|Year : 2017 | Volume
| Issue : 1 | Page : 13-19
Clinical utility of interleukin-18 in breast cancer patients: A pilot study
Reecha A Parikh, Toral P Kobawala, Trupti I Trivedi, Mahnaz M Kazi, Nandita R Ghosh
Division of Molecular Endocrinology, Department of Cancer Biology, Gujarat Cancer Research Institute, Ahmedabad, Gujarat, India
|Date of Submission||11-Aug-2016|
|Date of Acceptance||20-Dec-2016|
|Date of Web Publication||23-Feb-2017|
Nandita R Ghosh
301, Division of Molecular Endocrinology, Department of Cancer Biology, Gujarat Cancer Research Institute, Asarwa, Ahmedabad, Gujarat 380016
Source of Support: None, Conflict of Interest: None
Aim: The aim of this study is to analyze the protein expression of interleukin 18 (IL-18) in patients with untreated breast cancer and further to evaluate its clinical efficacy in predicting treatment outcome.
Methods: In the present study, a total of 50 untreated patients with invasive ductal carcinoma of breast were included in the study. Expression of IL-18 was studied by immunohistochemistry method. Statistical analysis was carried out using Statistical Package for Social Sciences statistical software and P ≤ 0.05 was considered statistically significant.
Results: Seventy-two percent of the breast cancer patients showed the presence of cytoplasmic and/or nuclear IL-18 immunoreactivity. IL-18 expression was significantly and positively correlated with the stromal response (χ2 = 3.97, r = 0.282, P = 0.044). Further, the IL-18 immunoreactivity was significantly higher in patients with HER2 amplification as compared to luminal B (χ2 = 2.82, r = −0.523, P = 0.047) breast cancer patients. Moreover, a trend of increased IL-18 expression was observed in estrogen/progesterone receptor (ER/PR) negative patients as compared to ER/PR positive patients (χ2 = 3.41, r = −0.282, P = 0.066).
Conclusion: IL-18 could be used as a potential predictive marker and guide clinicians for recommendations to newer treatment. It might serve as a potential therapeutic target to establish novel treatment approaches along with the current treatment protocol used.
Keywords: Breast cancer, cytokine, immunohistochemistry, interleukin-18
|How to cite this article:|
Parikh RA, Kobawala TP, Trivedi TI, Kazi MM, Ghosh NR. Clinical utility of interleukin-18 in breast cancer patients: A pilot study. Cancer Transl Med 2017;3:13-9
|How to cite this URL:|
Parikh RA, Kobawala TP, Trivedi TI, Kazi MM, Ghosh NR. Clinical utility of interleukin-18 in breast cancer patients: A pilot study. Cancer Transl Med [serial online] 2017 [cited 2018 Mar 20];3:13-9. Available from: http://www.cancertm.com/text.asp?2017/3/1/13/200855
| Introduction|| |
Breast cancer is the second most common cancer in women and in the world according to Globocan 2012. Traditional clinicopathological factors such as tumor size, grade, stage, estrogen receptor (ER), progesterone receptor (PR), and HER/2 neu protein (HER2) status provide important information for patients and treating clinicians; it is apparent, these factors are not able to accurately predict for all patients. Changes in the cytokine levels mediated by the tumor both directly and indirectly are important parameters that affect the path of disease; it is likely, they are involved in the mechanism of tumor cell evasion of the immunosurveillance system. In breast carcinogenesis, many cytokines are expressed by the cancer cells or are produced in the primary or metastatic microenvironment. Interleukins (ILs) are type of cytokines that have been studied in breast cancer patients. The IL-1, IL-8, IL-11, and IL-13 have shown to favor tumor growth while IL-2, IL-10, IL-12 mainly interfere with cell-mediated immune response.
