumors cells. The level of serum IL-8 in cancer has been evaluated in numerous studies (3,14 17). The studies that evaluated the prognostic and predictive significance of these serum interleukin levels in cancer patients have showed conflicting results (3,6 8,14 16,18 20). 163

2 164 Derin et al. In this study, our aim was to evaluate the significance of serum IL-12 and IL-8 levels in breast cancer patients. Materials and Methods Patients The study included 48 patients with pathologically verified breast carcinoma. They were consecutively admitted to the Institute of Oncology, Istanbul, Turkey, during a 10-mo period, September 2004 to July Tumors were classified by the ptnm system according to the diagnostic criteria of the American Joint Committee on Cancer 2002 classification system. The clinical characteristics of patients are summarized in Table 1. Serum samples were obtained at baseline and after two cycles of chemotherapy. None of the patients had received previous therapy including immunomodulators, cytokines, or steroids. Further exclusion criteria were to be pregnant, severe infections, and known allergic diseases. Thirty-nine patients were treated with adjuvant or neoadjuvant systemic chemotherapy. Nine patients had systemic metastases at presentation, and they received combination chemotherapy and palliative therapy. Twenty-one healthy persons were enrolled as controls. Measurement of Serum IL-12 and IL-8 Levels Blood samples were obtained from patients with breast carcinoma and from healthy controls by venipuncture. The sera were collected following centrifugation and were frozen immediately at 20 C until analysis. IL-8 (Biosource International, Inc., California, USA) is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA). A monoclonal antibody specific for IL-8 has been coated onto wells of the microtiter strips provided. Samples, including standards of known IL-8 content, are pipetted into the wells followed by the addition of a second biotinylated monoclonal antibody. During the first incubation, the IL-8 antigen binds to the immobilized antibody on one site, and to the solution phase biotinylated antibody on a second site. After removal of excess second antibody, streptavin peroxidase (enzyme) is added. Following a second incubation and washing to remove all unbound enzyme, a substrate solution is added, which is acted upon by the bound enzyme to produce color. The intensity of color was proportional to the concentration of IL-8 antigen in the samples. The amount of the product was quantified by an automated ELISA reader (Rayto, RT-1904C). The results were expressed as picogram per milliliter (pg/ml). IL-12 (Biosource International, Inc., California, USA) is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA). A monoclonal antibody specific for IL-12 has been coated onto the wells of the microtiter strips provided. Samples including standards of known IL-12 content are pipetted into the wells followed by the addition of a second biotinylated monoclonal antibody. During the first incubation, the IL-12 antigen binds to the immobilized antibody on one site, and to the solution-phase biotinylated antibody on a second site. After removal of excess second antibody, sterptavidin peroxidase (enzyme) is added. Following a second incubation and washing to remove all unbound enzyme, a substrate solution is added, which is acted upon by the bound enzyme to produce color. The intensity of color was proportional to the concentration of IL-12 antigen in the samples. The amount of the product was quantified by an automated ELISA reader (Rayto, RT-1904C). The results were expressed as picogram per milliliter (pg/ml). Statistical Analysis Data analyses were performed using SPSS software (SPSS 11, Chicago, IL, USA). The Wilcoxon signed ranks test was used for assessment of statistical significance. Correlations were calculated with Spearman s correlation test. Two-sides p values of <0.05 were considered to be significant. Results The levels of serum IL-12 and IL-8 in patients with breast carcinoma and in healthy controls are shown in Table 2. There was no significant difference in the serum IL-12 and IL-8 levels between patients with breast carcinoma and healthy controls (p = and p = 0.365, respectively).

3 Serum IL-8 and IL-12 in Breast Cancer 165 Table 1 Patient and Disease Characteristics Parameter n % 95% CI* No. of patients Age (median, range) 49 (24 74) < Histology Invasive ductal carcinoma Invasive medullary carcinoma Invasive lobular carcinoma Mixed Tumor size (cm) <2 cm cm Unknown 9 Nodal status Positive Negative Unknown 9 Number of involved nodes 1 3 nodes Metastasis Absent Present Nuclear grade Grade I II Grade III Unknown 6 Histological grade Grade I II Grade III Unknown 6 ER status Negative Positive Unknown 4 PR status Negative Positive Unknown 5 HER-2 Negative Positive * 95% CI: 95% confidence interval.

