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Clinical significance of serum interleukin-18 (IL-18) levels in patients with gastric cancer
Biomedicine & Pharmacotherapy 70 (2015) 19–23
Available online at
ScienceDirect www.sciencedirect.com
Original article
Clinical significance of serum interleukin-18 (IL-18) levels in patients with gastric cancer Faruk Tas *, Ceren Tilgen Yasasever, Senem Karabulut, Didem Tastekin, Derya Duranyildiz Institute of Oncology, University of Istanbul, Istanbul, Turkey
A R T I C L E I N F O
A B S T R A C T
Article history: Received 5 December 2014 Accepted 30 December 2014
An inappropriate production of interleukin-18 (IL-18) contributes to the pathogenesis of malignancies and may influence the clinical outcome of patients. The objective of this study was to determine the clinical significance of the serum levels of IL-18 in patients with gastric cancer. A total of 63 patients with a pathologically confirmed diagnosis of gastric cancer were enrolled into this study. Serum IL-18 concentrations were determined by the solid-phase sandwich Elisa method. Age- and sex-matched 30 healthy controls were included in the analysis. The median age at diagnosis was 62 years, range 28 to 82 years. The baseline serum IL-18 levels of the gastric cancer patients were a significantly higher than those in the control group (median values 1436.4 vs. 638.4 pg/mL, respectively, P < 0.001). The known clinical variables including age of patient, gender, site of lesion, histology, histological grade, stage of disease, and serum tumor markers such as LDH, CEA, and CA 19.9 were not found to be correlated with serum IL-18 concentrations (P > 0.05). Moreover, no correlation was found between serum IL-18 level and response to chemotherapy (P = 0.34). Serum IL-18 concentration was also found no prognostic role on survival (P = 0.21). In conclusion, serum levels of IL-18 may have a good diagnostic marker in patients with gastric cancer. However, its predictive and prognostic values were not determined. ß 2015 Elsevier Masson SAS. All rights reserved.
Keywords: Serum IL-18 Gastric cancer Diagnostic Predictive
1. Introduction Gastric cancer displays multifactorial etiology and its genetic and immunological background has not yet been fully elucidated. In vitro trials showed that cultured gastric cancer cell lines produce excessive levels of cytokines and growth factors with pleiotropic biological activities. Among them, interleukins function as an autocrine and paracrine factor that drives many cellular processes such as tumor growth, invasion, angiogenesis and metastasis [1–8]. Interleukin-18 (IL-18), a member of the IL-1 family, is synthesized as an inactive preform, and then converted to a bioactive form as a result of cleavage by an IL-1b-converting enzyme [1–8]. It is expressed in many cell types including macrophages, Kupffer cells, dendritic cells and some tumor cells. In addition to multiple biological activities by its capacity of stimulating immunity, it also exerts antitumor effects that are mediated by enhancement of NK cell activity, reduction of tumorigenesis, induction of apoptosis, and inhibition of angiogenesis in tumor cells. Recent data suggest that an inappropriate
* Corresponding author at: Institute of Oncology, Istanbul University, Capa, 34390 Istanbul, Turkey. Tel.: +90 212 534 80 78; fax: +90 212 534 80 78. E-mail address: [email protected] (F. Tas). http://dx.doi.org/10.1016/j.biopha.2014.12.040 0753-3322/ß 2015 Elsevier Masson SAS. All rights reserved.
production of IL-18 contributes to the pathogenesis of malignancies and may influence the clinical outcome of patients [1–8]. Although IL-18 expression has been investigated in a number of human tumors, its possible clinical significance remains unclear in gastric cancer, because, to date, there are a few studies addressing the clinical significance of IL-18 expression in gastric cancer [1– 4]. Increased IL-18 expressions were also detected in gastric cancer cell lines with increased gastric cancer cell proliferation and metastatic potential [1–4]. Therefore, there is an unsatisfactory understanding of the molecular function of IL-8 and the possible clinical significance of IL-18 has remained unclear in patients with gastric cancer. Although most available findings were provided from preclinical trials using gastric cancer tissue sections, so far, a few clinical studies to investigate the clinical significance of IL-8 in serum or plasma in gastric cancer patients [4–8]. Thus, the significance of the serological levels of IL-8 in gastric cancer patients is not known yet. Therefore, we evaluated the soluble serum levels of IL-18 in gastric cancer patients, and assessed associations with the prognosis, various known clinical variables, and response to chemotherapy, in order to examine whether these are potential new biomarkers, for use in the treatment of gastric cancer in this study.
