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Cancer Biomarkers 13 (2013) 417–425 DOI 10.3233/CBM-140392 IOS Press
IL-17A is elevated in sera of patients with poorly differentiated ovarian papillary serous cystadenocarcinoma Mahyar Malekzadeha, Alamtaj Samsami Dehaghanib, Abbas Ghaderia,c and Mehrnoosh Doroudchia,c,∗ a
Institute for Cancer Research, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran Department of Obstetrics and Gynecology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran c Department of Immunology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran b
Abstract. BACKGROUND: IL-17A a member of IL-17 family of cytokines is an inflammatory cytokine produced by a subset of CD4+ T cells that links innate and adaptive immunity. IL-17A has been shown to be a key mediator of inflammation in autoimmune diseases, transplant rejection and cancers. OBJECTIVE: To investigate the level of IL-17A in sera of southern Iranian patients with papillary serous cystadenocarcinoma of ovary and compare it with age-matched women of the same region. METHODS: In this study we investigated IL-17A and CA125 levels in sera of 26 patients with ovarian serous cystadenocarcinoma and 62 healthy age matched women by commercial ELISA assays. RESULTS: Fifteen (58%) and 16 (61.5%) out of 26 patients showed elevated IL-17A (1.25 ± 2.25 pg/ml) and CA125 (218 ± 224.69 IU/ml) in their sera, respectively. No healthy individual had detectable IL-17A or elevated CA125 in their sera (8.85 ± 2.86 IU/ml). The mean IL-17A levels in poorly differentiated tumors (3.33 ± 2.36 pg/ml) was significantly higher than that of well differentiated (0.14 ± 0.38 pg/ml) and moderately differentiated (undetectable) tumors. There was also a positive correlation between IL-17A and CA125 in sera of patients and controls when grouped together (r = 0.37, p = 0.005). CONCLUSION: Elevation of IL-17A in a high percentage of ovarian serous cystadenocarcinoma and lack of this cytokine in healthy individuals makes it a specific candidate in diagnosis, follow up or immunotherapy. Keywords: Serous cystadenocarcinoma, ovarian tumor, IL-17A, CA125
1. Introduction Despite low frequency, ovarian cancer is the deadliest gynecologic malignancy and the fifth most common cause of cancer death in women [1]. Ovarian serous cystadenocarcinomas are the most common form of malignant ovarian serous tumors [2]. Ovarian cancer is the eighth most frequent cancer in Iran [3]. The incidence of ovarian cancer in Iran is reported to be 3.9 per ∗ Corresponding author: Mehrnoosh Doroudchi, Department of Immunology, School of Medicine, Shiraz University of Medical Sciences, P.O. Box: 71345-3119, Shiraz, Iran. Tel./Fax: +98 711 235 1575; E-mail: [email protected].
100,000; which is much lower than that of USA and Australia [4]. The early diagnosis is hampered by the limited and non-specific symptoms until the advanced stages of the disease [4,5]. Moreover, the lack of reliable and cost-effective biomarker has been a hurdle in attainment of a consensus on early diagnostic or screening method. One of the best-known biomarkers for ovarian tumors, CA125, has not been very promising due to the low sensitivity and specificity [6–9]. One of the disadvantages of this biomarker is its elevation in almost 1–2% of normal population [10]. Although increasing the number of biomarkers increases the sensitivity of diagnosis, the specificity will be decreased in
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return [11]. Some other studies have shown that different settings of multiple biomarkers are elevated with high sensitivity and specificity in stage I ovarian cancer [12–15]. However, the clinical value of these multiplex biomarkers in most lethal cases with delayed symptoms is yet unclear [14]. The triggering, manipulation and exploitation of the immune system by tumor cells are part of the criteria of most tumors. The changes in the mediators and cellular profile of the immune system during tumor establishment and progression, mostly via tumor stroma, can provide valuable information on the process of tumorigenesis. In addition, there may be signature profiles of the immune changes by which immunodiagnosis or immunotherapeutic methods can be developed [15]. In this regard, autoantibodies to several tumor markers as well as serum autoantibodies signature for 65 ovarian cancer specific antigens with an average sensitivity and high specificity are reported [16,17]. It has also been shown that different subsets of T lymphocytes are recruited to the ovarian tumor under instruction of chemokines produced by macrophages in tumor stroma [18,19]. High percentages of T regulatory and Th17 cells in TILs from ovarian cancer patients are already shown [18,20]. Previous reports have shown the adverse effect of Treg recruitment to the ovarian tumor environment associated with poor prognosis for the patient [18]. Conversely, there is evidence suggesting a better outcome for epithelial ovarian cancer when the balance between Treg and Th17 cells is in favor of the latter subset [19,21]. However, the issue is still controversial as other investigators suggested that IL17A could promote progression of gynecological (and other) tumors [22–25]. In addition to the expression of IL-17 mRNA in