COLORECTAL CARCINOMA: EVALUATION OF SYSTEMIC VALUES OF IL–1 AND IL–33 IN PATIENTS WITH AND WITHOUT THROMBOCYTOSIS KOLOREKTALNI KARCINOM: PROCENA SISTEMSKIH VREDNOSTI IL–1 I IL–33 KOD PACIJENATA SA I BEZ TROMBOCITOZE

Background/Aim. Reactive thrombocytosis, as a paraneoplastic syndrome, is often observed in cancer patients. A variety of tumor-related humoral factors and cytokines con-tribute to tumor-stimulated thrombopoiesis. However, the exact role of these cytokines in the pathogenesis of thrombocytosis remains unclear. The aim of this study was to analyze systemic values of cytokines and clinical-pathological characteristics in colorectal carcinoma (CRC) patients with and without thrombocytosis. Methods. Fifty nine CRC patients were involved in this study and divided into two groups according to the number of platelets. We recorded and analyzed the data about: age, gender, size of the cancer, localization, metastasis, vascular or lymph vessel invasion, nuclear grade, histological differentiation rate, tumor, nodus, metastasis (TNM) stage and concentration of cytokines [interleukin (IL)-1, IL-33, IL-12, IL-17 and interferon (IFN)-?] in both groups. Results. CRC patients with thrombocytosis had significantly higher nuclear grade of the cancer (p = 0.002); higher percentage of detectable metastatic lesions in the liver (p = 0.002), lung (p = 0.001), peritoneal carcinomatosis (p = 0.001), detectable invasion of blood (p = 0.012) and lymph vessels (p = 0.010). Concentrations of tumor markers [alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA) and cancer antigen 19-9 (CA19-9)] and se-rum values of IL-1 a nd I L-33 were significantly higher in CRC patients with thrombocytosis. IL-1/IL-12 (p = 0.016), IL-1/IFN-? (p = 0.007), IL-1/IL-17 (p = 0.006), IL-33/IL- 12 (p = 0.001), IL-33/IFN-? (p = 0.001), IL-33/IL-17 (p = 0.002), and IL-33/IL-1 (p = 0.006) ratios were significantly higher in CRC patients with thrombocytosis in comparison to CRC patients without thrombocytosis. Analysis of Receiver Operating Characteristic (ROC) curves showed that values of IL-1 [area under curve (AUC) = 0.718; 95% confidence interval (CI): 0.567?0.868; sensitivity 69.2%, specificity 62.9%] and IL-33 (AUC = 0.763; 95% CI: 0.614? 0.911; sensitivity 84.6%, specificity 65.7%)], could be serve as possible markers for paraneoplastic thrombocytosis in CRC patients. Conclusion. IL-1 a nd I L-33 significantly correlated to high thrombocyte number in patients with more aggressive CRC.


Introduction
Colorectal cancer (CRC) is among the leading causes of mortality and morbidity throughout the world 1 .Overall, CRC ranks third in terms of incidence but second in terms of mortality 2 .The incidence of colorectal cancer is increasing due to ageing and unhealthy life style 3 .Although the distribution of CRC varies widely (worldwide incidence and mortality, 10.2 % and 9.2% of all cancers, respectively), more than two-thirds of all cases and more than half of all deaths happen in countries with high human development index (HDI) 2 .Frequently consumption of red or processed meat, alcohol drinks, and body fatness increase the risk of CRC, whereas physical activity is protective [4][5][6] .
Several recent studies have reported that thrombocytosis may be associated with the poor prognosis of CRC 10 .Thrombocytosis is associated with shorter overall, disease-free and cancer-specific survival.Overall survival is reduced in patients with thrombocytosis regardless of their clinical tumor stage, and ethnicity.Also, thrombocytosis is significantly related to female patients, colon tumor location, T3-4 stage, lymph node positivity, metastasis, undifferentiated histology and lymphatic involvement 11,12 .Interleukin (IL)-33, an IL-1 family member, plays an essential role in the regulation of immune response after cellular stress or damage 13,14 .Recent study revealed that IL-33 by biding to its receptor ST2 inhibits host anti-tumor immunity, remodels tumor stroma and enhances angiogenesis, thereby promoting the development of CRC 15 .Patients with metastatic CRC, with higher expression of IL-33 in cancer tissues were significantly associated with poorer survival 14,16 .IL-1 is secreted by different cell types, such as myeloid cells.Previous studies confirmed the importance of IL-1 in different processes, such as tumorigenesis, invasiveness and progression of tumor cells, as well as invasiveness of CRC, activation or inhibition of anti-tumor immune response 17 .However, the exact role of these cytokines in the pathogenesis of reactive paraneoplastic thrombocytosis remains unclear.
The aim of this study was to analyze clinical-pathological characteristics of disease and systemic values of IL-1 and IL-33, as well as their correlation with pro-inflammatory cytokines (IL-12, IFN-γ and IL-17), in CRC patients with and without thrombocytosis.