Among various ILs, IL-18 is also found to be an important regulator of innate and acquired immune response. It is a member of IL-1 family and was discovered as an interferon-γ inducing factor. This cytokine is mainly produced by activated macrophages and dendritic cells. IL-18 induces various biological activities through T helper type 1- and T helper type 2-mediated responses. It exerts antitumor effect by the enhancement of natural killer cell activity, reduction of tumorigenesis, induction of apoptosis, and inhibition of angiogenesis in tumor cells. In addition, studies have implicated its contribution in the pathogenesis and clinical outcome in patients with various cancers. Recent clinical studies associated this marker with prognosis in patients with gastric carcinoma, hematological malignancies, and also breast cancer. Nicolini et al. reported that elevated serum level of IL-18 was found in breast cancer patients over controls, and the values were found to be higher in advanced stages and in metastatic breast cancer patients. Results of Yang et al. suggest IL-18 as an important factor inducing breast cancer cell migration through downregulation of claudin-12 and activation of the p38 MAPK pathway. In their study, Li et al. have shown that leptin-induced IL-18 expression was regulated through PI3K-AKT/ATF-2 signaling in breast cancer cells and eventually lead to invasion and metastasis of these cells.
Therefore, the aim of this study was to investigate the distribution and potential role of IL-18 protein expression in breast cancer patients using immunohistochemical staining technique, and correlate results with established clinicopathological parameters such as age, tumor size, tumor stage, histopathology grade, lymph node involvement, molecular subtype, treatment, development of recurrence, and metastasis and also to evaluate clinical efficacy of IL-18 protein expression as predictive marker in breast cancer patients.
| Methods|| |
In this retrospective study, a total of 50 untreated female patients with histologically confirmed invasive ductal carcinoma of breast were included. In the criteria for patient selection, only the histopathologically confirmed invasive ductal breast cancer patients without any prior history of anticancer treatment were included and patients positive for Human Immunodeficiency Virus, hepatitis B surface antigen, and hepatitis C virus were excluded from the study. Written consent of all patients was obtained before treatment administration. Detailed clinical and pathological history including treatment given, disease status, and follow-up details (appearance of recurrence or metastases, survival time) were obtained from the case files. Histopathological details such as tumor size, lymph node status, diseases stage, histological grade, stromal response, molecular subtypes (luminal A and B, HER2 amplified and triple negative breast cancer [TNBC]) were evaluated and reported by the pathologists of our institute. Luminal A subtype comprised tumors with ER +, PR +/−, and HER2−, luminal B subtype were tumors with ER +, PR +/−, and HER2+; HER2 amplified subtype comprised ER −, PR −, and HER2+ tumors, whereas the TNBC subtype was those tumors negative for all the three receptors. The patients were followed for a minimum period of 12 months or until death within that period. Paraffin-embedded tissue blocks of 50 patients histopathologically confirmed as invasive ductal carcinoma were retrieved from the tissue repository.
Four micron thick sections were cut from the formalin-fixed paraffin-embedded tissue blocks using Leica microtome and mounted on aminopropyl triethoxy silane-coated glass slides. Immunohistochemical staining for IL-18 was performed using MACH4 HRP polymer detection system (Biocare, USA). Sections known to exhibit high expression of the protein were used as positive controls, while negative controls were obtained by omission of the primary antibody. The sections were first deparaffinized by passing through xylene and rehydrated in graded alcohol. Slides were then immersed in methanol with 3% H2O2, to quench the endogenous peroxidase activity. Antigenicity was retrieved by heating the sections in 10 mM sodium citrate buffer (pH 6.0) for 20 min in a pressure cooker and then allowed to cool at room temperature. Blocking was performed to reduce nonspecific background staining by applying the background punisher (provided in kit). The excess blocking serum was removed from the sections, and the primary antibody IL-18 (Rabbit Polyclonal; Santa Cruz Biotechnology Inc., USA, sc-7954; diluted 1:50) was added to the sections and incubated at 4°C, overnight, in a moist chamber. Next day, the slides were washed in tris buffer saline (TBS), and the sections were allowed to react with the MACH4 HRP-polymer (provided in kit) for 30 min at room temperature. Next, the specific immune reaction was identified using diaminobenzidine mixture. Sections were then lightly counterstained with hematoxylin. To destain the unwanted hematoxylin, the sections were given a single dip in acid-alcohol. All the steps were performed with intermediate washes of TBS/distilled water. In the final step, sections were dehydrated and cleaned by passing through graded alcohol and xylene. Finally, the stained sections were mounted with DPX and observed under a light microscope.