ovarian tumors, high proportions of Th17 cells have also been observed in the peripheral blood and tumordraining lymph nodes of patients with advanced ovarian cancer [26,27]. As far as the current knowledge is concerned, the role of IL-17 and Th17 cells in antitumor immunity remains a matter of debate. In some tumor models, expression of IL-17 can boost antitumor immunity by promoting the development of Agspecific cytotoxic T cells [28,29], while in other studies the tumor-derived Th17 cells not only did not induce tumor cells death but also promoted tumor growth in vitro [30]. It is worth mentioning that the Th17 cells are both recruited and generated in the tumor environment by stromal cells [22,30]. In their study, Miyahara et al. showed that incubation of naïve and memory T cells from patients with ovarian cancer with tu-
mor cells, APCs, or both can result in the generation and expansion of Th17 lymphocytes [20]. Moreover, IL-17 production by tumor cells is also suggested [31, 32]. Kato et al. [26] reported that a considerable proportion of ovarian carcinomas naturally express IL-17 and its expression significantly correlates with the increased vascularity [33]. In addition to the role IL-17A may play in the tumor suppression/progression, it represents an attractive target for immunotherapy of the tumors. There are several anti-interleukin 17 (IL-17) antibodies that have shown significant promise in recent Phase II trials in autoimmune diseases such as psoriasis [34,35]. A phase III study is currently underway to evaluate the safety and efficacy of ixekizumab (Ly2439821) humanized anti-IL-17A monoclonal antibody in patients with moderate to severe psoriasis (clinicaltrials.gov/show/NCT01597245). The aim of this study was to investigate the level of IL-17A in sera of southern Iranian patients with papillary serous cystadenocarcinoma of ovary and compare it with age-matched women of the same ethnicity. Therefore, the concentration of IL-17A was measured in sera of 26 patients with ovarian papillary serous cystadenocarcinomas and was compared with that of 62 normal healthy age-matched women by ELISA assay.
2. Subjects and methods 2.1. Subjects This study was approved by the ethics committee of Shiraz University of Medical Science (SUMS). The participants were informed about the aim of this study as well as safety and security measures before their consents were obtained. The patients group consisted of 26 patients with papillary serous cystadenocarcinoma of ovary (49 ± 16 yrs, range = 20–72 yrs). The patients were selected from among the cohort of ovarian cancer cases who were referred to our laboratory by collaborating Gynecologist from the hospitals affiliated to the Shiraz School of Medicine since 2002. All patients were visited by the Gynecologist but the pathological reports were considered as definite diagnosis. The findings were reported as cystic, exuberant excrescent tumors with finger like papillae and fibrovascular core covered by multilayered cuboidal or columnar epithelium and hyperchromatic nuclei with prominent nucleoli, concentric lamellated calcifications (psammomma bodies) were often present in poorly differentiated tumors. The complete pathologi-
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cal criteria of the tumors were gathered from patient’s hospital files and included histological tumor type, tumor size, histological grade, fallopian tube invasion, myometrial invasion, endometrial invasion, lymphatic invasion, and TNM staging. All the samples were taken before surgery and before chemotherapy. A questionnaire for the demographic criteria of the patients and controls was completed for at the time of sampling. Cases with autoimmune diseases, chronic infectious diseases, liver diseases, accompanying cancers, and any patient with previously received treatment (surgery, chemotherapy or radiotherapy) were excluded from the study. The pathological and demographical data of patients were gathered from patient’s hospital files and are shown in Table 1. The control subjects included 62 healthy age-matched women (54 ± 10 yrs, range = 21–71 yrs) with no sign of inflammatory or autoimmune diseases. In addition to the age/sex-matched healthy women, the IL17A levels were measured in the sera of a sum of 279 healthy individuals aged 20 to 91 yrs (117 female and 162 male). 2.2. Sampling Two ml blood was taken from patients and controls by venipuncture method after informed consent. The sera were collected by centrifugation and were kept in 50 ul volumes in −20◦ C. The sera were not thawed before use.
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Table 1 Pathological characteristics of ovarian cancer patients Pathological characteristics Tumor type Papillary serous cystadenocarcinoma Tumor size < 3 cm > 3 cm Missing Stage I II III IV Missing Fallopian tube involved Yes No Missing Myometrium involved Yes No Missing Endometrium involved Yes No Missing Histological grade Well differentiated Moderately differentiated Poorly differentiated Missing
No. of cases (%) 26 (100) 6 13 7 2 0 4 4 16 4 7 15 1 14 11 1 14 11 8 3 5 10
each test. Since the detection limit of the assay was 0.5 pg/ml, all OD’s below this limit (which were all negative in controls) were considered zero for numerical analysis.