Patients
Fifty nine CRC patients were involved in this study, after confirmed diagnosis of CRC with endoscopic and histopathological examination.Excluded criteria for patients were: not well-defined pathology, inadequate clinical document available or diagnosed CRC, previously treated with chemotherapy or radiation, active bleeding.After being included, all patients signed informed consent.In the study, we recorded and analyzed the data about: age, gender, size of the cancer, localization, metastasis, vascular or lymph vessel invasion and clinical TNM stage.In addition, pathological features (nuclear grade and histological differentiation rate) were analyzed in accordance to the American Joint Committee on Cancer (AJCC, 2010) classification.Thrombocytosis was defined as platelet count more or equal than 450×10 9 /L, similar to the related study 18 .

Ethic approvals
Study was performed at the Center for Gastroenterology, Clinical Center of Kragujevac and Center for Molecular Medicine and Stem Cell Research, Faculty of Medical Sciences, University of Kragujevac, Serbia.The researches were allowed by both institutions by obtaining Ethical approvals and made according to the Declaration of Helsinki and the Principle of Good Clinical Practice.

Measurement of cytokine concentration in serum
From each patient one blood sample in the volume of 10 ml was taken preoperatively, before surgery.Serum was separated by centrifugation and stored at -80 °C until testing.
The test was performed using commercial sensitive enzyme-linked immunosorbent assay kits (ELISA, R&D Systems, Minneapolis, MN, USA) particular for human cytokines, such as IL-1, IL-33, IL-12, IL-17 and IFN-γ.The micro titer plates (MTP), with 96-wells, were coated with 100μl of capture antibody in carbonate/bicarbonate buffer (pH 9.6) at the recommended concentrations, overnight.MTP were washed with washing buffer (0.05% Tween-20 in PBS).Briefly, standard recombinant IL-1, IL-12, IL-17, IL-33, IFN-γ or serum samples were incubated in plates for 2 hours before adding 100μl of detection antibody and incubating for 1 hour at room temperature covered with an adhesive sealing film.We have washed MTP and added 100μl of streptavidin peroxidase for 1 hour, and then 100μl of substrate reagent (prepared by mixing equal volumes of Color A and Color B reagents) for 20 minutes.The reaction was stopped with 50μl of stop solution (4 mol/L sulfuric acid) and using a microplate reader (Biochrom, Anthos Zenyth 200, UK), we have read the absorbance at 450 nm.As stated in the instructions of the manufacturer, concentrations of cytokines were estimated by interpolation of a standard curve (made from a series of previously established concentrations of cytokine) and were presented as pg/ml of sera.All measurements were performed in duplicate and in accordance with the manufacturer's recommendations.

Statistical analysis
SPSS software version 20.0 was applied for statistical analyses.All values were reported as means ± SEM.Student's t-test, the Mann-Whitney U-test or Kruskal-Wallis test were used, in order to assess statistical significance of differences between the means of two groups.Associations between thrombocytosis and tumor characteristics were evaluated using the chi-square test.Possible correlation between the markers of interest and thrombocytosis in CRC patients, were analyzed with Pearson's or Spearman's tests, as appropriate, and was determined as weak, moderate or strong (0.1-0.3, 0.3-0.5, 0.5-1.0,respectively), positive or negative.P-value ≤ 0.05 was considered significant.

Results
Fifty nine adult patients (n=59) with diagnosed CRC were recruited in this study.
Patients with diagnosed CRC were divided in two groups according to the number of platelets.The first group includes 35 CRC patients with the number of platelets < patients with the number of platelets ≥ 450×10 9 /L 18,19 (group with thrombocytosis).
Clinical and pathologic characteristics of these patients are presented in Table 1.