Scoring of the immunohistochemically stained sections was done by semiquantitative method on the basis of staining positivity and staining intensity. All the sections were scored independently in a blinded fashion using semi-quantitative score ranging from negative (no staining or < 10% of cells stained) to 3+ (1+, staining in 11%–30% of the cells: weak; 2+, staining in 31%–50% of cells: moderate; and 3+, staining in > 50% of cells: intense). For statistical analysis, the score 1+, 2+, and 3+ were clubbed in the positive group.
The data were analyzed using Statistical Package for Social Sciences (version 23, SPSS Inc., USA). Two-tailed Chi-square test and spearman's correlation were used to determine the correlation between the IL-18 expression and various clinicopathological parameters of breast cancer patients. For case of < 5 patients in the cells of 2 × 2 tables, Yate's continuity correction value along with its two-tailed significance was taken into consideration. P ≤ 0.05 were considered to be statistically significant.
| Results|| |
The detailed clinicopathological characteristics of the studied breast cancer patients are shown in [Table 1].
The age of the patients included in the study ranged from 30 to 85 years with median age of 50 years. Sixty-six percent (33/50) of patients were in early stage of disease (stage I + stage II) while 34% (17/50) were in advanced stage of disease (stage III + stage IV). Fifty-two percent (26/50) of patients had ER + while 48% (24/50) of patients were ER −. Thirty-two percent (16/50) of patients had PR +, while 68% (34/50) of patients were PR −. Thirty-six percent (18/50) of patients had HER2+ and 64% (32/50) of patients were HER/2–. On the basis of molecular classification, 38% (19/50) of patients were of luminal A subtype, 14% (7/50) were of luminal B subtype, 22% (11/50) of patients had HER2 amplification, while rest of the patients were TNBC patients. In treatment, majority 96% (48/50) of patients underwent modified radical mastectomy surgery. Out of 50 breast cancer patients, 4% (2/50) of patients were lost to follow-up. From rest of the patients, 14% (7/50) patients developed local recurrence/metastasis and 82% (41/50) were diseases free (with no recurrence/metastasis).
The IL-18 expression was found in cytoplasmic and/or nucleus of breast cancer cells. Seventy-two percent (36/50) patients with breast cancer showed presence of IL-18 immunoreactivity with 38% (19/36) showing 1+, 24% (12/36) showing 2+, and 10% (5/36) exhibiting 3+ immunoreactive score. All 72% (26/36) patients with presence of IL-18 expression showed cytoplasmic staining while only 28% (10/36) showed cytoplasmic and nuclear staining. A representative figure showing expression of IL-18 in breast carcinoma and negative control with absence of IL-18 expression has been shown in [Figure 1]a and [Figure 1]b, respectively.
|Figure 1: (a) Positive expression of interleukin-18 in breast cancer patients. (b) Negative control|
Click here to view
When IL-18 expression was correlated with various clinicopathological characteristics, it was observed that there was significant positive correlation between IL-18 expression and stromal response (χ = 3.97, r = 0.282, P = 0.044) [Figure 2]. The next important observation was that patients with HER/2 neu protein overexpression were significantly associated to high IL-18 protein expression when compared to breast cancer patients with luminal B molecular subtype (χ = 2.82, r = −0.523, P = 0.047) [Figure 3]. However, IL-18 expression did not correlate with the rest of the clinicopathological parameters [Table 2].
|Figure 3: Correlation of interleukin-18 with HER2 amplification and luminal B|
Click here to view
|Table 2: Correlation of interleukin-18 expression with clinicopathological parameters|
Click here to view
Moreover, an increased trend of IL-18 positivity was observed in ER −, PR −, and ER/PR − patients as compared to their respective counterparts. Further, when the patients were subgrouped into luminal A + luminal B, and HER2 amplification + TNBC, a trend of higher positive IL-18 expression was observed in the subgroup of patients with HER2 amplification + TNBC tumors when compared to sub group of patients with luminal A + luminal B tumor (χ = 2.94, r = 0.243, P = 0.080). However, IL-18 expression was not significantly different between subgroup of patients with HER2 amplified and luminal A (χ = 0.913, r = −0.256, P = 0.339), luminal A and TNBC (χ = 0.253, r = −0.166, P = 0.615), luminal B and TNBC (χ = 1.065, r = −0.341, P = 0.0302) as well as between HER2 amplified and TNBC (χ = 0.070, r = −0.171, P = 0.791) patients.