2.3. IL-17A ELISA 2.4. CA125 ELISA The IL-17A ELISA assay was performed by a biotin-avidin commercial ELISA assay according to manufacturer’s instructions (eBioscience BMS2017, USA). Briefly, sera were diluted 1:2 and added to the wells. The standard sera containing known concentrations of IL-17A were added to control wells. Then 50 µl of Biotin-labeled Anti-Human IL-17A antibody was added to the wells and the plate was incubated for 2 hrs in the room temperature with frequent shaking in 100 rpm. The wells were then washed by washing buffer for 4 times and then 100 µl of Streptavidin enzyme conjugate was added to each well. After 1 hr of incubation, the plate was washed and 100 µl of the substrate TMB was added. After 10 minutes of incubation in the dark, the stop solution was added and the optical densities (OD) were measured by an ELISA reader (Anthos, Austria). The ODs were then transformed to concentration by using the standard curve obtained in
The CanAg CA125 ELISA assay was performed by a biotin-avidin commercial ELISA assay according to manufacturer’s instructions (Fujirebio Diagnostics AB, Sweden). Briefly, 25 µl of CA125 calibrators containing known concentrations of CA125, controls and patients and controls sera were added to the wells. Then 100 µl of Biotin-labeled Anti-CA125 antibody was added to the wells and the plate was incubated for 2 hrs in the room temperature with frequent shaking in 100 rpm. The wells were then washed by washing buffer for 3 times and then 100 µl of Tracer working solution was added to each well and incubated for 1 hr in room temperature with constant shaking. After the incubation, the plate was washed 6 times and 100 µl of the substrate TMB HRP-Substrate was added to each well. After 30 minutes of incubation in the dark at room temperature, the absorbances were measured
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Fig. 1. IL-17A concentration was elevated in sera of patients with ovarian serous cystadenocarcinoma compared to healthy controls. (Colours are visible in the online version of the article; http://dx. doi.org/10.3233/CBM-140392)
at 620 nm by an ELISA reader (Anthos, Austria). The ODs were then transformed to concentration by using the standard curve obtained in the test. 2.5. Statistical analysis Using SPSS software (version 11.5, SPSS Inc., Chicago, IL., USA) Mann-Whitney rank sign test was used to compare the mean level of IL-17A between groups. Kruskal-Wallis test was used to compare IL17A and CA125 between multiple groups. Pearson correlation and Spearman’s Rho were used to correlate IL-17A and CA125 levels in patients and in the mixed group of patients and controls.
3. Results Fifteen (58%) out of 26 patients showed elevation of IL-17A in their sera while none of the 62 healthy women showed IL-17 elevation. The mean concentration of IL-17A among patients was found to be 1.25 ± 2.25 pg/ml while no control individual had IL-17A in the limit of detection of our test (Student t-test, equal variances not assumed, p = 0.005; Fig. 1). In addition, among the 279 healthy individuals, only two men aged 41 and 48 years returned positive with 1.19 and 1.37 pg/ml of IL-17A in their sera. Interestingly, the mean level of IL-17A among poorly differentiated tumors was 3.33 ± 2.36 pg/ml and was significantly higher than that of moderately (0.00 pg/ml) and well differentiated (0.14 ±
0.38 pg/ml) tumors (Kruskal Wallis test, p = 0.002; Fig. 2a). Comparison of CA125 levels between patients with different grades of the tumor showed a non-significant increase in both poorly and well differentiated grades (Fig. 2b). The mean concentration of CA125 in patients was 218.88 ± 224.69 IU/ml while the CA125 levels in control subjects was 8.85 ± 2.86 IU/ml (p < 0.0001). None of the healthy controls had CA125 levels in their sera above 35 IU/ml, while 16 out of 26 (61.5%) of the patients had CA125 levels above the consensus threshold of 35 IU/ml and 14 out of 26 (53.8%) patients had CA125 levels above 65 IU/ml [36,37]. There was no correlation between IL-17A levels and CA125 if only the patients were considered (p = 0.88 Figs 3a and 3b). However, there was a positive correlation between IL-17A and CA125 levels in sera of patients and controls when they grouped together (Pearson r = 0.37, p = 0.005; Figs 3c and d). There was also an increase in IL-17A among patients whose fallopian tubes were involved (2.19 ± 3.32 pg/ml) compared to that of patients with fallopian tubes free of tumor (0.19 ± 0.51 pg/ml) but due to the low number of data points the difference did not reach statistical significance. The two out 4 patients for whom fallopian tube involvement was positive, also showed elevation of IL-17A. However, among the seven patients for whom fallopian tube involvement was negative, only one patient had elevation of IL-17A. IL-17A level in patients with tumors less than 3 cm in diameter (1.52 ± 2.78 pg/ml) was not different from that of patients with tumors greater than 3 cm in diameter (1.56 ± 2.33 pg/ml). There was no difference in the IL-17A concentration regarding vascular and lymphatic involvement and stage of the tumors. Of the 26 patients in this study, the follow up results (up to 9 years) were available for 14 patients. The low number of cases hampered the statistical analysis of the survival rate with serum IL-17A and CA-125. However, in general patients with IL-17A showed a better long term survival (Fig. 4).
4. Discussion The first finding of this study was the elevation of IL-17A in sera of 58% of ovarian papillary serous cystadenocarcinoma patients, while none of the healthy age-matched women had detectable IL-17A in their sera. This percentage is similar to the finding of Kato et al. who found 64.7% of ovarian tumors express IL-17 mRNA [26].