20
F. Tas et al. / Biomedicine & Pharmacotherapy 70 (2015) 19–23
2. Material and methods 2.1. Patients and therapy This study included 63 consecutive patients admitted to Institute of Oncology, Istanbul University. All patients had histologically confirmed gastric cancer and had not received chemotherapy or chemoradiation in the last 6 months. The staging was determined according to the American Joint Committee on Cancer (AJCC) and International Union against Cancer (UICC) staging systems. The pretreatment evaluation included assessment of detailed clinical history and physical examination with a series of biochemistry tests including lactate dehydrogenase (LDH), complete blood cell counts including thrombocytes (PLTs), leukocytes (WBCs), hemoglobin (Hb) and serum tumor markers, CEA and CA 19.9. Those with Eastern Cooperative Oncology Group (ECOG) performance status 2 or less and appropriate blood chemistry tests received chemotherapy on an outpatient basis that included different combinations of fluorouracil, folinic acid, capecitabine, docetaxel, cisplatin, epirubicine, with/without radiotherapy depending on the stage of disease. Follow-up programs included clinical, laboratory, and radiological assessments performed at 8-week intervals during chemotherapy or every 12 weeks for no anticancer treatment. Response to treatment was determined according to the revised RECIST criteria version 1.1. For comparison of serum levels of IL-18, 30 age- and sexmatched healthy controls were included in the analysis. Informed consent was obtained from all patients and the study was reviewed and approved by our local ethical committee. 2.2. Measurement of serum IL-18 levels Serum samples were obtained on first admission before any adjuvant and metastatic treatment was given or follow-up patients. Blood samples were obtained from patients with gastric cancer and healthy controls by venipuncture and clotted at room temperature. The sera were collected following centrifugation and frozen immediately at 20 8C until analysis. Serum IL-18 (eBioscience, Inc., CA, USA) levels were determined by the solid-phase sandwich Elisa method. The IL-18 Elisa uses a double-antibody sandwich enzymelinked immunosorbent assay to determine the level of IL-18 in samples. Serum samples and standards were added to the wells, which were pre-coated with human IL-18 monoclonal antibody. Following incubation, IL-18 antibodies labeled with biotin and combined with Streptavidin-HRP were added to form immune complex and allowed to incubate. Unbound material was washed away and then Chromogen solution was added for the conversion of the colorless solution to a blue solution, the intensity of which was proportional to the amount of IL-18 in the sample. As the effect of the acidic stop solution, the color has become yellow. The colored reaction product was measured using an automated Elisa reader (Rayto, RT-1904C Chemistry Analyzer, Atlanta GA, USA). The results were expressed as pg/mL. 2.3. Statistical analysis Continuous variables were categorized using median values as cut-off point. Assessment of relationships, comparisons between various clinical/laboratory parameters and serum levels of IL-18 assay were accomplished using Mann-Whitney U test. Survival was calculated from the date of first admission to hospital to death resulting from any cause or to last contact with the patient or any family member. Kaplan-Meier method was used for estimation of
survival of patient and differences in survivals were assessed by the log-rank statistics. A P value 0.05 was considered significant. Statistical analysis was carried out using SPSS 16.0 software (SPSS Inc., Chicago, Illinois, USA). 3. Results A total of 63 patients with diagnosis of gastric cancer were enrolled in this study. The baseline histopathological characteristics and the demographic characteristics of the patients are listed in Table 1. The median age at diagnosis was 62 years, range 28 to 82 years. The baseline serum IL-18 levels of the gastric cancer patients were a significantly higher than those in the control group (median values 1436.4 vs. 638.4 pg/mL, respectively, P < 0.001) (Table 2). The known clinical variables including age of patient, gender, site of lesion, histology, histological grade, stage of disease, and serum levels of LDH, CEA, and CA 19.9 were not found to be correlated with serum IL-18 concentrations (P > 0.05) (Table 3). Moreover, no relationship was shown between serum IL-18 level and response to chemotherapy (P = 0.34). The median follow-up time was 25 weeks (range 1 to 164 weeks). At the end of the observation period, 35 patients (55.6%) were dead. The median survival for all patients was 42.0 4.2 weeks (95% CI = 33.8–50.2). The 1-year survival rates were 42.2% (95% CI = 28.3–56.1). The presence of metastasis (M1) (P = 0.03), antrum localization (P = 0.04), elevated erythrocyte sedimentation rate (P = 0.02), higher serum CEA levels (P = 0.01), elevated serum CA 19.9 concentrations (P = 0.04), and unresponsiveness to chemotherapy (P = 0.05) had statistically significant worse survival variables (Table 3). However, serum IL-18 concentrations were not associated with prognosis on outcome (P = 0.21) (Table 3 and Fig. 1). 4. Discussion IL-18, a proinflammatory cytokine, plays a central role in inflammation and immune response in many diseases. In addition to this situation, there is evidence suggesting that several proinflammatory gene products such as IL-18 have been liked with carcinogenesis, which proposes that inflammation is a risk factor for cancer progression. IL-18 activity in tumor cells show a very complex and ambiguous role depending on the cellular microenvironment. Although IL-18 expression and localization have been investigated in a number of human malignancies, its possible function still remain elusive in human gastric carcinoma. Therefore, the possible clinical significance of the IL-18 expression in gastric cancer remains unknown, because, to date, there are a few studies addressing the clinical significance of IL-18 expression in gastric cancer [1–4]. In a study expressed of IL-18 from 50 paraffin-embedded samples of gastric cancer tissue were analyzed by immunohistochemistry (IHC), the positive expression rate of IL-18 was 26% [3]. IL-18 was distributed in both the cytoplasm and nuclear compartment of the cancer cells. Adjacent normal gastric mucosa was not immunoreactive within anti-human IL-18 antibody. Although there was no significant association with gender, age, pathologic differentiation, depth of invasion, lymph node metastasis and TNM stage, IL-18 tended to be positively stained in the gastric carcinoma with distant metastasis (P = 0.001). However, significant difference was found between IL-18 expressions and the overall survival. The study demonstrated that the positive expression of IL-18 could play an important role in progression and metastasis of gastric cancer. Moreover, Kang et al. first measured the IL-18 concentration in sera of stomach cancer patients (n = 20)