Furthermore, among the 50 patients, 7 patients developed recurrence; among them, 4 patients had IL-18 positive score. Out of these 4 patients, 3 patients were with TNBC, and only 1 patient was with luminal A molecular subtype [Table 3].
| Discussion|| |
The current understanding of breast cancer supports the concept of multifactorial causation, involving genomic alteration of epithelial cells coupled with an altered microenvironment supportive of tumor growth.
In the tumor microenvironment, "inflammation", a beneficial response to restore the tissue damage, is activated which when unregulated becomes chronic and induces malignant cell transformation. This inflammatory response shares various molecular targets and signaling pathways with the carcinogenic process, such as apoptosis, increased proliferation rate, and angiogenesis and hence is now considered as one of the hallmarks of cancer. Moreover, the immune response comprises a series of events triggered in response to chronic inflammation, involving cells and soluble mediators, such as cytokines.
In recent decades, many advances have occurred in the understanding of the role of cytokines in breast cancer. The relationships between numerous cytokines and breast cancer and their role in effecting patient prognosis have been documented. However, the role of IL-18 expression by immunohistochemical technique, especially in patients with breast cancer remains yet to be explored. Therefore, the current study explores the role of IL-18 in untreated patients with breast cancer.
In the present study, the immunohistochemical localization of IL-18 was observed in the cytoplasm and nucleus of the breast cancer cells. In similarity with present observation, Ye et al. also found that IL-18 was distributed in both cytoplasmic and nuclear compartments of the gastric tumor cells while Orengo et al. have reported only cytoplasmic staining of IL-18 in epithelial ovarian cancer cells. Even in our previous study, IL-18 immunoreactivity was observed in cytoplasm and nucleus of thyroid cancer cells and it was not significantly associated with any of the clinicopathological parameters in the papillary thyroid cancer patients. Further, Chia et al. and Srabović et al. did not find any association between IL-18 expression and histopathological factors in hepatocellular cancer and breast cancer patients, respectively. These findings were in corroboration with the present study with exception to tumor stromal response. In the present study, IL-18 immunoreactivity significantly and positively correlated with stromal response. Sixty-three percent patients having absence of stromal response showed positive IL-18 expression, while 89% patients having presence of stromal response showed positive IL-18 expression. Since a great deal of evidence points toward stroma as one of the major regulators of tumor progression following the initial stages of tumor formation, therefore, stroma may also contribute to risk factors determining tumor formation. Besides, IL-18 also exerts pro-cancer effects when produced by cancer cells, acting to promote cell proliferation, and migration. Therefore, coexistence of high IL-18 expression with the presence of stromal response probably point toward the role of IL-18 in subset of breast cancer patients with aggressive phenotype.
Some studies showed the role of IL-18 as an angiogenesis mediator. Ye et al. detected a higher expression of IL-18 in invasive tumor in gastric cancer. These tissues have higher microvessel density which indicates the association of IL-18 with new blood vessel formation. However, the current study failed to find any association of IL-18 with vascular permeation in patients with breast cancer.
It is well established that breast cancers are currently clinically subtyped based on the ER, PR, and HER2 expressed by the tumor cells. This subtyping predicts prognosis and guides clinical recommendations for treatment. Breast cancer subtypes have been linked with different cytokine expression patterns, and ongoing efforts are looking to correlate these expression patterns with clinical outcomes and identify potential therapeutic options. There are no studies reporting a link between IL-18 protein expression and ER, PR, and HER2/neu protein levels. The current study is first to report the association of IL-18 and ER, PR, and HER2/neu protein levels. Interestingly, it was observed that patients ER −, PR −, and ER/PR − patients showed a higher IL-18 expression as compared to their respective counterparts. The next important observation was that patients with HER2/neu protein overexpression were significantly associated to high IL-18 protein expression when compared to breast patients with luminal B molecular subtype. Ninety-one percent of HER2/neu positive breast cancer patients showed IL-18 positivity whereas only 43% breast cancer patients in luminal B subtype had IL-18 positive tumors. This link between IL-18 protein expression and high risk/aggressive molecular subtypes could be one of the important finding that may aid clinicians to predict prognosis and guide clinical recommendations for treatment approach.
