IL-10 Gene Polymorphism and Influence of Chemotherapy on Cytokine Plasma Levels in Childhood Acute Lymphoblastic Leukemia Patients Carlos Hiroji Hiroki, Marla Karine Amarante, Diego Lima Petenuci, Alberto Yoichi Sakaguchi, Fausto Celso Trigo, Maria Angelica Ehara Watanabe, Carlos Eduardo Coral de Oliveira PII: DOI: Reference:

S1079-9796(15)00104-7 doi: 10.1016/j.bcmd.2015.06.004 YBCMD 1944

To appear in:

Blood Cells, Molecules, and Diseases

Received date: Revised date: Accepted date:

1 June 2015 11 June 2015 12 June 2015

Please cite this article as: Carlos Hiroji Hiroki, Marla Karine Amarante, Diego Lima Petenuci, Alberto Yoichi Sakaguchi, Fausto Celso Trigo, Maria Angelica Ehara Watanabe, Carlos Eduardo Coral de Oliveira, IL-10 Gene Polymorphism and Influence of Chemotherapy on Cytokine Plasma Levels in Childhood Acute Lymphoblastic Leukemia Patients, Blood Cells, Molecules, and Diseases (2015), doi: 10.1016/j.bcmd.2015.06.004

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ACCEPTED MANUSCRIPT IL-10 GENE POLYMORPHISM AND INFLUENCE OF CHEMOTHERAPY ON CYTOKINE PLASMA LEVELS IN CHILDHOOD ACUTE LYMPHOBLASTIC

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LEUKEMIA PATIENTS

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IL-10 POLYMORPHISM AND PLASMA LEVELS IN LEUKEMIA PATIENTS

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Carlos Hiroji Hiroki1, Marla Karine Amarante1, Diego Lima Petenuci1, Alberto Yoichi Sakaguchi1, Fausto Celso Trigo2, Maria Angelica Ehara Watanabe1, Carlos Eduardo

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Coral de Oliveira1

Laboratory of Study and Application of DNA Polymorphisms, Department of

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Pathological Sciences, State University of Londrina, Londrina/PR, Brazil. Londrina Cancer Hospital and University Hospital of State University of Londrina,

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Medical Clinic Department, Londrina/PR/ Brazil.

Corresponding Author: Dr. Carlos Eduardo Coral de Oliveira (PhD) Laboratory of Study and Application of DNA Polymorphisms Biological Sciences Center – Dept of Pathological Sciences State University of Londrina

ZIP: 86051-970

Londrina- Paraná – Brazil Phone: 55 43 33715629 Fax: 55 43 33715630

e-mail: [email protected]

ACCEPTED MANUSCRIPT ABSTRACT

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Acute Lymphoblastic Leukemia is the leading form of cancer in infancy, and compelling evidences suggest an involvement of altered immune competence on this malignancy pathogenesis. Interleukin 10 (IL-10) is a pleiotropic cytokine designated as an immunosuppressive molecule, but may act as an immunostimulant factor in cancer development and progression. An IL-10 single nucleotide polymorphism (SNP) rs1800896 has been associated with disease progression to ALL, and might influence cytokine expression. This study analyzed the IL-10 rs1800896 polymorphism and performed a case-control study to determine the significant associations with ALL susceptibility and prognosis. IL-10 plasma levels were determined and associated with genotypes and disease phase. The study consisted of 67 childhood ALL patients and 75 age-related healthy controls. The rs1800896 was not associated with ALL susceptibility or risk of relapse. No significant association was observed between different genotypes of the rs1800896 and plasma levels of IL-10. Cytokine plasma levels were significantly higher in diagnosis group (9.71pg/mL± 3.7), comparing to treatment (3.48pg/mL± 1.3; p=0.01) and remission phase (0.12pg/mL± 0.1; p=0.0001) groups. This work indicates that the IL-10 plasma expression is altered from ALL disease diagnosis and remission. Moreover, prospective studies will establish the functional role of IL-10 in immune modulation in childhood ALL.

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Key-words: Acute Lymphoblastic Leukemia; IL-10, polymorphism; plasma; prognosis.

ACCEPTED MANUSCRIPT INTRODUCTION

Acute Lymphoblastic Leukemia (ALL) is a malignant disease which affects both

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children and adults, with peak prevalence between 2 and 5 years of age. Lymphocyte

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precursors acquire genetic and epigenetic alterations, resulting in a continuous signal of proliferation and a blockage on the differentiation, leading to the malignancy [1].