F. Tas et al. / Biomedicine & Pharmacotherapy 70 (2015) 19–23
21
Table 1 (Continued )
Table 1 Patient and disease characteristics. Variables
n (%)
Number of patients
63 (100)
Age, years 60 < 60
35 (56) 28 (44)
Gender Male Female
25 (40) 38 (60)
Site of tumor Cardia Antrum Undetermined
21 (33) 27 (43) 15 (24)
Histology Adenocarcinoma Signet ring cell
42 (67) 21 (33)
Grade I–II III Undetermined
10 (16) 44 (70) 9 (14)
Tumor (T) stage 1–3 4 Unknown
14 (22) 22 (35) 27 (43)
Number of lymph node involvement 0–2 3 Unknown
12 (19) 13 (21) 38 (60)
Stage of disease Nonmetastatic Metastatic
32 (51) 31 (49)
Liver metastasisa Yes No
14 (45) 17 (55)
Curative surgeryb Yes No Unknown
17 (53) 9 (28) 6 (19)
Serum hemoglobin level Low (< 12 g/dL) Normal ( 12 g/dL)
35 (56) 28 (44)
Serum WBC count Normal (< 10,000) Elevated ( 10,000)
52 (83) 11 (17)
Serum platelet count Normal (< 350,000) Elevated (> 350,000)
54 (86) 9 (14)
Serum LDH level Normal (< 450 U/L) Elevated ( 450 U/L) Unknown
43 (68) 10 (16) 10 (16)
Erythrocyte sedimentation rate (ESR),/h Elevated ( 50) Normal (< 50) Unknown
16 (25) 10 (16) 37 (59)
Serum CEA level Normal (< 10 ng/mL) Elevated ( 10 ng/mL) Unknown
44 (70) 13 (21) 6 (9)
Serum CA 19.9 level Normal (< 40 IU/mL) Elevated ( 40 IU/mL) Unknown
32 (51) 25 (40) 6 (9)
Response to chemotherapy Responsive (SD + CR + PR) Non-responsive (PD)
13 (43) 17 (57)
Variables
n (%)
Last status Alive Dead
28 (44) 35 (56)
CR: complete response; PR: partial response; SD: stable disease; PD: progressive disease; LDH: lactate dehydrogenase; WBC: leukocyte. a In metastatic patients. b In nonmetastatic patients.
[4]. IL-18 levels in patients were three times higher than those of normal controls (P < 0.05). Next, they investigated IL-18 expression in tumor tissues from stomach cancer patients (n = 10) by IHC. The distinctive IL-18 expression was shown in all of the tumor tissues. Adjacent nontransformed stomach epithelium was stained as a negative control, but there was no expression of IL-18. Those results imply that IL-18 seems to play important role in the pathogenesis of stomach cancer. In order to determine serum IL-18 levels and their clinical significance in patients with gastric carcinoma, firstly, Kawabata et al. used peripheral blood samples measured by Elisa in 94 patients [5]. The mean serum IL-18 level for patients was significantly higher compared with healthy controls (P < 0.01). When the patients were subdivided into groups, it was found that the serum IL-18 level in patients with stages II and III disease was significantly higher compared with the level found in healthy volunteers (P < 0.01). However, there was no significant difference in the serum IL-18 level between healthy controls and patients with stages I and IV. Patients with IL-18 levels equal to or greater than the mean levels standard deviation in the healthy volunteers experienced a significantly lower survival rate compared with patients who had lower levels (P < 0.05). The serum IL-18 level was also identified as an independent postoperative prognostic factor in multivariate survival analysis (P = 0.01). Contrarily, a lack of any discernible difference in clinicopathological factors between the two groups. Fifty-one Thai patients with gastric cancer were also studied in serum with IL-18 levels measured by Elisa [6]. The IL-18 level in patients group was significantly higher than in control group (P = 0.0001). A significant clinical correlation was found between hematocrit level, serum albumin, and serum Il-18 levels in gastric cancer patients. However, age of patient was not correlated. For survival, a significant difference was shown between IL-18 serum levels in patients. These findings demonstrated that serum IL-18 levels may be the useful biological markers of diagnostic and prognostic markers in patients with gastric cancer. Additionally, in another trial, serum IL-18 levels were measured by Elisa in 44 Iranian patients with gastric cancer [7]. The mean IL-18 serum level in patients with gastric cancer was significantly higher than that in control group (P < 0.002). Therefore, increasing serum IL-18 level may have clinical importance as a diagnostic marker in patients with gastric cancer. In a novel study, diagnostic emphasis was placed on Elisa method as defined in the serum of IL-18 in 192 patients with diseases gastroduodenal zone [8]. This study revealed that an increase in the concentration of IL-18 correlated with tumor stage, the level of VEGF, and total antibodies to CagA Helicobacter pylori. The data allow to recommend that the use of IL18 in serum for diagnostic gastric cancer in addition to benign gastric pathologies. The differences in the findings with IL-18 probably reflect the differences in source, kinetics of expression or destruction, and possibly signals including their expression and release. Additionally, these contradicting results of these studies might be attributable to other several factors. So far, no consensus exists on which tumors and/or sera and methods should be used for testing IL-18 expressions. During recent decades, IHC has become a
F. Tas et al. / Biomedicine & Pharmacotherapy 70 (2015) 19–23
22
Table 2 The values of serum IL-18 levels in gastric cancer patients and healthy controls. Marker
Patients (n = 63)
Controls (n = 30)
P
Median
Range
Median
Range
IL-18 (pg/mL)
1436.4
578.4–4100.2
638.4
103.2–9558.6
< 0.001
IL-18: interleukin-18.