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a
b
Fig. 2. Comparison of IL-17A and CA125 levels in sera of patients based on histological grades. (a) CA125 was elevated in poorly differentiated and well differentiated ovarian serous cystadenocarcinoma tumors compared to moderately differentiated tumors (Kruskal-Wallis test, p = 0.157). (b) IL-17A was significantly elevated in poorly differentiated ovarian serous cystadenocarcinoma tumors compared to moderately and well differentiated tumors (Kruskal-Wallis test, p = 0.002). (Colours are visible in the online version of the article; http://dx.doi. org/10.3233/CBM-140392)
A very interesting finding was the increase of IL17A in patients with poorly differentiated tumors. This finding, although needs confirmation in a setting with a larger sample size and low missing numbers, is consistent with the reported correlation of IL-17 mRNA expression in ovarian tumors with tumor angiogenesis and progression [26]. In an interesting study by Charles et al. the interplay between TNF related inflammatory network and IL-17A production by CD4+ T cells promoted tumor growth in a murine model of ovarian cancer [25]. Moreover, treatment with infliximab in patients with ovarian cancer is reported to result in decreased IL-17A levels [25]. The elevation of IL-17A in the poorly differentiated tumors, which are in more advanced phase of tumor progression, hampers the relevance of this cytokine in early diagnosis of ovarian papillary serous cystadenocarcinoma. However, with the advent of immunotherapy of au-
toimmune diseases with humanized anti-IL-17A, there may be the opportunity of its application in the therapy of ovarian papillary serous cystadenocarcinoma, as well [34,35,38]. CA125 levels were elevated in well differentiated and poorly differentiated but not moderately differentiated tumors. CA125 is a well studied ovarian tumor marker with low specificity and average sensitivity [6, 9]. The sensitivity of CA125 as a diagnostic marker in cancer is reported to be around 53% with about 1–2% of healthy individuals showing levels above 35 IU/ml cut off value [39]. As IL-17A was not detectable in healthy women, its elevation in 58% of patients makes it a very specific target for ovarian (and maybe other) cancer(s) immunotherapy. We observed a positive correlation between IL-17A levels and CA125 levels in patients and controls (Fig. 3d). What is more promising is the lack of IL-17A elevation in our control group of
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a
c
b
d
Fig. 3. Correlation between IL-17A levels (pg/ml) and CA125 (IU/ml). (a and b) IL-17A and CA125 correlation in sera of patients with ovarian serous cystadenocarcinoma did not reach the significant level. (c and d) IL-17A and CA125 showed a significant correlation in sera of patients and controls when grouped together (Spearmann’s Rho = 0.37, p = 0.005).
Fig. 4. Kaplan-Meier curve illustrating 9-year survival rate in patients with ovarian serous cystadenocarcinoma with (n = 5) and without (n = 9) IL-17A in their sera. (Colours are visible in the online version of the article; http://dx.doi.org/10.3233/CBM-140392)
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healthy women. In our hands and by testing more than 279 healthy individuals of different ages and genders, IL-17A is not elevated in general population [Supplementary Fig. 1 [40,41]. However, it remains to be identified if IL-17A increases in the sera of patients with non-malignant ovarian conditions. Several previous studies have shown the infiltration and expansion of Th17 cells in the ovarian tumors [19– 21,30]. The role of IL-17A and Th17 cells in ovarian tumors is still controversial. While most of studies point to the anti-tumor effect of Th17 cells and reduction in the Th17 TILs in advanced ovarian cancer [19, 21], a study indicated a pro-angiogenic effect of IL17A producing tumor cells in the tumor microenvironment [33]. We observed a slight increase in the serum IL-17A in well differentiated tumors while a significant increase was evident in poorly differentiated tumors (Fig. 2a). It is therefore worth investigating to see if different sources of IL-17A (Th17 vs. tumor cell) are involved in the production of this cytokine in different phases of tumor progression. Moreover, the effect of cellular source of IL-17A and the accompanying cytokines and soluble factors in the tumor milieu on the anti-tumor or pro-tumor function of IL-17A would worth exploring. We also suggest a larger study for evaluation of prognosis of the patients with ovarian papillary serous cystadenocarcinoma with elevated IL-17A levels in their sera. In conclusion, our study reports the elevation of IL17A in sera of patients with ovarian papillary serous cystadenocarcinoma. Considering the high percentage (58%) of IL-17A positive cases and absence of IL-17A in sera of healthy age-matched women, this cytokine can be a candidate for immunotherapy of this type of ovarian tumor.
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Acknowledgements The authors would like to thank all individuals who participated in this study and made this study possible. This work was performed in and was financially supported by a grant (ICR-100-502) from Shiraz Institute for Cancer Research, Shiraz University of Medical Sciences, Shiraz, Iran. The authors have no conflict of interest.
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Supplementary materials
Supplementary Fig. 1.
