The Use of the Cytokines EMAP-II, IL-19 and IL-10 as Biomarkers to Determine Prognosis of Non – Hodgkin’s Lymphoma

Copyright: © 2016 Saber MM, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The Use of the Cytokines EMAP-II, IL-19 and IL-10 as Biomarkers to Determine Prognosis of Non – Hodgkin’s Lymphoma Manal Mohamed Saber*


Introduction
Non-Hodgkin's lymphoma (NHL) is a group of lympho proliferative malignant disorders with heterogeneous histological and clinical features [1]. NHL is characterized by abnormal proliferation or accumulation of B or T lymphocytes [2]. Many clinical and laboratory parameters had been employed in the prognostic definition of NHL [3].
EMAP-II is a molecule with pleiotropic activities toward endothelial cells, monocytes/macrophages, and neutrophils [19,20]. It was initially identified in the supernatant of murine methylcholanthrene A-induced fibrosarcomas, which induced tissue factor procoagulant activity in endothelial cells [21]. Hypoxia was found to be an inducer of the release and processing of EMAP-II in prostate adenocarcinoma cells [22]. Endothelial cell-specific apoptosis was induced by EMAP-II [23]. Another function of EMAP-II has been described in tumors: Its expression induced apoptosis of lymphocytes in solid tumors. This suggests an immunosuppressive role of EMAP-II in growing tumors [24,25].
IL-19 is a member of the IL-10 family of cytokines. This cytokine is produced by many cells such as monocytes, macrophages, B cells, endothelial and epithelial cells [26]. High IL-19 expression in tumor tissues had been associated with advanced tumor stage, high tumor metastasis and poor clinical outcome [14,15]. IL-19 directly affected cancer cell proliferation and migration and indirectly affected tumor progression by inducing the expression of cytokines and chemokine [14].
IL-10 is an immunosuppressive cytokine produced by many different cells of the immune system, including T and B lymphocytes, macrophages, monocytes, dendritic cells, and NK cells [27] but it is also produced by neoplastic B lymphocytes [10,27]. IL-10 suppresses antigen presenting cells thereby allowing tumor cells to escape immune system [8][9][10]. In addition, IL-10 may increase bcl-2 expression and protect malignant cells from apoptosis [28]. Elevated IL-10 levels have been found in patients with NHL and elevated levels were associated with poor prognosis [29][30][31][32].
The diagnosis of NHL was confirmed by immunophenotyping and histopathology.
The controls were thirty, apparently healthy subjects, matched for age and sex with the patient group. Their ages ranged from 21 to 70 years and this group included 15 males and 15 females.

NHL treatment
Patients received treatment strategies as follows: patients with early-stage NHL received CHOP chemotherapy (cyclophosphamide, doxorubicin, vincristine, prednisone) (6-8 cycles) [33] and patients with advanced stage NHL received CHOP in combination with rituximab [34]. Involved-field radiation therapy was considered consolidation treatment for patients in early and intermediate stages or for those presenting with bulky disease. CR was defined as the absence of clinical and radiographic evidence of disease, or disease-related symptoms, and appropriate laboratory results. Partial remission was defined as the decrease by at least 50% of tumor mass. Primary treatment failure was defined as progressive disease during initial treatment, failure to achieve complete remission or partial remission after initial therapy, or progressive disease within 5 months after complete remission [35].

Measurement of serum EMAP-II, IL-19, IL-10
Serum IL-10, IL-19, and EMAP-II levels were measured by solidphase enzyme-linked immunosorbent assay using 96 well microplates in accordance with the manufacturer's instructions (Sunred Biological Technology Co., Ltd, Shanghai). A monoclonal antibody specific for EMAP-II, IL-19 and IL-10 has been coated onto the wells of the microtiter strips provided. Optical densities at 450 nm were determined using a microtiter plate reader. Granting to the data supplied by the producer of the ELISA kits, the lower detection limits for EMAP-II, IL-19 and IL-10 were 0.073 pg/ml, 1.3 pg/ml, and 0.095 pg/ml, respectively.

Statistical method
The gathered data were coded, tabulated, and statistically analyzed using SPSS program (Statistical Package for Social Sciences) software version 20. Descriptive statistics were made out for numerical data by mean, standard deviation, and minimum and maximum of the orbit, while they were done for categorical data by number and percent. Nonparametric quantitative data are transformed into the logarithm before analysis (WBCs, Platelets, ALT, AST, IL-10, IL-19, EMAP-II). Analyses were done between more than two groups using one way ANOVA test for quantitative data between groups, followed by post HOC Tukey's correction between each two groups. Analyses were done between the two groups using the independent sample t test for quantitative data, Chi square test was employed for qualitative data between groups. The correlation between two quantitative variables was performed by using Pearson's correlation coefficient, and non-parametric Spearman's correlation coefficient for non-parametric ordinal data. Correlation coefficient ranges from (0-1): -weak (r=0-0.24), fair (r=0.25-0.49), moderate (r=0.5-0.74), strong (r=0.75-1).