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Although some modification in genome, like gene NOTCH1 [2], TEL-AML1 [3], Philadelphia chromosome [4], and exposure to carcinogenic factors, like aromatic hydrocarbons [5], ionizing radiation [6], air pollution [7] have been described to

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influence the ALL pathogenesis, they explain a few percentage of the cases. Neoplastic cells are able to modulate the tumor microenvironment to its favor by

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producing cytokines, recruiting regulatory cells, like Tregs and Myeloid Derived Suppressor Cells (MDSC), which help the tumor escape immunological surveillance [8]. One of the cytokines related to tumor tolerance is Interleukin-10 (IL-10), an anti-

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inflammatory molecule expressed by cells of innate and adaptive immune system [9]. It

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acts inhibiting the production of inflammatory cytokine, as IL-1β, IL-6, IL-12 and TNFα, and expression of MHC II, CD80 and CD86. Moreover, it interferes with NF-kB and AP-1 activation in T CD4+, monocytes and antigen-presenting cells, but induces their

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activation in T CD8+, and promotes Bcl-2 expression in CD34+ B cells [10]. A polymorphism located in the promoter region of IL-10 gene, at position -1082, results in a allele (G) which is described to increase IL-10 expression [11]. Many

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studies have analyzed IL-10 polymorphisms and its expression in different models, like inflammatory, infectious, auto-immune diseases and cancer [12, 13]. In cancer, these polymorphisms have been associated with increased risk in several types, including cervical [14], gastric [15], colorectal [16], B-cell lymphoma [17] and lung [18]. However, a study showed that IL-10-deficient mice had increased levels of Treg and MSDC, associated with tumor development [19], demonstrating a pleiotropic function of IL-10 in cancer. Although

IL-10

is

commonly

termed

as

an

anti-inflammatory,

immunosuppressive cytokine that favors tumor escape from immune surveillance, a wealth of evidence indicates that IL-10 also possesses some immunostimulating properties. In fact, the role of IL-10 in immune system regulation coupled with polymorphic regulation of its expression, presents ability of influencing positively and negatively the function of innate and adaptive immunity in different experimental

ACCEPTED MANUSCRIPT models, which makes it questionable to categorize this cytokine as a target of antiimmune escape therapeutic strategies against cancer [13, 20]. In this study, we genotyped the IL-10 (rs1800896) SNP and performed a case-

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control study, investigating its possible role in ALL susceptibility and prognosis.

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Plasma IL-10 levels were measured to assess possible functional correlation between

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the rs1800896 polymorphism and protein levels.

METHODS

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Study population

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Following approval from the Human Ethics Committee of the State University of Londrina (No. 214/09-CAAE (Presentation of Certificate of Appreciation for ethics) N°. 0164.0.268.000-09), 5 mL of peripheral blood was collected from ALL patients and

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healthy children as controls. A term of free informed consent was signed by the parents

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of the children donors. The case group consisted of sixty-seven patients (37 males and 30 females) (mean age 12.0 years) with confirmed childhood ALL diagnostic that were

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recruited from two institutions: Cancer Hospital of Londrina and University Hospital of Londrina. A total of seventy-five (36 males and 39 females), with similar age to case group (mean age 8.7 years) were used as control group. The control group consisted of healthy individuals, mainly free of inflammation processes and neoplasia, according to

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biochemical tests and clinical reports.

DNA extraction

Genomic DNA was isolated from 200uL EDTA-anti-coagulated peripheral blood leukocytes. Extraction was performed using Biopur Mini Spin Plus Kit (Biometrix Diagnostica, Curitiba, Brazil), according to manufacturer’s instructions. The samples were quantified by NanoDrop 2000c® Spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, USA), at wavelength of 260/280 to indicate DNA’s concentration and purity.