useful adjunctive method in diagnostic histopathology from tissues and tumor cell analysis and Elisa kits for sera. There are a number of drawbacks with IHC, the most important of which are lack of assay standardization and variance in the interpretation of the IHC staining. These drawbacks were also valid for determination of circulation of IL-18 in gastric cancer patients. Additionally, each of the studies was performed on a relatively small sample
Table 3 Distribution and survival comparisons of serum IL-18 levels on various clinical parameters in patients with gastric cancer. Serum IL-18 level Parameters
Distribution P value
Survival P value
Age of patients < 60/ 60 years
0.92
0.61
Gender Male/female
0.53
0.56
Site of tumor Cardia/antrum
0.60
0.04
Histology (p) Adenocarcinoma/Signet ring
0.25
0.22
Grade I–II/III
0.21
0.10
Tumor (T) stage 1–3/4
0.25
0.06
Number of lymph node involvement 0–2/ 3
0.27
0.21
Curative surgery Yes/No
0.20
0.36
Metastasis Yes/no
0.78
0.03
Liver metastasis Yes/no
0.26
0.11
Serum hemoglobin level Low/normal
0.85
0.34
Serum WBC count High/normal
0.06
0.30
Serum platelet count High/normal
0.35
0.51
Erythrocyte sedimentation rate High/normal
0.36
0.02
Serum LDH level High/normal
0.30
0.11
Serum CEA level High/normal
0.21
0.01
Serum CA 19.9 level High/normal
0.55
0.04
Response to chemotherapy Yes/no
0.34
0.05
Serum IL-18 level < median
–
0.21
WBC: leukocyte; LDH: lactate dehydrogenase; IL-18: interleukin-18.
size, which may have been insufficient to show significant differences. A standardized method remains to be established and validated in larger series of patients in prospective studies. Although prominent available findings were provided from preclinical trials using gastric cancer tissue sections, so far, only a few clinical study to investigate the clinical significance of IL-18 in serum or plasma in gastric cancer patients. Thus, the significance of the serological levels of IL-18 in gastric cancer patients is not known extensively yet. In the present study, we aimed the clinical significance of the serum IL-18 levels in patients with gastric cancer. A total of 63 patients with different stages of gastric cancer were studied in this study. Serum IL-18 concentrations were determined by the solid-phase sandwich Elisa method. The results demonstrated that the analysis of serum IL-18 was able to discriminate between the gastric cancer patients and healthy persons, indicating that IL-18 was a good serological diagnostic marker of gastric cancer. However, there were no significant associations between the levels of serum IL-18 and the tumor characteristics including stage, histology, grade, and serum tumor markers in this study. Additionally, we also found that serum levels of IL-18 were not associated with survival. Therefore, in this study, IL-18 levels in serum could not be useful prognostic marker to predict tumor prognosis in gastric cancer patients. Moreover, no link between serum IL-18 concentrations and chemosensitivity has raised the possibility of using IL-18 as predictors of response to chemotherapy in patients scheduled to undergone various chemotherapeutic regimens in our study. We showed that serum IL-18 levels may not be a potential predictor of clinical response to chemotherapy in gastric cancer patients. In conclusion, we found that serum levels of IL-18 may have only diagnostic role in gastric cancer patients. However, its predictive and prognostic values were not determined. However, the small sample size and short follow-up time of our study could be considered as significant limitation and might have influenced these results. However, our study contributes to the literature, because we performed it that contained all stages group of disease
Fig. 1. Overall survival curves in gastric cancer patients according to serum interleukin-18 levels (P = 0.21).
F. Tas et al. / Biomedicine & Pharmacotherapy 70 (2015) 19–23
preliminarily in literature. Further studies in a larger patient population are necessary to determine the potential clinical significance of this assay in patients with gastric cancer. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Role of the funding source: none.
References [1] Kim KE, Song H, Hahm C, Yoon SY, Park S, Lee H, et al. Expression of ADAM33 is a novel regulatory mechanism in IL-18-secreted process in gastric cancer. J Immunol 2009;182:3548–55.