Patients' characteristics
Patients' characteristics are listed in Table 1. The average age of the group was 46 years (range 13-75). In total, 64 patients (38 male, 26 female) were included. For 23 patients (35.9%), blood samples were obtained at initial diagnosis of the lymphoma. Nine patients (14.1%) were in complete remission of the lymphoma. 10 patients (15.6%) experienced a partial remission and 22 (34.4%) were in relapse. Patients and controls were assessed for their main clinical and laboratory characteristic. There were significant differences in regard to haemoglobin (Hb), platelets, AST, ALT, LDH, splenomegaly, hepatomegaly, lymph node enlargement (P<0.001) and creatinine (P=0.010) (Supplementary Table 1).
The Patients' characteristics in the different groups are summarized in Table 2. There was a significant difference in white blood cells (WBCs) (P=0.016), but not with other features (P>0.05). There was a significant difference in CD 23 (P=0.027) when comparing patients with relapse versus those in complete remission, but not with other immunophenotyping markers (Supplementary Table 2).

Serum EMAP-II levels in patients with NHL
Patients with NHL had significantly higher serum EMAP-II levels than the control group (P<0.001) ( Figure 1A). The mean serum EMAP-II level was 8379.37 ± 5223.75 pg/ml (470-27800) in the patient group and 102.66 ± 13.38 pg/ml (74-121) in the control group. EMAP-II showed significant negative correlation with WBCs (P=0.026), but not with other features (P>0.05) (Supplementary Table 3).
Next, serum EMAP-II levels were compared in different groups of  patients. The newly diagnosed patients showed serum EMAP-II levels ranging from 5900-27800 pg/ ml with a mean of 12475.65 ± 5164.58 pg/ ml. Patients with CR had EMAP-II values ranging from 470 to 4400 pg/ ml with a mean of 2096.66 ± 1354.81 pg/ml, and patients with partial remission had EMAP-II levels ranging from 4400 -14800 pg/ml with a mean of 7730 ± 3533.34 pg/ml. Samples of patients who were in relapse were also analyzed. Their EMAP-II values ranged from 3900-16000 pg/ml with a mean of 6962.27 ± 3204.85 pg/ml ( Figure 1B). P values were highly significant (P<0.001) in newly diagnosed patients when compared with CR, and relapse. In the same manner, p values were significant (P<0.001) in CR when compared with partial remission and relapse in NHL patients. There was a significant difference between EMAP-II levels in CR compared to PR (P=0.027).
The patients were split into high-EMAP-II groups (≥ 7000 pg/ ml vs. <7000 pg/ml). High serum EMAP-II levels were found in newly diagnosed patients not yet treated (P<0.001). There was a significant correlation between high serum EMAP-II levels and ALT (P=0.043) but not with other features (P>0.05) ( Table 3). There was no correlation between high EMAP-II levels and immunophenotyping markers (Supplementary Table 4). Those with CR and in relapse had a significantly lower value of EMAP-II ( P=0.002, P=0.017) respectively.   In 32 patients, we found levels of EMAP-II ≥ 7000 pg / ml in 22 patients (68.8%) in group I, 4 patients (12.5%) in group III, and 6 patients (18.8%) in group IV. In 32 NHL patients, we found levels of EMAP-II <7000 pg/ml in 1 (3.1%) patient in group I, in 9 (28.1%) patients in group II, in 6 (18.8%) patients in group III and 16 (50%) patients in group IV (Table 3).

Serum IL-19 levels in NHL
We compared the level of IL-19 in sera of patients and controls. There was a significantly higher mean serum IL-19 level in NHL patients (mean: 151.25 ± 140.41 pg/ml; range: 14-693 pg/ml) compared to the control group (mean: 7.99 ± 2.87 pg/ml; range: 2-13 pg/ml; P<0.001; Figure 2A). Interestingly, our results showed IL-19 in NHL was positively associated with AST (P=0.032), Bcl-2 (P=0.024), and negatively correlated with CD23 (P=0.015) (Supplementary Table 5 Figure 2B). P values were highly significant (P<0.001) in patients with relapse when compared with CR, and patients with no treatment. There was a significant difference between IL-19 levels in relapse compared to PR (P=0.006).   The patients were split into high IL-19 groups (≥ 100 pg/ml vs. <100 pg/ml). High serum IL-19 levels were found in patients in relapse (P<0.001). There was a significant correlation between high serum IL-19 levels and AST (P=0.002) but not with other features (P>0.05) ( Table  4). Newly diagnosed patients not yet treated had a significantly lower value of IL-19 (P<0.001).
Serum IL-10 was detected in patients with no treatment, and the mean concentration was 1524 ± 651.29 pg/ml (range 716-2870 pg/ml), which was significantly higher than that of CR patients with a mean of 335.77 ± 137.71 pg/ml (range 98 -523) pg/ml. Patients with partial remission had IL-10 levels ranging from 435 to 1520 pg/ml with a mean of 740.2 ± 331.3 pg/ml. Patients in relapse had IL-10 levels ranging from 439-1650 pg/ml with a mean of 719.22 ± 308.48 ( Figure 3B). P values   were highly significant (P<0.001) in patients with no treatment when compared with CR, partial remission and patients with relapse.
The patients were split into high IL-10 groups (≥ 700 pg/ml vs. <700 pg/ml). High serum IL-10 levels were found in newly diagnosed patients not yet treated (P<0.001). There was a significant correlation between high serum IL-10 levels and blood urea (P=0.020) but not with other features (P>0.05) ( Table 5). There was no correlation between high EMAP-II levels and immunophenotyping markers (Supplementary Table 7). Those with CR and in relapse had a significantly lower value of IL-10 (P=0.002, P=0.003) respectively.