IL-10 genotyping

ACCEPTED MANUSCRIPT Polymorphism analysis in the promoter region of IL-10 (rs 1800896) was performed by polymerase chain reaction (PCR). Samples were amplified in a final volume of 25uL containing 100ng of template DNA, 200mM Tris-Hcl (pH 8.4), 500mM KCl, 1.25mM

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dNTP, 50mM MgCl2, 1mg/mL bovine serum albumin (ultrapure, non-acetylated),

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2.5uM of each primer (forward 5’ CTCGCTGCAACCCAACTGGC 3’; reverse 5’CTTACCTATCCCTACTTCC 3’) (GenBank Accession Number U16720.1) and 5U/uL

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Taq DNA Polymerase (InvitrogenTM, Carlsbad, USA). PCR conditions were denaturation at 95°C for 15 minutes, 40 cycles of 95°C for 30 seconds, 61°C for 30 seconds, 72°C for 30 seconds and a final extension at 72°C for 10 minutes. PCR

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products were digested 5U of Mnl1 restriction enzyme (New England BioLabs Inc, Ipswich, USA) at 37°C for 4 hours, and genotypes were scored as homozygous wild

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type (106bp and 33bp), heterozygous (139bp, 106bp, and 33bp), and mutant homozygous (139bp, no restriction site).

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Plasma IL-10 quantification

Analysis was performed on blood plasma, using Human IL-10 ELISA Ready-SET-Go

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(e-Bioscience Inc, San Diego, USA). In brief, blood plasma from each group was added into the precoated plates with anti-human IL-10 monoclonal antibody and incubated overnight at 8ºC. Samples were washed, and biotinylated secondary antibody-HRP+Sav conjugated were added. 3,3’,5,5’-tetramethylbenzidine (TMB) Peroxidase substrate

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were added after wash, followed by 1M Phosphoric acid stop solution. The ELISA plate was read at absorbance OD of 450nm wavelength using ELX-300 Reader (BioTek Instruments Inc., VT, USA). The results were expressed in pg/mL for IL-10. Each experimental and control sample was assayed in two biological replicates. Assay sensibility was 2pg/mL. Data processing was performed using the analytical curvefitting software Gen5 (BioTek).

Statistical analysis

Genotype frequencies were submitted to chi-square test to detect a deviation from Hardy-Weinberg equilibrium. To compare the polymorphism frequency between groups, Odds Ratio (OR) analysis, with 95% confidence interval (CI), was performed. Cytokine levels were analyzed and compared to genotypes by t test (Mann-Whitney U

ACCEPTED MANUSCRIPT test). Statistical analysis was performed using Prism 5 software (GraphPad Software, San Diego, USA).

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RESULTS

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Clinical characteristics

The ALL diagnosis included age, leukocyte count, immunophenotyping, involvement of tissues other than bone marrow and responsiveness to the treatment. In addition, all

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patients were stratified into risk of relapse status by a hematologist-oncologist (Trigo, FC). Among them, 25 were classified as low risk and 42 as high risk of recurrence,

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according to the clinical and laboratorial findings at diagnosis, as defined by the Brazilian Group for Childhood Leukemia Treatment in the GBTLI LLA-1999 [21]. Most of our sample, both patients and controls, were predominantly Caucasian, a

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prevalent population in southern Brazil due to European colonization.

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IL-10 rs1800896 polymorphism in ALL patients and controls

The controls and ALL patients were both in Hardy-Weinberg equilibrium for the rs1800896 polymorphism (p=0.61). Allele frequencies and genotypes are summarized at Table 1. There were no significant differences between ALL patients and controls,

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comparing allele, genotypes and allele carrier frequencies.

In addition, genotypes were compared regarding risk of relapse status of ALL (Table 2); however, any association was found.

IL-10 plasma levels in ALL patients and controls

Concentrations of plasma IL-10 in each group are shown in Figure 1. We have observed that the concentrations of IL-10 were lower in ALL patients than in the controls (means and S.E.M., Control= 6.1pg/mL± 1.85; ALL= 2.03pg/mL± 0.76), but they were not statistically different (Figure 1a; p=0.25). In relation to recurrence status

ACCEPTED MANUSCRIPT of ALL patients, high risk group presented lower mean IL-10 plasma levels than low risk group (2.15pg/mL± 0.9 vs. 6.59pg/mL± 2.15), although difference was not significant (Figure 1b; p=0.19). Moreover, IL-10 plasma levels were compared between

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genotypes of rs1800896 polymorphism, and did not differentiate between them (Figure

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1c).

IL-10 plasma levels were also compared in ALL patients based on therapeutic

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regimen (Figure 2) at time of sampling: 11 of the patients were newly diagnosed (Diagnosis group); 34 were receiving chemotherapy (Treatment group), and 16 of them were in complete remission but receiving low-dose maintenance chemotherapy

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(Remission group). Interestingly, in the analysis of chemotherapy effect, IL-10 plasma levels were significantly higher in Diagnosis group (9.71pg/mL± 3.7), comparing to

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Treatment (3.48pg/mL± 1.3; p=0.01) and Remission (0.12pg/mL± 0.1; p=0.0001) groups. Plasma levels of IL-10 in the Treatment group were compared to control group (5.4pg/mL± 1.5; p=0.99). However, IL-10 values of Diagnosis group were significantly

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higher than controls (p=0.01).