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[2] Majima T, Ichikura T, Chochi K, Kawabata T, Tsujimoto H, Sugasawa H, et al. Exploitation of interleukin-18 by gastric cancers for their growth and evasion of host immunity. Int J Cancer 2006;118:388–95. [3] Ye ZB, Ma T, Li X, Jin XL, Xu HM. Expression and significance of intratumoral interleukin-12 and interleukin-18 in human gastric carcinoma. World J Gastroenterol 2007;13:1747–51. [4] Kang JS, Bae SY, Kim HR, Kim YS, Kim DJ, Cho BJ, et al. Interleukin-18 increases metastasis and immune escape of stomach cancer via the downregulation of CD70 and maintenance of CD44. Carcinogenesis 2009;30:1987–96. [5] Kawabata T, Ichikura T, Majima T, Seki S, Chochi K, Takayama E, et al. Preoperative serum interleukin-18 level as a postoperative prognostic marker in patients with gastric carcinoma. Cancer 2001;92:2050–5. [6] Thong-Ngam D, Tangkijvanich P, Lerknimitr R, Mahachai V, Theamboonlers A, Poovorawan Y. Diagnostic role of serum interleukin-18 in gastric cancer patients. World J Gastroenterol 2006;12:4473–7. [7] Haghshenas MR, Hosseini SV, Mahmoudi M, Saberi-Firozi M, Farjadian S, Ghaderi A. IL-18 serum level and IL-18 promoter gene polymorphism in Iranian patients with gastrointestinal cancers. J Gastroenterol Hepatol 2009;24:1119–22. [8] Matveeva LV, Mosina LM. Serum interleukin-18 level in precancerous conditions and gastric cancer. Eksp Klin Gastroenterol 2013;6:21–4.
Available online at
ScienceDirect www.sciencedirect.com
Original article
Clinical significance of serum interleukin-18 (IL-18) levels in patients with gastric cancer Faruk Tas *, Ceren Tilgen Yasasever, Senem Karabulut, Didem Tastekin, Derya Duranyildiz Institute of Oncology, University of Istanbul, Istanbul, Turkey
A R T I C L E I N F O
A B S T R A C T
Article history: Received 5 December 2014 Accepted 30 December 2014
An inappropriate production of interleukin-18 (IL-18) contributes to the pathogenesis of malignancies and may influence the clinical outcome of patients. The objective of this study was to determine the clinical significance of the serum levels of IL-18 in patients with gastric cancer. A total of 63 patients with a pathologically confirmed diagnosis of gastric cancer were enrolled into this study. Serum IL-18 concentrations were determined by the solid-phase sandwich Elisa method. Age- and sex-matched 30 healthy controls were included in the analysis. The median age at diagnosis was 62 years, range 28 to 82 years. The baseline serum IL-18 levels of the gastric cancer patients were a significantly higher than those in the control group (median values 1436.4 vs. 638.4 pg/mL, respectively, P < 0.001). The known clinical variables including age of patient, gender, site of lesion, histology, histological grade, stage of disease, and serum tumor markers such as LDH, CEA, and CA 19.9 were not found to be correlated with serum IL-18 concentrations (P > 0.05). Moreover, no correlation was found between serum IL-18 level and response to chemotherapy (P = 0.34). Serum IL-18 concentration was also found no prognostic role on survival (P = 0.21). In conclusion, serum levels of IL-18 may have a good diagnostic marker in patients with gastric cancer. However, its predictive and prognostic values were not determined. ß 2015 Elsevier Masson SAS. All rights reserved.
Keywords: Serum IL-18 Gastric cancer Diagnostic Predictive
1. Introduction Gastric cancer displays multifactorial etiology and its genetic and immunological background has not yet been fully elucidated. In vitro trials showed that cultured gastric cancer cell lines produce excessive levels of cytokines and growth factors with pleiotropic biological activities. Among them, interleukins function as an autocrine and paracrine factor that drives many cellular processes such as tumor growth, invasion, angiogenesis and metastasis [1–8]. Interleukin-18 (IL-18), a member of the IL-1 family, is synthesized as an inactive preform, and then converted to a bioactive form as a result of cleavage by an IL-1b-converting enzyme [1–8]. It is expressed in many cell types including macrophages, Kupffer cells, dendritic cells and some tumor cells. In addition to multiple biological activities by its capacity of stimulating immunity, it also exerts antitumor effects that are mediated by enhancement of NK cell activity, reduction of tumorigenesis, induction of apoptosis, and inhibition of angiogenesis in tumor cells. Recent data suggest that an inappropriate
* Corresponding author at: Institute of Oncology, Istanbul University, Capa, 34390 Istanbul, Turkey. Tel.: +90 212 534 80 78; fax: +90 212 534 80 78. E-mail address: [email protected] (F. Tas). http://dx.doi.org/10.1016/j.biopha.2014.12.040 0753-3322/ß 2015 Elsevier Masson SAS. All rights reserved.
production of IL-18 contributes to the pathogenesis of malignancies and may influence the clinical outcome of patients [1–8]. Although IL-18 expression has been investigated in a number of human tumors, its possible clinical significance remains unclear in gastric cancer, because, to date, there are a few studies addressing the clinical significance of IL-18 expression in gastric cancer [1– 4]. Increased IL-18 expressions were also detected in gastric cancer cell lines with increased gastric cancer cell proliferation and metastatic potential [1–4]. Therefore, there is an unsatisfactory understanding of the molecular function of IL-8 and the possible clinical significance of IL-18 has remained unclear in patients with gastric cancer. Although most available findings were provided from preclinical trials using gastric cancer tissue sections, so far, a few clinical studies to investigate the clinical significance of IL-8 in serum or plasma in gastric cancer patients [4–8]. Thus, the significance of the serological levels of IL-8 in gastric cancer patients is not known yet. Therefore, we evaluated the soluble serum levels of IL-18 in gastric cancer patients, and assessed associations with the prognosis, various known clinical variables, and response to chemotherapy, in order to examine whether these are potential new biomarkers, for use in the treatment of gastric cancer in this study.