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Cancer Biomarkers 13 (2013) 417–425 DOI 10.3233/CBM-140392 IOS Press
IL-17A is elevated in sera of patients with poorly differentiated ovarian papillary serous cystadenocarcinoma Mahyar Malekzadeha, Alamtaj Samsami Dehaghanib, Abbas Ghaderia,c and Mehrnoosh Doroudchia,c,∗ a
Institute for Cancer Research, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran Department of Obstetrics and Gynecology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran c Department of Immunology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran b
Abstract. BACKGROUND: IL-17A a member of IL-17 family of cytokines is an inflammatory cytokine produced by a subset of CD4+ T cells that links innate and adaptive immunity. IL-17A has been shown to be a key mediator of inflammation in autoimmune diseases, transplant rejection and cancers. OBJECTIVE: To investigate the level of IL-17A in sera of southern Iranian patients with papillary serous cystadenocarcinoma of ovary and compare it with age-matched women of the same region. METHODS: In this study we investigated IL-17A and CA125 levels in sera of 26 patients with ovarian serous cystadenocarcinoma and 62 healthy age matched women by commercial ELISA assays. RESULTS: Fifteen (58%) and 16 (61.5%) out of 26 patients showed elevated IL-17A (1.25 ± 2.25 pg/ml) and CA125 (218 ± 224.69 IU/ml) in their sera, respectively. No healthy individual had detectable IL-17A or elevated CA125 in their sera (8.85 ± 2.86 IU/ml). The mean IL-17A levels in poorly differentiated tumors (3.33 ± 2.36 pg/ml) was significantly higher than that of well differentiated (0.14 ± 0.38 pg/ml) and moderately differentiated (undetectable) tumors. There was also a positive correlation between IL-17A and CA125 in sera of patients and controls when grouped together (r = 0.37, p = 0.005). CONCLUSION: Elevation of IL-17A in a high percentage of ovarian serous cystadenocarcinoma and lack of this cytokine in healthy individuals makes it a specific candidate in diagnosis, follow up or immunotherapy. Keywords: Serous cystadenocarcinoma, ovarian tumor, IL-17A, CA125
1. Introduction Despite low frequency, ovarian cancer is the deadliest gynecologic malignancy and the fifth most common cause of cancer death in women [1]. Ovarian serous cystadenocarcinomas are the most common form of malignant ovarian serous tumors [2]. Ovarian cancer is the eighth most frequent cancer in Iran [3]. The incidence of ovarian cancer in Iran is reported to be 3.9 per ∗ Corresponding author: Mehrnoosh Doroudchi, Department of Immunology, School of Medicine, Shiraz University of Medical Sciences, P.O. Box: 71345-3119, Shiraz, Iran. Tel./Fax: +98 711 235 1575; E-mail: [email protected].
100,000; which is much lower than that of USA and Australia [4]. The early diagnosis is hampered by the limited and non-specific symptoms until the advanced stages of the disease [4,5]. Moreover, the lack of reliable and cost-effective biomarker has been a hurdle in attainment of a consensus on early diagnostic or screening method. One of the best-known biomarkers for ovarian tumors, CA125, has not been very promising due to the low sensitivity and specificity [6–9]. One of the disadvantages of this biomarker is its elevation in almost 1–2% of normal population [10]. Although increasing the number of biomarkers increases the sensitivity of diagnosis, the specificity will be decreased in
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return [11]. Some other studies have shown that different settings of multiple biomarkers are elevated with high sensitivity and specificity in stage I ovarian cancer [12–15]. However, the clinical value of these multiplex biomarkers in most lethal cases with delayed symptoms is yet unclear [14]. The triggering, manipulation and exploitation of the immune system by tumor cells are part of the criteria of most tumors. The changes in the mediators and cellular profile of the immune system during tumor establishment and progression, mostly via tumor stroma, can provide valuable information on the process of tumorigenesis. In addition, there may be signature profiles of the immune changes by which immunodiagnosis or immunotherapeutic methods can be developed [15]. In this regard, autoantibodies to several tumor markers as well as serum autoantibodies signature for 65 ovarian cancer specific antigens with an average sensitivity and high specificity are reported [16,17]. It has also been shown that different subsets of T lymphocytes are recruited to the ovarian tumor under instruction of chemokines produced by macrophages in tumor stroma [18,19]. High percentages of T regulatory and Th17 cells in TILs from ovarian cancer patients are already shown [18,20]. Previous reports have shown the adverse effect of Treg recruitment to the ovarian tumor environment associated with poor prognosis for the patient [18]. Conversely, there is evidence suggesting a better outcome for epithelial ovarian cancer when the balance between Treg and Th17 cells is in favor of the latter subset [19,21]. However, the issue is still controversial as other investigators suggested that IL17A could promote progression of gynecological (and other) tumors [22–25]. In addition to the expression of IL-17 mRNA in