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DISCUSSION

Interleukin-10

(IL-10)

is

a

multifunctional

cytokine

with

both

immunosuppressive and anti-angiogenic functions. In consequence, IL-10 can have both tumor-promoting and tumor-inhibiting properties. Raised levels of serum and

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peritumoral IL-10 have been reported in many malignancies, which have been interpreted in support of a role for IL-10 in tumor escape from the immune response [22].

The influence of IL-10 expression at the ALL pathogenesis has been target of several

studies.

IL-10-3575T>A

(rs1800890)

and

-1082A>G

(rs1800896)

polymorphisms has been studied concerning a potential implication in terms of some cancer risks, but the results from single studies are contradictory [23, 24]. The present study demonstrates that there are no significant differences in genotype distribution and allele frequency of IL-10 rs1800896 polymorphism between ALL patients and age-matched controls. The allelic and genotypic distribution rs1800896 did not differ in ALL patients of high risk of relapse status than that of low risk. These results indicated an absence of association between this polymorphism and ALL susceptibility or recurrence.

ACCEPTED MANUSCRIPT Convincing evidences for an impairment on immune system is possibly involved in childhood leukemogenic process [25-28], and IL-10 might represent an engaged cytokine. Previously, Schulz, Munker, Ertl, Holler and Kolb [29] have evaluated the

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autocrine production of cytokines by leukemic cells, and demonstrated that ALL blasts

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express the mRNA for IL-10 and TNF-α. Chang, Zhou, Buffler, Chokkalingam, Metayer and Wiemels [30] showed that serum levels of IL-10 in children neonatal who

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developed ALL during childhood were significantly lower than in controls. Published data regarding IL-10 expression levels in leukemic patients present conflicting results. At the time of diagnosis, IL-10 expression was higher in bone

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marrow ALL blasts [31, 32], Treg cells from peripheral blood of ALL patients [33], and plasma/serum [29, 34, 35]. Contrariwise, Mazur, Mertas, Sonta-Jakimczyk, Szczepanski

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and Janik-Moszant [36] reported no differences in IL-10 concentrations among treated ALL children and controls.

Furthermore, it is reasonable that high IL-10 expression could be related to a

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suppression of anti-tumor response, which would contribute to the evolution of disease.

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Wu, Qing, Wu, Zhu and Zhou [33] found an increased number of CD4+CD25+ Treg, an immunoregulatory cell, in patients with ALL comparing to controls, and those cells

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showed higher IL-10 and TGF-β expression, and lower IL-2 expression. In our study, IL-10 plasma levels in ALL patients were not different from those in controls. Similarly, differences between IL-10 plasma levels in low and high risk of relapse groups were not significant. However, despite the absence of statistical

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association, there is a slightly difference in IL-10 expression between ALL patients and controls, which could indicate a necessity of more studies. It is known that rs1800896 polymorphism, within the IL-10 gene promoter region, might influence IL-10 expression and plasma levels [37, 38]. It was described that G-allele carrier patients have a better response to ALL treatment which includes glucocorticoids, leading to a better outcome [39, 40]. Franchimont, Martens, Hagelstein, Louis, Dewe, Chrousos, Belaiche and Geenen [41] have shown that the binding capacity for dexamethasone can be upregulated by high IL-10 production. Moreover, patients carrying haplotypes signatures of high expression for IL-10, including G allele of rs1800896 polymorphism, have less relapses and better survival [42]. de Deus, Lugo and Muniz [37] have shown a lower survival among patients with IL-10 AA genotype and the concomitant occurrence of IL-10 AA and TNF AA genotypes. In fact, Winkler, Taschik, Haubitz, Eyrich, Schlegel and Wiegering [42]

ACCEPTED MANUSCRIPT concluded that gene-polymorphisms of the regulatory/anti-inflammatory cytokines, TGF-β and IL-10, but not of the pro-inflammatory cytokines, IFN-γ and TNF-α, have an impact on prognosis and risk group of ALL. The reduced capacity to produce

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proinflammatory cytokines at diagnosis may serve as another important functional risk

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factor. These data may help in further risk stratification and adaptation of therapyintensity in pediatric patients with ALL.