20
F. Tas et al. / Biomedicine & Pharmacotherapy 70 (2015) 19–23
2. Material and methods 2.1. Patients and therapy This study included 63 consecutive patients admitted to Institute of Oncology, Istanbul University. All patients had histologically confirmed gastric cancer and had not received chemotherapy or chemoradiation in the last 6 months. The staging was determined according to the American Joint Committee on Cancer (AJCC) and International Union against Cancer (UICC) staging systems. The pretreatment evaluation included assessment of detailed clinical history and physical examination with a series of biochemistry tests including lactate dehydrogenase (LDH), complete blood cell counts including thrombocytes (PLTs), leukocytes (WBCs), hemoglobin (Hb) and serum tumor markers, CEA and CA 19.9. Those with Eastern Cooperative Oncology Group (ECOG) performance status 2 or less and appropriate blood chemistry tests received chemotherapy on an outpatient basis that included different combinations of fluorouracil, folinic acid, capecitabine, docetaxel, cisplatin, epirubicine, with/without radiotherapy depending on the stage of disease. Follow-up programs included clinical, laboratory, and radiological assessments performed at 8-week intervals during chemotherapy or every 12 weeks for no anticancer treatment. Response to treatment was determined according to the revised RECIST criteria version 1.1. For comparison of serum levels of IL-18, 30 age- and sexmatched healthy controls were included in the analysis. Informed consent was obtained from all patients and the study was reviewed and approved by our local ethical committee. 2.2. Measurement of serum IL-18 levels Serum samples were obtained on first admission before any adjuvant and metastatic treatment was given or follow-up patients. Blood samples were obtained from patients with gastric cancer and healthy controls by venipuncture and clotted at room temperature. The sera were collected following centrifugation and frozen immediately at 20 8C until analysis. Serum IL-18 (eBioscience, Inc., CA, USA) levels were determined by the solid-phase sandwich Elisa method. The IL-18 Elisa uses a double-antibody sandwich enzymelinked immunosorbent assay to determine the level of IL-18 in samples. Serum samples and standards were added to the wells, which were pre-coated with human IL-18 monoclonal antibody. Following incubation, IL-18 antibodies labeled with biotin and combined with Streptavidin-HRP were added to form immune complex and allowed to incubate. Unbound material was washed away and then Chromogen solution was added for the conversion of the colorless solution to a blue solution, the intensity of which was proportional to the amount of IL-18 in the sample. As the effect of the acidic stop solution, the color has become yellow. The colored reaction product was measured using an automated Elisa reader (Rayto, RT-1904C Chemistry Analyzer, Atlanta GA, USA). The results were expressed as pg/mL. 2.3. Statistical analysis Continuous variables were categorized using median values as cut-off point. Assessment of relationships, comparisons between various clinical/laboratory parameters and serum levels of IL-18 assay were accomplished using Mann-Whitney U test. Survival was calculated from the date of first admission to hospital to death resulting from any cause or to last contact with the patient or any family member. Kaplan-Meier method was used for estimation of
survival of patient and differences in survivals were assessed by the log-rank statistics. A P value 0.05 was considered significant. Statistical analysis was carried out using SPSS 16.0 software (SPSS Inc., Chicago, Illinois, USA). 3. Results A total of 63 patients with diagnosis of gastric cancer were enrolled in this study. The baseline histopathological characteristics and the demographic characteristics of the patients are listed in Table 1. The median age at diagnosis was 62 years, range 28 to 82 years. The baseline serum IL-18 levels of the gastric cancer patients were a significantly higher than those in the control group (median values 1436.4 vs. 638.4 pg/mL, respectively, P < 0.001) (Table 2). The known clinical variables including age of patient, gender, site of lesion, histology, histological grade, stage of disease, and serum levels of LDH, CEA, and CA 19.9 were not found to be correlated with serum IL-18 concentrations (P > 0.05) (Table 3). Moreover, no relationship was shown between serum IL-18 level and response to chemotherapy (P = 0.34). The median follow-up time was 25 weeks (range 1 to 164 weeks). At the end of the observation period, 35 patients (55.6%) were dead. The median survival for all patients was 42.0 4.2 weeks (95% CI = 33.8–50.2). The 1-year survival rates were 42.2% (95% CI = 28.3–56.1). The presence of metastasis (M1) (P = 0.03), antrum localization (P = 0.04), elevated erythrocyte sedimentation rate (P = 0.02), higher serum CEA levels (P = 0.01), elevated serum CA 19.9 concentrations (P = 0.04), and unresponsiveness to chemotherapy (P = 0.05) had statistically significant worse survival variables (Table 3). However, serum IL-18 concentrations were not associated with prognosis on outcome (P = 0.21) (Table 3 and Fig. 1). 4. Discussion IL-18, a proinflammatory cytokine, plays a central role in inflammation and immune response in many diseases. In addition to this situation, there is evidence suggesting that several proinflammatory gene products such as IL-18 have been liked with carcinogenesis, which proposes that inflammation is a risk factor for cancer progression. IL-18 activity in tumor cells show a very complex and ambiguous role depending on the cellular microenvironment. Although IL-18 expression and localization have been investigated in a number of human malignancies, its possible function still remain elusive in human gastric carcinoma. Therefore, the possible clinical significance of the IL-18 expression in gastric cancer remains unknown, because, to date, there are a few studies addressing the clinical significance of IL-18 expression in gastric cancer [1–4]. In a study expressed of IL-18 from 50 paraffin-embedded samples of gastric cancer tissue were analyzed by immunohistochemistry (IHC), the positive expression rate of IL-18 was 26% [3]. IL-18 was distributed in both the cytoplasm and nuclear compartment of the cancer cells. Adjacent normal gastric mucosa was not immunoreactive within anti-human IL-18 antibody. Although there was no significant association with gender, age, pathologic differentiation, depth of invasion, lymph node metastasis and TNM stage, IL-18 tended to be positively stained in the gastric carcinoma with distant metastasis (P = 0.001). However, significant difference was found between IL-18 expressions and the overall survival. The study demonstrated that the positive expression of IL-18 could play an important role in progression and metastasis of gastric cancer. Moreover, Kang et al. first measured the IL-18 concentration in sera of stomach cancer patients (n = 20)