4 166 Derin et al. Table 2 Distribution of Serum IL-12 and IL-8 Values in Patients with Breast Carcinoma and Healthy Controls Patients (n = 48) Controls (n = 21) Median Range Median Range p IL IL Table 3 Distribution of IL-12 and IL-8 Values in Patients with Breast Cancer as a Function of Different Variables Parameter IL-12 IL-8 Age (<50 vs 50) ns ns Tumor size (<3cm vs 3cm) ns ns Node (negative vs positive) ns ns Metastasis (absent vs present) ns Histology (invasive ductal vs others) ns ns Nuclear grade (grade I, II vs grade III) ns ns Histological grade (grade I, II vs grade III) ns ns ER status (negative vs positive) ns ns PR status (negative vs positive) ns ns HER-2 status (negative vs positive) ns ns ns, not significant. Table 3 shows the correlations between baseline serum levels of IL-12 and IL-8 and clinicopathological factors. None of the prognostic parameters analyzed, including age, tumor size, number of nodes, receptors, histology, grade, or chemotherapy, were significantly correlated with the serum IL-12 or IL- 8 level. The only exception was the presence of metastasis; serum IL-8 levels were elevated in the metastatic group compared with non-metastatic disease (p = 0.047). Table 4 shows the differences of the serum levels of IL-12 and IL-8 due to the effects of chemotherapy in breast cancer patients. There was no significant difference in the serum IL-12 and IL-8 levels at presentation and after chemotherapy (p = and p = 0.124, respectively). The patients treated with combined chemotherapy including anthracycline were 23, and the patients treated with combined chemotherapy including anthracycline and taxane were 14. Adding a taxane Table 4 Distribution of Serum Values of IL-12 and IL-8 in Patients with Breast Carcinoma and in Healthy Controls to anthracycline-based combination chemotherapy did not alter serum levels of IL-12 and IL-8 (p = 0.61 and p = 0.87, respectively) Discussion Before chemotherapy After chemotherapy Median Range Median Range p IL IL There exists strong evidence that tumor growth can be actively controlled by the host immune system (21) and interleukins are known to play a significant role in immune response regulation. In this study, we investigated serum IL-12 and IL- 8 levels in breast cancer patients. The results were evaluated in relation to tumors prognostic parameters and chemotherapy. IL-12, which is produced primarily by monocytes/macrophages and by B-cells, is an important cytokine in the activation of cell-mediated immunity. IL-12 has a potent antitumor activity, and is used in the immunotherapy of cancer. It appears that patients with elevated blood concentrations of IL-12 have a higher survival rate than patients with low concentrations (22). It has been measured in different cancer types to detect a relationship between serum levels and tumor progression or diagnosis. In the literature, the reports concerning IL-12 levels in cancer patients have been contradictory. The serum levels of IL-12 were found to be decreased in colon cancer (6), gastrointestinal tumors (7), and breast cancer (8). In patients with ovarian cancer, elevated levels were discovered in ascitic fluid samples, and were found to be correlated with progression (19). In another study, Lissoni et al. found abnormally high levels of IL-12 in metastatic cancer patients (18). In concordance with the two last studies, Kovacs found that the serum levels of IL-12 were elevated during

5 Serum IL-8 and IL-12 in Breast Cancer 167 the progression of cancer (20). However, a subgroup of this study, consisting of breast cancer patients, did not show any positive correlation. In our study, there was no significant difference in the serum IL-12 levels of breast cancer patients and control group. None of the prognostic parameters analyzed, including tumor size, nodal status, nuclear and histological grade, ER, PR and HER-2 status, histology, or metastasis, was significantly correlated with serum IL-12 levels. The serum values were not altered after chemotherapy. Angiogenesis is a critical step of metastasis, in which IL-8 has an important role. The role of IL-8 in breast cancer was investigated by examining the differential expression of the IL-8 receptors CXR-1 and CXR-2 on endothelial and tumor cells in breast cancer tissue. All breast cancer cells expressed the IL-8 receptors, whereas only 50% of benign breast tissue samples expressed either CXR-1 or CXR-2 (23). Serum IL-8 measurement in breast cancer was rarely documented (3,14,16). Elevated serum IL-8 serum concentrations were found in breast cancer patients compared with healthy women and this elevation was also correlated with the clinical stage (3,14). The study group concluded that increased serum IL- 8 level in breast cancer patients has an independent prognostic significance for post-relapse survival (14) and it can be used to diagnose breast cancer and to identify patients with a poor prognosis who may benefit from more aggressive management (3). However, another study reported no significant difference in IL-8 levels between patients with breast carcinoma and healthy controls (15). Our results are similar to the results of this last study; there is no significant difference in IL-8 levels between breast cancer patients and healthy controls. None of the prognostic parameters are correlated with serum IL-8 levels. However, we found that serum IL-8 levels are elevated in patients with metastatic breast carcinoma (p = 0.047). The results of the study by De Larco et al. (16) showed a strong correlation between the metastatic potential of breast carcinoma cell lines and their ectopic expression of IL-8. The observation of Benoy et al. (14) supports this finding. They clearly determined the independent prognostic significance of circulating IL-8 levels in patients presenting with metastatic breast cancer. In our study, in correlation with those other studies, we found that serum IL-8 levels are elevated significantly in the metastatic breast cancer group (p = 0.047). Potential changes in the serum levels of cytokines in cancer after chemotherapy were investigated. Kovacs found that there was no alteration in serum level of IL-12 and IL-16 in cancer patients after chemoradiotherapy (20). In contrast, therapy consisted 5-FU was reported to lead to increased serum levels of IL-12 in cancer patients (24). We could not determine any significant differences in serum IL-12 and IL-8 levels before and after chemotherapy. The potential influence of the chemotherapeutic agent used on the serum levels of cytokines was also investigated. Yokoe et al. found no change in the plasma levels of IL-8 in recurrent breast cancer patients after anthracycline-based chemotherapy (17). Similarly, Pusztai et al. found that in breast cancer patients, anthracycline-containing chemotherapy did not change serum IL-8 and IL-12 levels, but that a 3-wk paclitaxel chemotherapy significantly increased serum IL-8 and IL-16, but not IL-12 (14). In ovarian carcinoma, a significant increase in serum IL-8 levels was demonstrated following paclitaxel-containing chemotherapy (25). In our study, we found no change in the serum levels of IL-8 and IL-12 after either anthracycline or anthracycline and taxane containing chemotherapy. These findings are partially correlated with those found in the literature. In conclusion, the significantly higher serum levels of IL-8 in metastatic breast cancer patients are correlated with the literature. We can hypothesize that IL-8 can be used as non-invasive predictive indicator of metastases and for follow up of breast cancer. However, our sample size is small, and larger scale research in this field is needed in order to learn more about the role of cytokines in this common disease of women. References 1. Suri C, et al. Increased vascularization in mice overexpressing angiopoietin-1. Science 1998; 182: Teicher BA, et al. Potentiation of cytotoxic cancer therapies by TNP-470 alone and with other anti-angiogenic agents. Int J Cancer 1994; 57: Kozlowski L, Zakrzewska I, Tokajuk P, Wojtukiewicz MZ. Concentration of interleukin-6 (IL-6), interleukin-8 (IL-8)