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Interestingly, our data suggest an absence of association of IL-10 polymorphism and cytokine plasma levels in childhood ALL. It is important to mention that not all ALL patients and controls (47.9% and 53.1%, respectively) presented detectable levels

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of plasma IL-10. To answer the question of biological importance on how this polymorphism might affect IL-10 expression in childhood ALL, intracellular protein

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evaluation and/or mRNA quantitation studies will have to be performed. The immunosuppressive effect of cytotoxic drugs, basic therapeutic agents in the treatment of childhood acute leukemias, requires monitoring of the immune system

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following cessation of therapy [36]. Indeed our results demonstrated high IL-10 levels

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in patients before chemotherapy compared to treatment (p=0.01) and remission group (p=0.0001). Notwithstanding, mean plasma concentration of IL-10 during treatment was

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comparable to controls (p=0.99).

In newly diagnosed ALL patients, Horacek, Kupsa, Vasatova, Jebavy and Zak [43] have used biochip array technology and reported differences in serum levels of IL8, IL-3, IL-4 and other adhesion molecules, but not in IL-10, compared to healthy

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controls. After 1 year from the cessation of therapy, ALL patients did not present differences in IL-10 serum levels in comparison to healthy controls [36]. In addition, a prospective study on spontaneous IL-10 production in the serum of 96 patients, including ALL patients, admitted for allogeneic bone marrow transplantation (BMT) indicated that higher serum IL-10 levels at the time of admission and prior to any preparative treatment correlates with a subsequent low incidence of acute graft versus host disease (GVHD) as compared to patients with low IL-10 production, suggesting a role for it in maintaining immunobalance in the setting of allogenic BMT [44]. Although our study focused on a relatively small number of individuals, its findings contribute to the evidence that the effects of a deregulated immune function in childhood ALL are important when predicting prognosis. However, further molecular and functional analyses are needed to determine whether the association of IL-10 plasma levels may be influenced by polymorphism in its gene. Similarly, the precise

ACCEPTED MANUSCRIPT role of IL-10 polymorphism and plasma levels in ALL development need to be elucidated in larger cohorts of subjects with ALL to provide a better understanding of

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the IL-10 function in leukemia pathophysiology.

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ACKNOWLEDGMENTS

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The authors would like to acknowledge Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação Araucária do Paraná, Secretaria da Ciência, Tecnologia e Ensino Superior (SETI), Fundo Estadual para a Infância e Adolescência (FIA/PR), Secretaria da Família e Desenvolvimento Social (SEDS) and Pro-reitoria de Pós-Graduação da Universidade Estadual de Londrina (PROPPG-UEL).

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REFERENCES

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[1] C.H. Pui, L.L. Robison, A.T. Look, Acute lymphoblastic leukaemia, Lancet, 371 (2008) 1030-1043. [2] A.P. Weng, A.A. Ferrando, W. Lee, J.P.t. Morris, L.B. Silverman, C. Sanchez-Irizarry, S.C. Blacklow, A.T. Look, J.C. Aster, Activating mutations of NOTCH1 in human T cell acute lymphoblastic leukemia, Science, 306 (2004) 269-271. [3] H. Mori, S.M. Colman, Z. Xiao, A.M. Ford, L.E. Healy, C. Donaldson, J.M. Hows, C. Navarrete, M. Greaves, Chromosome translocations and covert leukemic clones are generated during normal fetal development, Proceedings of the National Academy of Sciences of the United States of America, 99 (2002) 8242-8247. [4] R. Ren, Mechanisms of BCR-ABL in the pathogenesis of chronic myelogenous leukaemia, Nature reviews. Cancer, 5 (2005) 172-183. [5] N.C. Deziel, R.P. Rull, J.S. Colt, P. Reynolds, T.P. Whitehead, R.B. Gunier, S.R. Month, D.R. Taggart, P. Buffler, M.H. Ward, C. Metayer, Polycyclic aromatic hydrocarbons in residential dust and risk of childhood acute lymphoblastic leukemia, Environmental research, 133 (2014) 388-395. [6] D.L. Preston, S. Kusumi, M. Tomonaga, S. Izumi, E. Ron, A. Kuramoto, N. Kamada, H. Dohy, T. Matsuo, T. Matsui, et al., Cancer incidence in atomic bomb survivors. Part III. Leukemia, lymphoma and multiple myeloma, 1950-1987, Radiation research, 137 (1994) S6897. [7] T. Filippini, J.E. Heck, C. Malagoli, C. Del Giovane, M. Vinceti, A review and metaanalysis of outdoor air pollution and risk of childhood leukemia, Journal of environmental science and health. Part C, Environmental carcinogenesis & ecotoxicology reviews, 33 (2015) 36-66. [8] F. Chen, X. Zhuang, L. Lin, P. Yu, Y. Wang, Y. Shi, G. Hu, Y. Sun, New horizons in tumor microenvironment biology: challenges and opportunities, BMC medicine, 13 (2015) 45. [9] M. Saraiva, A. O'Garra, The regulation of IL-10 production by immune cells, Nature reviews. Immunology, 10 (2010) 170-181. [10] K.W. Moore, R. de Waal Malefyt, R.L. Coffman, A. O'Garra, Interleukin-10 and the interleukin-10 receptor, Annual review of immunology, 19 (2001) 683-765. [11] C.J. Edwards-Smith, J.R. Jonsson, D.M. Purdie, A. Bansal, C. Shorthouse, E.E. Powell, Interleukin-10 promoter polymorphism predicts initial response of chronic hepatitis C to interferon alfa, Hepatology, 30 (1999) 526-530.