Table 1
There were no differences in age or gender distribution between two groups.
Concentrations of tumor markers AFP, CEA and CA 19-9 were significantly higher in CRC patients with thrombocytosis compared to CRC patients without thrombocytosis (Table 1).
Evaluation of nuclear grade of CRC was also made in patients with the presence or absence of thrombocytosis.Nuclear grade was based on evaluation of the size and shape of the nucleus in cells of colorectal cancer and the percentage of tumor cells that are in the process of dividing or growing 20 .Results revealed that CRC patients with thrombocytosis had significantly higher nuclear grade of colorectal cancer in comparison to patients with normal number of platelets (p=0.002; Figure 1A).Next, CRC patients with/without thrombocytosis were analyzed regarded detection of lymph and blood vessels invasion.
Significantly higher percentage of CRC patients with diagnosed thrombocytosis had detectable invasion of blood and lymph vessels in comparison to CRC patients without thrombocytosis, respectively (p=0.012; p=0.010; Figure 1B, 1C).CRC patients with thrombocytosis had advanced TNM stage (III or IV), while CRC patients without thrombocytosis mostly had TNM stage I or II, but this difference did not reach statistical significance (data not shown).Further, CRC patients with/without thrombocytosis were analyzed on the basis of detection metastatic lesions in liver and lung as well as peritoneal carcinomatosis.Significantly higher percentage of CRC patients with thrombocytosis had detectable metastatic lesions in liver and lung as well as peritoneal carcinomatosis, respectively, in comparison to CRC patients without thrombocytosis (p=0.002;p=0.001; p=0.001; Figure 1D, 1E, 1F).

Figure 1
Serum concentrations of cytokines of interest were measured in CRC patients with/without thrombocytosis.Results revealed that CRC patients with diagnosed thrombocytosis had significantly higher serum concentration of IL-1 in comparison to CRC patients with normal number of platelets (p=0.022; Figure 2A).Serum levels of IL-33 were significantly increased in CRC patients with diagnosed thrombocytosis compared to CRC patients without thrombocytosis (p=0.001; Figure 2B).Moreover, strong positive correlation was detected between serum values of IL-1 and IL-33 (r=0.879;p=0.001, Figure 2C).