F. Tas et al. / Biomedicine & Pharmacotherapy 70 (2015) 19–23
21
Table 1 (Continued )
Table 1 Patient and disease characteristics. Variables
n (%)
Number of patients
63 (100)
Age, years 60 < 60
35 (56) 28 (44)
Gender Male Female
25 (40) 38 (60)
Site of tumor Cardia Antrum Undetermined
21 (33) 27 (43) 15 (24)
Histology Adenocarcinoma Signet ring cell
42 (67) 21 (33)
Grade I–II III Undetermined
10 (16) 44 (70) 9 (14)
Tumor (T) stage 1–3 4 Unknown
14 (22) 22 (35) 27 (43)
Number of lymph node involvement 0–2 3 Unknown
12 (19) 13 (21) 38 (60)
Stage of disease Nonmetastatic Metastatic
32 (51) 31 (49)
Liver metastasisa Yes No
14 (45) 17 (55)
Curative surgeryb Yes No Unknown
17 (53) 9 (28) 6 (19)
Serum hemoglobin level Low (< 12 g/dL) Normal ( 12 g/dL)
35 (56) 28 (44)
Serum WBC count Normal (< 10,000) Elevated ( 10,000)
52 (83) 11 (17)
Serum platelet count Normal (< 350,000) Elevated (> 350,000)
54 (86) 9 (14)
Serum LDH level Normal (< 450 U/L) Elevated ( 450 U/L) Unknown
43 (68) 10 (16) 10 (16)
Erythrocyte sedimentation rate (ESR),/h Elevated ( 50) Normal (< 50) Unknown
16 (25) 10 (16) 37 (59)
Serum CEA level Normal (< 10 ng/mL) Elevated ( 10 ng/mL) Unknown
44 (70) 13 (21) 6 (9)
Serum CA 19.9 level Normal (< 40 IU/mL) Elevated ( 40 IU/mL) Unknown
32 (51) 25 (40) 6 (9)
Response to chemotherapy Responsive (SD + CR + PR) Non-responsive (PD)
13 (43) 17 (57)
Variables
n (%)
Last status Alive Dead
28 (44) 35 (56)
CR: complete response; PR: partial response; SD: stable disease; PD: progressive disease; LDH: lactate dehydrogenase; WBC: leukocyte. a In metastatic patients. b In nonmetastatic patients.
[4]. IL-18 levels in patients were three times higher than those of normal controls (P < 0.05). Next, they investigated IL-18 expression in tumor tissues from stomach cancer patients (n = 10) by IHC. The distinctive IL-18 expression was shown in all of the tumor tissues. Adjacent nontransformed stomach epithelium was stained as a negative control, but there was no expression of IL-18. Those results imply that IL-18 seems to play important role in the pathogenesis of stomach cancer. In order to determine serum IL-18 levels and their clinical significance in patients with gastric carcinoma, firstly, Kawabata et al. used peripheral blood samples measured by Elisa in 94 patients [5]. The mean serum IL-18 level for patients was significantly higher compared with healthy controls (P < 0.01). When the patients were subdivided into groups, it was found that the serum IL-18 level in patients with stages II and III disease was significantly higher compared with the level found in healthy volunteers (P < 0.01). However, there was no significant difference in the serum IL-18 level between healthy controls and patients with stages I and IV. Patients with IL-18 levels equal to or greater than the mean levels standard deviation in the healthy volunteers experienced a significantly lower survival rate compared with patients who had lower levels (P < 0.05). The serum IL-18 level was also identified as an independent postoperative prognostic factor in multivariate survival analysis (P = 0.01). Contrarily, a lack of any discernible difference in clinicopathological factors between the two groups. Fifty-one Thai patients with gastric cancer were also studied in serum with IL-18 levels measured by Elisa [6]. The IL-18 level in patients group was significantly higher than in control group (P = 0.0001). A significant clinical correlation was found between hematocrit level, serum albumin, and serum Il-18 levels in gastric cancer patients. However, age of patient was not correlated. For survival, a significant difference was shown between IL-18 serum levels in patients. These findings demonstrated that serum IL-18 levels may be the useful biological markers of diagnostic and prognostic markers in patients with gastric cancer. Additionally, in another trial, serum IL-18 levels were measured by Elisa in 44 Iranian patients with gastric cancer [7]. The mean IL-18 serum level in patients with gastric cancer was significantly higher than that in control group (P < 0.002). Therefore, increasing serum IL-18 level may have clinical importance as a diagnostic marker in patients with gastric cancer. In a novel study, diagnostic emphasis was placed on Elisa method as defined in the serum of IL-18 in 192 patients with diseases gastroduodenal zone [8]. This study revealed that an increase in the concentration of IL-18 correlated with tumor stage, the level of VEGF, and total antibodies to CagA Helicobacter pylori. The data allow to recommend that the use of IL18 in serum for diagnostic gastric cancer in addition to benign gastric pathologies. The differences in the findings with IL-18 probably reflect the differences in source, kinetics of expression or destruction, and possibly signals including their expression and release. Additionally, these contradicting results of these studies might be attributable to other several factors. So far, no consensus exists on which tumors and/or sera and methods should be used for testing IL-18 expressions. During recent decades, IHC has become a
F. Tas et al. / Biomedicine & Pharmacotherapy 70 (2015) 19–23
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Table 2 The values of serum IL-18 levels in gastric cancer patients and healthy controls. Marker
Patients (n = 63)
Controls (n = 30)
P
Median
Range
Median
Range
IL-18 (pg/mL)
1436.4
578.4–4100.2
638.4
103.2–9558.6
< 0.001
IL-18: interleukin-18.