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

SC R

IP

T

[12] M. Ansari, G. Sauty, M. Labuda, V. Gagne, C. Laverdiere, A. Moghrabi, D. Sinnett, M. Krajinovic, Polymorphisms in multidrug resistance-associated protein gene 4 is associated with outcome in childhood acute lymphoblastic leukemia, Blood, 114 (2009) 1383-1386. [13] J. Trifunovic, L. Miller, Z. Debeljak, V. Horvat, Pathologic patterns of interleukin 10 expression--a review, Biochemia medica, 25 (2015) 36-48. [14] K. Matsumoto, A. Oki, T. Satoh, S. Okada, T. Minaguchi, M. Onuki, H. Ochi, S. Nakao, M. Sakurai, A. Abe, H. Hamada, H. Yoshikawa, Interleukin-10 -1082 gene polymorphism and susceptibility to cervical cancer among Japanese women, Japanese journal of clinical oncology, 40 (2010) 1113-1116. [15] T. Yu, Q. Lu, X.L. Ou, D.Z. Cao, Q. Yu, Clinical study on gastric cancer susceptibility genes IL-10-1082 and TNF-alpha, Genetics and molecular research : GMR, 13 (2014) 1090910912. [16] L.D. Miteva, N.S. Stanilov, T.S. Deliysky, S.A. Stanilova, Significance of -1082A/G polymorphism of IL10 gene for progression of colorectal cancer and IL-10 expression, Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine, 35 (2014) 12655-12664. [17] H.Y. Cao, P. Zou, H. Zhou, Genetic association of interleukin-10 promoter polymorphisms and susceptibility to diffuse large B-cell lymphoma: a meta-analysis, Gene, 519 (2013) 288-294. [18] C.M. Shih, Y.L. Lee, H.L. Chiou, W.F. Hsu, W.E. Chen, M.C. Chou, L.Y. Lin, The involvement of genetic polymorphism of IL-10 promoter in non-small cell lung cancer, Lung cancer, 50 (2005) 291-297. [19] T. Tanikawa, C.M. Wilke, I. Kryczek, G.Y. Chen, J. Kao, G. Nunez, W. Zou, Interleukin10 ablation promotes tumor development, growth, and metastasis, Cancer research, 72 (2012) 420-429. [20] S. Mocellin, F.M. Marincola, H.A. Young, Interleukin-10 and the immune response against cancer: a counterpoint, Journal of leukocyte biology, 78 (2005) 1043-1051. [21] M.B. Cazé, D; Santos, MEF, Referential study of a chemotherapy protocol for acute lymphocytic leukemia in childhood, Rev HCPA, 30 (2010) 7. [22] W.M. Howell, M.J. Rose-Zerilli, Interleukin-10 polymorphisms, cancer susceptibility and prognosis, Familial cancer, 5 (2006) 143-149. [23] Z.M. Dai, J. Liu, X.M. Cao, Y. Zhang, M. Wang, X.H. Liu, C.J. Li, Z.J. Dai, W.G. Zhang, Association Between Interleukin-10-3575T>A (rs1800890) Polymorphism and Cancer Risk, Genetic testing and molecular biomarkers, (2015). [24] Y. Zhang, Z.G. Xia, J.H. Zhu, M.B. Chen, T.M. Wang, W.X. Shen, J. He, Association of Interleukin-10 -3575T>A and -1082A>G polymorphisms with non-Hodgkin lymphoma susceptibility: a comprehensive review and meta-analysis, Molecular genetics and genomics : MGG, (2015). [25] M. Greaves, Childhood leukaemia, BMJ, 324 (2002) 283-287. [26] M. Greaves, Infection, immune responses and the aetiology of childhood leukaemia, Nature reviews. Cancer, 6 (2006) 193-203. [27] L.J. Kinlen, Epidemiological evidence for an infective basis in childhood leukaemia, British journal of cancer, 71 (1995) 1-5. [28] D.P. Strachan, Hay fever, hygiene, and household size, BMJ, 299 (1989) 1259-1260. [29] U. Schulz, R. Munker, B. Ertl, E. Holler, H.J. Kolb, Different types of human leukemias express the message for TNF-alpha and interleukin-10, European journal of medical research, 6 (2001) 359-363. [30] J.S. Chang, M. Zhou, P.A. Buffler, A.P. Chokkalingam, C. Metayer, J.L. Wiemels, Profound deficit of IL10 at birth in children who develop childhood acute lymphoblastic leukemia, Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology, 20 (2011) 1736-1740. [31] S. Wu, R. Gessner, A. von Stackelberg, R. Kirchner, G. Henze, K. Seeger, Cytokine/cytokine receptor gene expression in childhood acute lymphoblastic leukemia: correlation of expression and clinical outcome at first disease recurrence, Cancer, 103 (2005) 1054-1063.