ovarian tumors, high proportions of Th17 cells have also been observed in the peripheral blood and tumordraining lymph nodes of patients with advanced ovarian cancer [26,27]. As far as the current knowledge is concerned, the role of IL-17 and Th17 cells in antitumor immunity remains a matter of debate. In some tumor models, expression of IL-17 can boost antitumor immunity by promoting the development of Agspecific cytotoxic T cells [28,29], while in other studies the tumor-derived Th17 cells not only did not induce tumor cells death but also promoted tumor growth in vitro [30]. It is worth mentioning that the Th17 cells are both recruited and generated in the tumor environment by stromal cells [22,30]. In their study, Miyahara et al. showed that incubation of naïve and memory T cells from patients with ovarian cancer with tu-
mor cells, APCs, or both can result in the generation and expansion of Th17 lymphocytes [20]. Moreover, IL-17 production by tumor cells is also suggested [31, 32]. Kato et al. [26] reported that a considerable proportion of ovarian carcinomas naturally express IL-17 and its expression significantly correlates with the increased vascularity [33]. In addition to the role IL-17A may play in the tumor suppression/progression, it represents an attractive target for immunotherapy of the tumors. There are several anti-interleukin 17 (IL-17) antibodies that have shown significant promise in recent Phase II trials in autoimmune diseases such as psoriasis [34,35]. A phase III study is currently underway to evaluate the safety and efficacy of ixekizumab (Ly2439821) humanized anti-IL-17A monoclonal antibody in patients with moderate to severe psoriasis (clinicaltrials.gov/show/NCT01597245). The aim of this study was to investigate the level of IL-17A in sera of southern Iranian patients with papillary serous cystadenocarcinoma of ovary and compare it with age-matched women of the same ethnicity. Therefore, the concentration of IL-17A was measured in sera of 26 patients with ovarian papillary serous cystadenocarcinomas and was compared with that of 62 normal healthy age-matched women by ELISA assay.
2. Subjects and methods 2.1. Subjects This study was approved by the ethics committee of Shiraz University of Medical Science (SUMS). The participants were informed about the aim of this study as well as safety and security measures before their consents were obtained. The patients group consisted of 26 patients with papillary serous cystadenocarcinoma of ovary (49 ± 16 yrs, range = 20–72 yrs). The patients were selected from among the cohort of ovarian cancer cases who were referred to our laboratory by collaborating Gynecologist from the hospitals affiliated to the Shiraz School of Medicine since 2002. All patients were visited by the Gynecologist but the pathological reports were considered as definite diagnosis. The findings were reported as cystic, exuberant excrescent tumors with finger like papillae and fibrovascular core covered by multilayered cuboidal or columnar epithelium and hyperchromatic nuclei with prominent nucleoli, concentric lamellated calcifications (psammomma bodies) were often present in poorly differentiated tumors. The complete pathologi-
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cal criteria of the tumors were gathered from patient’s hospital files and included histological tumor type, tumor size, histological grade, fallopian tube invasion, myometrial invasion, endometrial invasion, lymphatic invasion, and TNM staging. All the samples were taken before surgery and before chemotherapy. A questionnaire for the demographic criteria of the patients and controls was completed for at the time of sampling. Cases with autoimmune diseases, chronic infectious diseases, liver diseases, accompanying cancers, and any patient with previously received treatment (surgery, chemotherapy or radiotherapy) were excluded from the study. The pathological and demographical data of patients were gathered from patient’s hospital files and are shown in Table 1. The control subjects included 62 healthy age-matched women (54 ± 10 yrs, range = 21–71 yrs) with no sign of inflammatory or autoimmune diseases. In addition to the age/sex-matched healthy women, the IL17A levels were measured in the sera of a sum of 279 healthy individuals aged 20 to 91 yrs (117 female and 162 male). 2.2. Sampling Two ml blood was taken from patients and controls by venipuncture method after informed consent. The sera were collected by centrifugation and were kept in 50 ul volumes in −20◦ C. The sera were not thawed before use.
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Table 1 Pathological characteristics of ovarian cancer patients Pathological characteristics Tumor type Papillary serous cystadenocarcinoma Tumor size < 3 cm > 3 cm Missing Stage I II III IV Missing Fallopian tube involved Yes No Missing Myometrium involved Yes No Missing Endometrium involved Yes No Missing Histological grade Well differentiated Moderately differentiated Poorly differentiated Missing
No. of cases (%) 26 (100) 6 13 7 2 0 4 4 16 4 7 15 1 14 11 1 14 11 8 3 5 10
each test. Since the detection limit of the assay was 0.5 pg/ml, all OD’s below this limit (which were all negative in controls) were considered zero for numerical analysis.