Figure 4 Discussion
In the present study, we have showed that CRC patients with diagnosed thrombocytosis had significantly higher nuclear grade compared to CRC patients with normal number of platelets (Figure 1).Significantly higher percentage of CRC patients with thrombocytosis had detectable metastatic lesions in lung and liver and peritoneal carcinomatosis, as well as blood and lymph vessels invasion compared to CRC patients without thrombocytosis (Figure 1).Higher concentrations of tumor markers AFP, CEA, CA 19-9 were detected in CRC patients with diagnosed thrombocytosis (Table 1).In line with the revelation of previous studies that increased concentrations of tumor markers in patients with CRC indicate on more aggressive type of cancer and can be treated as poor prognostic factor, presented data indicate on more severe form of CRC in patients with thrombocytosis 21 .
Reactive thrombocytosis is an elevated platelet count (≥ 450×10 9 /L) develops secondary to another disorder 19 .Previous studies investigated relation between cancer and thrombocytosis [10][11][12][22][23][24] . Sasaki t al. have shown that cancer-specific survival of CRC patients with thrombocytosis was significantly shorter compared to CRC patients without diagnosed thrombocytosis 22 .Moreover, thrombocytosis indicates adverse prognosis in CRC and also may serve as clinically useful, cost-effective, noninvasive marker to facilitate risk assessment and guide postoperative management 11,12 .The platelet count is also treated as valuable prognostic marker for the survival in patients with metastasis of colorectal cancer 23 .Furthermore, pretreatment hematologic abnormalities, such as anemia and thrombocytosis, can be also considered as useful prognostic markers in patients with colorectal cancer 24 .In line with these studies, presented data implicate on severe and more progressive form of colorectal cancer in patients with diagnosed thrombocytosis.
Several factors such as iron deficiency, acute infection and chronic inflammatory disorders can be causes of reactive thrombocytosis 19 .Lately, cancer is more often associated with paraneoplastic thrombocytosis 7 .A variety of tumor-related humoral factors and cytokines influence thrombopoiesis in patients with tumor and contribute to tumorstimulated thrombopoiesis 8,9 .In order to investigate potential role of soluble molecules on thrombocytosis development, we have further analyzed systemic concentrations of cytokines of interest.IL-1 is potent cytokine involved in variety of pro-inflammatory processes, but also in tumorigenesis and tumor progression 25 .Besides direct stimulating effect on tumor cell proliferation, it has also been shown that colon cancer cell-derived IL-1α may upregulate angiogenesis by modulating stromal cells within the tumor microenvironment 15 .In this study, significantly higher systemic level of IL-1 was measured in CRC patients with diagnosed thrombocytosis compared to CRC patients with normal number of platelets (Figure 2).Previous studies investigate the importance of IL-1 in thrombocytopoiesis [26][27][28] .Yang et al. revealed stimulative effect of IL-1β on megakaryocyte colony forming units production.Moreover, they confirmed that megakaryocytic cells have IL-1 receptors on their surface 26 .The other study has shown that a single dose of IL-1 was able to stimulate an increase in platelet production for 3 weeks. 27.Also, administration of recombinant human IL-1β to C57B1/6 male mice consequently induced a remarkable thrombocytosis, about 2.3 times higher in IL-1β treated mice than in control mice 28 .These results are in line with our finding, suggesting on important role of IL-1 in thrombocytosis development.
IL-33, member of IL-1 family is mostly expressed by mucosal epithelial cells 29 .
Previous studies confirmed important role of IL-33 in CRC pathogenesis 13,14 .It is known that IL-33 activates core stem cell genes, recruits macrophages into the cancer microenvironment and stimulates them to produce prostaglandin E2, which all promote carcinogenesis of CRC and metastasis of cancers 16,30 .To our knowledge, this is the first study describing significant increment of IL-33 in sera of CRC patients with diagnosed thrombocytosis compared to CRC patients without thrombocytosis (Figure 2).In line with our result are different animal and clinical studies suggesting on importance of IL-33 in pathogenesis of thrombocytosis.Talabot et al. described that CMV/IL33 mice with IL-33 overexpression, characterized as increased local or systemic levels of pro-inflammatory mediators such as IL-1b, Cxcl-1, G-CSF, and IL-6, also suffer from anemia, thrombocytosis, and dysregulation of myelopoiesis 31 .The other study has shown that ST2, membrane receptor for IL-33 is expressed in bone marrow mainly on endothelial, mesenchymal, and early myeloid cells.Activation of IL-33/ST2 signaling pathway on these cells stimulates production of different soluble molecules that further promote development and proliferation of myeloid cells 32 .Previous studies have confirmed that thrombocytosis correlates with a shorter overall survival (OS) and poorer disease free survival (DFS) 33,34 .Except platelet number, other platelet-associated indicators, such as plateletcrit (PCT), mean platelet volume (MPV) and platelet distribution width (PDW), may also correlate with poorer OS of CRC patient and can be used as potentional prognostic factors 35 .To our knowledge, some studies revealed that development and progression of CRC is associated with alterations in serum IL-1 level but it`s significance is not well defined 36, 37 , whille significane of serum IL-33 as marker of CRC survival has not been explored yet.
Moreover, our study showed predomination of IL-1 and IL-33 over pro-inflammatory cytokines IL-12, IFN-γ and IL-17 (Figure 3), known for their crucial role in antitumor immune response 38 .Interesting fact is that IL-33 also predominates over IL-1 in sera of CRC patients with thrombocytosis (Figure 3).Predomination of IL-1 and IL-33 over mediators of potent antitumor immunity in patients with thrombocytosis can partially explain more severe and progressive disease diagnosed in these patients.
Strong positive correlation detected between serum values of IL-1 and IL-33 implicated on potential united effect of these cytokines in pathogenesis of thrombocytosis in CRC patients.This simultaneous effect can be realized in at least two manners: 1) IL-1 may directly affect megakaryocytes and stimulates production of platelets; 2) IL-33 may indirectly stimulate stromal cells in bone marrow to produce mediators that can further stimulates thrombopoiesis in CRC patients (Figure 5).

Figure 5
This proposed mechanism is supported by analysis of Receiver Operating Characteristic (ROC) curves of IL-33 and IL-1 revealed that these cytokines could be used as posible markers of paraneoplastic thrombocytosis in CRC patients (Figure 4).

Conclusion
Presented data revealed that IL-1 and IL-33 are significantly correlated to high thrombocyte number in patients with more aggressive CRC.

Declaration of interest
The authors declare that they have no competing interests.Online First October, 2019

Figure 1 CRC patients with thrombocytosis have severe and advanced diseaseFigure 1 .
Figure 1

Figure 2 .
Figure 2. (A) CRC patients with diagnosed thrombocytosis had significantly higher serum concentration of IL-1 in comparison to CRC patients with normal number platelets count (p=0.022).(B) Serum levels of IL-33 were also significantly increased in CRC patients with diagnosed thrombocytosis compared to CRC patients without thrombocytosis (p=0.001).(C) strong positive correlation was detected between serum values of IL-1 and IL-33 (r=0.879;p=0.001).

Figure 4 ReceiverFigure 4 .
Figure 4 Receiver Operating Characteristic (ROC) curves analyses of serum IL-1 and IL-33 levels for prediction of paraneoplastic thrombocytosis in CRC patients