6 168 Derin et al. and interleukin-10 (IL-10) in blood serum of breast cancer patients. Rocz Akad Med Bialymst 2003; 48: Trinchieri G. Interleukin-12: a proinflammatory cytokine with immunoregulatory functions that bridge innate resistance and antigen-specific adaptive immunity. Annu Rev Immunol 1995; 13: Blanks RE, Patel PM, Selby PJ. Interleukin-12: a new clinical player in cytokine therapy. Br J Cancer 1995; 71: O Hara RJ, et al. Advanced colorectal cancer is associated with impaired Interleukin 12 and enhanced Interleukin 10 production. Clin Cancer Res 1998; 4: Nakayama Y, et al. Serum levels of interleukin-12 in patients with gastrointestinal cancer. Anticancer Res 2000; 20: Merendino RA, et al. Interleukin-12 and interleukin-10 production by mononuclear phagocytic cells from breast cancer patients. Immunol Lett 1999; 68: Voest EE, et al. Inhibition of angiogenesis in vivo by interleukin 12. J Natl Cancer Inst 1995; 87: Cavallo F, et al. Immune events associated with the cure of established tumors and spontaneous metastases by local and systemic interleukin-12. Cancer Res 1999; 59: Strieter RM, et al. Interleukin-8. A corneal factor that induces neovascularization. Am J Pathol 1992; 141: Belperio JA, et al. CXC chemokines in angiogenesis. J Leukocyte Biol 2000; 68: Kunz M, et al. Anoxia-induced up-regulation of interleukin-8 in human malign melanoma. A potential mechanism for high tumor aggressiveness. Am J Pathol 1999; 155: Benoy IH, et al. Increased serum interleukin-8 in patients with early and metastatic breast cancer correlates with early dissemination and survival. Clin Cancer Res 2004; 10: Pusztai L, et al. Changes in plasma levels of inflammatory cytokines in response to paclitaxel chemotherapy. Cytokines 2004; 25: De Larco JE, et al. A potential role for interleukin-8 in the metastatic phenotype of breast carcinoma cells. Am J Pathol 2001; 158: Yokoe T, Lino Y, Morishita Y. Trends of IL-6 and IL-8 levels in patients with recurrent breast cancer: preliminary report. Breast Cancer 2000; 7: Lissoni P, et al. Interleukin-12 in early or advanced cancer patients. Eur J Cancer 1997; 33: Zeimet AG, et al. Ascitic interleukin-12 is an independent prognostic factor in ovarian cancer. J Clin Oncol 1998; 16: Kovacs E. The serum levels of IL-12 and IL-16 in cancer patients: relation to the tumour stage and previous therapy. Biomed Pharmacother 2001; 55: Urosevic M, Dummer R. HLA-G and IL-10 expression in human cancer. Sem Cancer Biol 2003; 13: Lissoni P, et al. Physiopathology of IL-12 in early or advanced cancer patients and their variations with surgery and immunotherapy. J Biol Regul Homeost Agents 1998; 12: Miller LT, et al. Expression of interleukin-8 receptors on tumor cells and vascular endothelial cells in human breast cancer tissue. Anticancer Res 1998, 18: Okamoto M, et al. Induction of cytokines and killer cell activities by cisplatin and 5-fluorouracil in head and neck patients. Anticancer Drugs 2000; 11: Uslu R, et al. Predictive value of serum interleukin-8 in ovarian cancer patients treated with paclitaxel-containing regimens. Int J Gynecol Cancer 2005; 15:

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