2.3. IL-17A ELISA 2.4. CA125 ELISA The IL-17A ELISA assay was performed by a biotin-avidin commercial ELISA assay according to manufacturer’s instructions (eBioscience BMS2017, USA). Briefly, sera were diluted 1:2 and added to the wells. The standard sera containing known concentrations of IL-17A were added to control wells. Then 50 µl of Biotin-labeled Anti-Human IL-17A antibody was added to the wells and the plate was incubated for 2 hrs in the room temperature with frequent shaking in 100 rpm. The wells were then washed by washing buffer for 4 times and then 100 µl of Streptavidin enzyme conjugate was added to each well. After 1 hr of incubation, the plate was washed and 100 µl of the substrate TMB was added. After 10 minutes of incubation in the dark, the stop solution was added and the optical densities (OD) were measured by an ELISA reader (Anthos, Austria). The ODs were then transformed to concentration by using the standard curve obtained in
The CanAg CA125 ELISA assay was performed by a biotin-avidin commercial ELISA assay according to manufacturer’s instructions (Fujirebio Diagnostics AB, Sweden). Briefly, 25 µl of CA125 calibrators containing known concentrations of CA125, controls and patients and controls sera were added to the wells. Then 100 µl of Biotin-labeled Anti-CA125 antibody was added to the wells and the plate was incubated for 2 hrs in the room temperature with frequent shaking in 100 rpm. The wells were then washed by washing buffer for 3 times and then 100 µl of Tracer working solution was added to each well and incubated for 1 hr in room temperature with constant shaking. After the incubation, the plate was washed 6 times and 100 µl of the substrate TMB HRP-Substrate was added to each well. After 30 minutes of incubation in the dark at room temperature, the absorbances were measured
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Fig. 1. IL-17A concentration was elevated in sera of patients with ovarian serous cystadenocarcinoma compared to healthy controls. (Colours are visible in the online version of the article; http://dx. doi.org/10.3233/CBM-140392)
at 620 nm by an ELISA reader (Anthos, Austria). The ODs were then transformed to concentration by using the standard curve obtained in the test. 2.5. Statistical analysis Using SPSS software (version 11.5, SPSS Inc., Chicago, IL., USA) Mann-Whitney rank sign test was used to compare the mean level of IL-17A between groups. Kruskal-Wallis test was used to compare IL17A and CA125 between multiple groups. Pearson correlation and Spearman’s Rho were used to correlate IL-17A and CA125 levels in patients and in the mixed group of patients and controls.
3. Results Fifteen (58%) out of 26 patients showed elevation of IL-17A in their sera while none of the 62 healthy women showed IL-17 elevation. The mean concentration of IL-17A among patients was found to be 1.25 ± 2.25 pg/ml while no control individual had IL-17A in the limit of detection of our test (Student t-test, equal variances not assumed, p = 0.005; Fig. 1). In addition, among the 279 healthy individuals, only two men aged 41 and 48 years returned positive with 1.19 and 1.37 pg/ml of IL-17A in their sera. Interestingly, the mean level of IL-17A among poorly differentiated tumors was 3.33 ± 2.36 pg/ml and was significantly higher than that of moderately (0.00 pg/ml) and well differentiated (0.14 ±
0.38 pg/ml) tumors (Kruskal Wallis test, p = 0.002; Fig. 2a). Comparison of CA125 levels between patients with different grades of the tumor showed a non-significant increase in both poorly and well differentiated grades (Fig. 2b). The mean concentration of CA125 in patients was 218.88 ± 224.69 IU/ml while the CA125 levels in control subjects was 8.85 ± 2.86 IU/ml (p < 0.0001). None of the healthy controls had CA125 levels in their sera above 35 IU/ml, while 16 out of 26 (61.5%) of the patients had CA125 levels above the consensus threshold of 35 IU/ml and 14 out of 26 (53.8%) patients had CA125 levels above 65 IU/ml [36,37]. There was no correlation between IL-17A levels and CA125 if only the patients were considered (p = 0.88 Figs 3a and 3b). However, there was a positive correlation between IL-17A and CA125 levels in sera of patients and controls when they grouped together (Pearson r = 0.37, p = 0.005; Figs 3c and d). There was also an increase in IL-17A among patients whose fallopian tubes were involved (2.19 ± 3.32 pg/ml) compared to that of patients with fallopian tubes free of tumor (0.19 ± 0.51 pg/ml) but due to the low number of data points the difference did not reach statistical significance. The two out 4 patients for whom fallopian tube involvement was positive, also showed elevation of IL-17A. However, among the seven patients for whom fallopian tube involvement was negative, only one patient had elevation of IL-17A. IL-17A level in patients with tumors less than 3 cm in diameter (1.52 ± 2.78 pg/ml) was not different from that of patients with tumors greater than 3 cm in diameter (1.56 ± 2.33 pg/ml). There was no difference in the IL-17A concentration regarding vascular and lymphatic involvement and stage of the tumors. Of the 26 patients in this study, the follow up results (up to 9 years) were available for 14 patients. The low number of cases hampered the statistical analysis of the survival rate with serum IL-17A and CA-125. However, in general patients with IL-17A showed a better long term survival (Fig. 4).
4. Discussion The first finding of this study was the elevation of IL-17A in sera of 58% of ovarian papillary serous cystadenocarcinoma patients, while none of the healthy age-matched women had detectable IL-17A in their sera. This percentage is similar to the finding of Kato et al. who found 64.7% of ovarian tumors express IL-17 mRNA [26].