Further, some researchers have observed that expression of IL-18 was significantly associated with poor prognosis. However, IL-18 was not found to be associated with prognosis in patients with gastric cancer, esophageal carcinoma, and lung cancer.,,, Few reports exist that IL-18 is expressed at higher levels in patients with breast cancer metastasis, especially bony metastasis, compared with patients with nonmetastatic tumors, thus making it a potential detection tool for bone involvement in cancer.,, In the present study, the total follow-up period of the enrolled breast cancer patients was only 12 months; hence, it was not possible to determine the prognostic significance of IL-18. However, 7 breast cancer patients developed recurrence within the period of 12 months, among them 57% (4 out of 7) patients had IL-18 positive tumors, out of these 4, 3 patients had TNBC molecular subtype, which is one of the aggressive phenotype of breast cancer. Hence, together it may indicate that in addition to molecular subtype, IL-18 may emerge as one of the additional promising predictive biomarker of prognosis in breast cancer patients.
It may be concluded from the above study that, the study of IL-18 could be used as a potential predictive marker of prognosis and guide clinicians for recommendations to newer treatment. That it might serve as a potential therapeutic target to establish novel treatment approaches along with the current treatment protocol used. However, larger patient series with longer follow-up is fundamental for a definite conclusion.
Financial support and sponsorship
This study was supported by the Gujarat Cancer Society and Gujarat Cancer Research Institute.
Conflict of interest
There are no conflicts of interest.
| References|| |
Zardawi SJ, O'Toole SA, Sutherland RL, Musgrove EA. Dysregulation of hedgehog, Wnt and Notch signalling pathways in breast cancer. Histol Histopathol
2009; 24: 385–98.
Nicolini A, Carpi A, Rossi G. Cytokines in breast cancer. Cytokine Growth Factor Rev
2006; 17 (5): 325–37.
Carpi A, Nicolini A, Antonelli A, Ferrari P, Rossi G. Cytokines in the management of high risk or advanced breast cancer: an update and expectation. Curr Cancer Drug Targets
2009; 9 (8): 888–903.
Gracie JA, Robertson SE, McInnes IB. Interleukin-18. J Leukoc Biol
2003; 73 (2): 213–24.
Akahira JI, Konno R, Ito K, Okamura K, Yaegashi N. Impact of serum interleukin-18 level as a prognostic indicator in patients with epithelial ovarian carcinoma. Int J Clin Oncol
2004; 9 (1): 42–6.
Lebel-Binay S, Berger A, Zinzindohoue F, Cugnenc P, Thiounn N, Fridman WH. Interleukin-18: biological properties and clinical implications. Eur Cytokine Netw
2000; 11 (1): 15–26.
Srabović N, Mujagić Z, Mujanović-Mustedanagić J, Muminović Z, Čičkušić E. Interleukin 18 expression in the primary breast cancer tumour tissue. Med Glas (Zenica)
2011; 8 (1): 109–15.
Yang Y, Cheon S, Jung MK, Song SB, Kim D, Kim HJ, Park H, Bang SI, Cho D. Interleukin-18 enhances breast cancer cell migration via down-regulation of claudin-12 and induction of the p38 MAPK pathway. Biochem Biophys Res
2015; 459 (3): 379–86.
Li K, Wei L, Huang Y, Wu Y, Su M, Pang X, Wang N, Ji F, Zhong C, Chen T. Leptin promotes breast cancer cell migration and invasion via IL-18 expression and secretion. Int J Oncol
2016; 48 (6): 2479–87.
Hawsawi NM, Ghebeh H, Hendrayani SF, Tulbah A, Al-Eid M, Al-Tweigeri T, Ajarcem D, Alaiya A, Dermime S, Aboussekhra A. Breast carcinoma-associated fibroblasts and their adjacent counterparts display tumor-associated features. Cancer Res
2008; 68 (8): 2717–25.
Landskron G, De la Fuente M, Thuwajit P, Thuwajit C, Hermoso MA. Chronic inflammation and cytokines in the tumor microenvironment. J Immunol Res
2014; 2014: 149185.