Discussion
It has been postulated that cytokines play important roles in the pathogenesis of lymphomas [36] and disease progression [37]. Serum concentration of the cytokines may be used as a marker of prognosis in cancer [5]. In this study, we chose to measure serum EMAP-II, IL-19, and IL-10 levels in NHL patient. There have been no studies on EMAP-II and IL-19 that analyzed the serum levels of these cytokines in NHL patients.  Low: IL-10 was <700 pg/ml; High: ≥ 700 pg/ml; Statistically significant differences (P ≤ 0.05) are indicated with an asterisk (* ). Highly statistically significant differences (P ≤ 0.001) are indicated with asterisks ( ** ).  The present study showed elevated serum levels of EMAP-II in patients with NHL. In a previous study, serum EMAP-II levels were significantly higher in patients than in controls [13]. There were no significant associations between serum EMAP-II levels and various clinicopathologic and phenotypic parameters which are consistent with previously reported findings [13]. However, Serum EMAP-II levels showed a significant negative correlation with WBCs. Higher EMAP-II level ≥ 7000 pg/ml showed significant correlation with ALT. Patients who achieved CR had a lower serum EMAP-II level than those who achieved PR or did not respond to therapy. This notice is consistent with that of Sen et al. [13], who reasoned that high serum EMAP levels is of possible predictive value. In the present study, an association between higher pretreatment serum levels of EMAP-II in newly diagnosed patients with NHL was observed. These data suggest the idea that EMAP-II may have a role as a biomarker of treatment response and outcome in NHL. Elevation of EMAP-II levels in patients with NHL might be linked to factors controlling its release, such as hypoxia, as a result of vascular insufficiency [22].
There was a high IL-19 concentration in sera of patients with NHL compared to controls. Our results seemed to be uniform with other works which showed upregulation of IL-19 in tumors compared to normal tissues and an association of high IL-19 expression in tumor tissues with advanced tumor stage [14].
In the present study, patients had a high level IL-19 level at the time of diagnosis, which was associated with higher AST levels, CD23, and Bcl2. In former studies, Bcl-2 and CD23 positive patients were related with poor prognosis [38,39]. In our present subject, IL-19 levels were statistically significantly higher in patients with relapse. This determination was confirmed by other studies which demonstrated the association between high IL-19 expression and high tumor metastasis and poor clinical outcome [14,40]. This involves the meaning of using IL 19 for the early detection of recurrence, suggesting earlier treatment and longer endurance.
Former subjects have shown that increased serum IL-10 levels influence the forecast of various subtypes of lymphoma [17,41,42]. The present study showed elevated serum levels of IL-10 in patients with NHL at diagnosis when likened with the levels in healthy controls, and after treatment, those levels decreased substantially which is in conformity with previous results [41]. The drop in IL-10 levels after treatment can be explained by a therapy-induced disruption in IL-10 signalling. IL-10 may affect antitumor immunity and, proliferation and resistance to apoptosis [27]. So, it is easy to explain the correlation between IL-10 treatment response.
In this study, none of the parameters analyzed was significantly correlated with serum IL-10 levels. Even so, we found a statistically significant correlation between serum IL-10 levels and WBCs levels and blood urea level. Furthermore, serum IL-10 levels showed a significant correlation with the response to therapy. Patients who achieved CR had a lower median serum IL-10 level than those who achieved PR or with relapse. This observation confirmed that IL-10 is a strong predictor of treatment response in NHL, suggesting a key role for IL-10 in the pathogenesis and progression of this disease.
In this article, a strong association was demonstrated between EMAP-II and IL-10 in NHL. This finding is supported by higher serum levels of EMAP-II and IL-10 before therapy and its decrease significantly thereafter in all patients.
In summary, it was observed that pretreatment serum levels of EMAP-II and IL-10 were associated with relevant laboratory findings in patients with NHL and that these cytokines decreased significantly after treatment for the disease. Higher IL-19 serum levels seem to be associated with treatment failure and relapse. We, therefore, suggest, introducing them into routine panel, for the useful follow up of NHL patients, assessment of their response to therapy and early detection of recurrence for an improved survival.