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a
b
Fig. 2. Comparison of IL-17A and CA125 levels in sera of patients based on histological grades. (a) CA125 was elevated in poorly differentiated and well differentiated ovarian serous cystadenocarcinoma tumors compared to moderately differentiated tumors (Kruskal-Wallis test, p = 0.157). (b) IL-17A was significantly elevated in poorly differentiated ovarian serous cystadenocarcinoma tumors compared to moderately and well differentiated tumors (Kruskal-Wallis test, p = 0.002). (Colours are visible in the online version of the article; http://dx.doi. org/10.3233/CBM-140392)
A very interesting finding was the increase of IL17A in patients with poorly differentiated tumors. This finding, although needs confirmation in a setting with a larger sample size and low missing numbers, is consistent with the reported correlation of IL-17 mRNA expression in ovarian tumors with tumor angiogenesis and progression [26]. In an interesting study by Charles et al. the interplay between TNF related inflammatory network and IL-17A production by CD4+ T cells promoted tumor growth in a murine model of ovarian cancer [25]. Moreover, treatment with infliximab in patients with ovarian cancer is reported to result in decreased IL-17A levels [25]. The elevation of IL-17A in the poorly differentiated tumors, which are in more advanced phase of tumor progression, hampers the relevance of this cytokine in early diagnosis of ovarian papillary serous cystadenocarcinoma. However, with the advent of immunotherapy of au-
toimmune diseases with humanized anti-IL-17A, there may be the opportunity of its application in the therapy of ovarian papillary serous cystadenocarcinoma, as well [34,35,38]. CA125 levels were elevated in well differentiated and poorly differentiated but not moderately differentiated tumors. CA125 is a well studied ovarian tumor marker with low specificity and average sensitivity [6, 9]. The sensitivity of CA125 as a diagnostic marker in cancer is reported to be around 53% with about 1–2% of healthy individuals showing levels above 35 IU/ml cut off value [39]. As IL-17A was not detectable in healthy women, its elevation in 58% of patients makes it a very specific target for ovarian (and maybe other) cancer(s) immunotherapy. We observed a positive correlation between IL-17A levels and CA125 levels in patients and controls (Fig. 3d). What is more promising is the lack of IL-17A elevation in our control group of
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a
c
b
d
Fig. 3. Correlation between IL-17A levels (pg/ml) and CA125 (IU/ml). (a and b) IL-17A and CA125 correlation in sera of patients with ovarian serous cystadenocarcinoma did not reach the significant level. (c and d) IL-17A and CA125 showed a significant correlation in sera of patients and controls when grouped together (Spearmann’s Rho = 0.37, p = 0.005).
Fig. 4. Kaplan-Meier curve illustrating 9-year survival rate in patients with ovarian serous cystadenocarcinoma with (n = 5) and without (n = 9) IL-17A in their sera. (Colours are visible in the online version of the article; http://dx.doi.org/10.3233/CBM-140392)
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healthy women. In our hands and by testing more than 279 healthy individuals of different ages and genders, IL-17A is not elevated in general population [Supplementary Fig. 1 [40,41]. However, it remains to be identified if IL-17A increases in the sera of patients with non-malignant ovarian conditions. Several previous studies have shown the infiltration and expansion of Th17 cells in the ovarian tumors [19– 21,30]. The role of IL-17A and Th17 cells in ovarian tumors is still controversial. While most of studies point to the anti-tumor effect of Th17 cells and reduction in the Th17 TILs in advanced ovarian cancer [19, 21], a study indicated a pro-angiogenic effect of IL17A producing tumor cells in the tumor microenvironment [33]. We observed a slight increase in the serum IL-17A in well differentiated tumors while a significant increase was evident in poorly differentiated tumors (Fig. 2a). It is therefore worth investigating to see if different sources of IL-17A (Th17 vs. tumor cell) are involved in the production of this cytokine in different phases of tumor progression. Moreover, the effect of cellular source of IL-17A and the accompanying cytokines and soluble factors in the tumor milieu on the anti-tumor or pro-tumor function of IL-17A would worth exploring. We also suggest a larger study for evaluation of prognosis of the patients with ovarian papillary serous cystadenocarcinoma with elevated IL-17A levels in their sera. In conclusion, our study reports the elevation of IL17A in sera of patients with ovarian papillary serous cystadenocarcinoma. Considering the high percentage (58%) of IL-17A positive cases and absence of IL-17A in sera of healthy age-matched women, this cytokine can be a candidate for immunotherapy of this type of ovarian tumor.
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Acknowledgements The authors would like to thank all individuals who participated in this study and made this study possible. This work was performed in and was financially supported by a grant (ICR-100-502) from Shiraz Institute for Cancer Research, Shiraz University of Medical Sciences, Shiraz, Iran. The authors have no conflict of interest.
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Supplementary materials
Supplementary Fig. 1.
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