useful adjunctive method in diagnostic histopathology from tissues and tumor cell analysis and Elisa kits for sera. There are a number of drawbacks with IHC, the most important of which are lack of assay standardization and variance in the interpretation of the IHC staining. These drawbacks were also valid for determination of circulation of IL-18 in gastric cancer patients. Additionally, each of the studies was performed on a relatively small sample
Table 3 Distribution and survival comparisons of serum IL-18 levels on various clinical parameters in patients with gastric cancer. Serum IL-18 level Parameters
Distribution P value
Survival P value
Age of patients < 60/ 60 years
0.92
0.61
Gender Male/female
0.53
0.56
Site of tumor Cardia/antrum
0.60
0.04
Histology (p) Adenocarcinoma/Signet ring
0.25
0.22
Grade I–II/III
0.21
0.10
Tumor (T) stage 1–3/4
0.25
0.06
Number of lymph node involvement 0–2/ 3
0.27
0.21
Curative surgery Yes/No
0.20
0.36
Metastasis Yes/no
0.78
0.03
Liver metastasis Yes/no
0.26
0.11
Serum hemoglobin level Low/normal
0.85
0.34
Serum WBC count High/normal
0.06
0.30
Serum platelet count High/normal
0.35
0.51
Erythrocyte sedimentation rate High/normal
0.36
0.02
Serum LDH level High/normal
0.30
0.11
Serum CEA level High/normal
0.21
0.01
Serum CA 19.9 level High/normal
0.55
0.04
Response to chemotherapy Yes/no
0.34
0.05
Serum IL-18 level < median
–
0.21
WBC: leukocyte; LDH: lactate dehydrogenase; IL-18: interleukin-18.
size, which may have been insufficient to show significant differences. A standardized method remains to be established and validated in larger series of patients in prospective studies. Although prominent available findings were provided from preclinical trials using gastric cancer tissue sections, so far, only a few clinical study to investigate the clinical significance of IL-18 in serum or plasma in gastric cancer patients. Thus, the significance of the serological levels of IL-18 in gastric cancer patients is not known extensively yet. In the present study, we aimed the clinical significance of the serum IL-18 levels in patients with gastric cancer. A total of 63 patients with different stages of gastric cancer were studied in this study. Serum IL-18 concentrations were determined by the solid-phase sandwich Elisa method. The results demonstrated that the analysis of serum IL-18 was able to discriminate between the gastric cancer patients and healthy persons, indicating that IL-18 was a good serological diagnostic marker of gastric cancer. However, there were no significant associations between the levels of serum IL-18 and the tumor characteristics including stage, histology, grade, and serum tumor markers in this study. Additionally, we also found that serum levels of IL-18 were not associated with survival. Therefore, in this study, IL-18 levels in serum could not be useful prognostic marker to predict tumor prognosis in gastric cancer patients. Moreover, no link between serum IL-18 concentrations and chemosensitivity has raised the possibility of using IL-18 as predictors of response to chemotherapy in patients scheduled to undergone various chemotherapeutic regimens in our study. We showed that serum IL-18 levels may not be a potential predictor of clinical response to chemotherapy in gastric cancer patients. In conclusion, we found that serum levels of IL-18 may have only diagnostic role in gastric cancer patients. However, its predictive and prognostic values were not determined. However, the small sample size and short follow-up time of our study could be considered as significant limitation and might have influenced these results. However, our study contributes to the literature, because we performed it that contained all stages group of disease
Fig. 1. Overall survival curves in gastric cancer patients according to serum interleukin-18 levels (P = 0.21).
F. Tas et al. / Biomedicine & Pharmacotherapy 70 (2015) 19–23
preliminarily in literature. Further studies in a larger patient population are necessary to determine the potential clinical significance of this assay in patients with gastric cancer. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Role of the funding source: none.
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