Esquivel-Velázquez M, Ostoa-Saloma P, Palacios-Arreola MI, Nava-Castro KE, Castro JI, Morales-Montor J. The role of cytokines in breast cancer development and progression. J Interferon Cytokine Res
2015; 35 (1): 1–6.
Barak V, Kalickman I, Nisman B, Farbstein H, Fridlender ZG, Baider L, Kaplan A, Stephanos S, Peretz T. Changes in cytokine production of breast cancer patients treated with interferons. Cytokine
1998; 10 (12): 977–83.
Ye ZB, Ma T, Li H, Jin XL, Xu HM. Expression and significance of intratumoral interleukin-12 and interleukin-18 in human gastric carcinoma. World J Gastroenterol
2007; 13 (11): 1747–51.
Orengo AM, Fabbi M, Miglietta L, Andreani C, Bruzzone M, Puppo A, Cristoforoni P, Centurioni MG, Gualco M, Salvi S, Boccardo S. Interleukin (IL) 18, a biomarker of human ovarian carcinoma, is predominantly released as biologically inactive precursor. Int J Cancer
2011; 129 (5): 1116–25.
Kobawala TP, Trivedi TI, Gajjar KK, Patel DH, Patel GH, Ghosh NR. Role of Interleukin-18 in thyroid tumorigenesis. Int J Cancer Ther Oncol
2016; 4 (4): 431.
Chia CS, Ban K, Ithnin H, Singh H, Krishnan R, Mokhtar S, Malihan N, Seow HF. Expression of interleukin-18, interferon-γ and interleukin-10 in hepatocellular carcinoma. Immunol Lett
2002; 84 (3): 163–72.
Mao Y, Keller ET, Garfield DH, Shen K, Wang J. Stromal cells in tumor microenvironment and breast cancer. Cancer Metastasis Rev
2013; 32 (1–2): 303–15.
Park CC, Morel JC, Amin MA, Connors MA, Harlow LA, Koch AE. Evidence of IL-18 as a novel angiogenic mediator. J Immunol
2001; 167 (3): 1644–53.
Onitilo AA, Engel JM, Greenlee RT, Mukesh BN. Breast cancer subtypes based on ER/PR and Her2 expression: comparison of clinicopathologic features and survival. Clin Med Res
2009; 7 (1–2): 4–13.
Lippitz BE. Cytokine patterns in patients with cancer: a systematic review. Lancet Oncol
2013; 14 (6): e218–28.
Szaflarska A, Szczepanik A, Siedlar M, Czupryna A, Sierżęga M, Popiela T, Zembala M. Preoperative plasma level of IL-10 but not of proinflammatory cytokines is an independent prognostic factor in patients with gastric cancer. Anticancer Res
2009; 29 (12): 5005–12.
Tsuboi K, Miyazaki T, Nakajima M, Fukai Y, Masuda N, Manda R, Fukuchi M, Kato H, Kuwano H. Serum interleukin-12 and interleukin-18 levels as a tumor marker in patients with esophageal carcinoma. Cancer Lett
2004; 205 (2): 207–14.
Naumnik W, Chyczewska E, Kovalchuk O, Tałałaj J, Izycki T, Panek B. Serum levels of interleukin-18 (IL-18) and soluble interleukin-2 receptor (sIL-2R) in lung cancer. Rocz Akad Med Bialymst
2004; 49: 246–51.
Günel N, Coşkun U, Sancak B, Günel U, Hasdemir O, Bozkurt Ş. Clinical importance of serum interleukin 18 and nitric oxide activities in breast carcinoma patients. Cancer
2002; 95 (3): 663–7.
Merendino RA, Gangemi S, Ruello A, Bene A, Losi E, Lonbardo G, Purello-Dambrosio F. Serum levels of interleukin-18 and sICAM-1 in patients affected by breast cancer: preliminary considerations. Int J Biol Markers
2001; 16 (2): 126–9.
Sabel MS, Su G, Griffith KA, Chang AE. Intratumoral delivery of encapsulated IL-12, IL-18 and TNF-α in a model of metastatic breast cancer. Breast Cancer Res Treat
2010; 122 (2): 325–36.
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]