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[32] C. Kebelmann-Betzing, G. Korner, L. Badiali, D. Buchwald, A. Moricke, A. Korte, J. Kochling, S. Wu, D. Kappelmeier, K. Oettel, G. Henze, K. Seeger, Characterization of cytokine, growth factor receptor, costimulatory and adhesion molecule expression patterns of bone marrow blasts in relapsed childhood B cell precursor all, Cytokine, 13 (2001) 39-50. [33] C.P. Wu, X. Qing, C.Y. Wu, H. Zhu, H.Y. Zhou, Immunophenotype and increased presence of CD4(+)CD25(+) regulatory T cells in patients with acute lymphoblastic leukemia, Oncology letters, 3 (2012) 421-424. [34] E. Bien, A. Balcerska, E. Adamkiewicz-Drozynska, M. Rapala, M. Krawczyk, J. Stepinski, Pre-treatment serum levels of interleukin-10, interleukin-12 and their ratio predict response to therapy and probability of event-free and overall survival in childhood soft tissue sarcomas, Hodgkin's lymphomas and acute lymphoblastic leukemias, Clinical biochemistry, 42 (2009) 1144-1157. [35] B.-J.A. Drabko K, Kowalczyk, JR, Serum concentration of IL-2, IL-4, IL-10 and TNF-α in children with acute lymphoblastic leukemia – possible role of oxidative stress, Centr Eur J Immunol, 33 (2008) 4. [36] B. Mazur, A. Mertas, D. Sonta-Jakimczyk, T. Szczepanski, A. Janik-Moszant, Concentration of IL-2, IL-6, IL-8, IL-10 and TNF-alpha in children with acute lymphoblastic leukemia after cessation of chemotherapy, Hematological oncology, 22 (2004) 27-34. [37] D.M. de Deus, K.A. Lugo, M.T. Muniz, Influence of IL10 (G1082A) and TNFalpha (G308A) Polymorphisms on the Survival of Pediatric Patients with ALL, Leukemia research and treatment, 2012 (2012) 692348. [38] A. Lesiak, M. Zakrzewski, K. Przybylowska, M. Rogowski-Tylman, A. Wozniacka, J. Narbutt, Atopic dermatitis patients carrying G allele in -1082 G/A IL-10 polymorphism are predisposed to higher serum concentration of IL-10, Archives of medical science : AMS, 10 (2014) 1239-1243. [39] M. Lauten, T. Matthias, M. Stanulla, C. Beger, K. Welte, M. Schrappe, Association of initial response to prednisone treatment in childhood acute lymphoblastic leukaemia and polymorphisms within the tumour necrosis factor and the interleukin-10 genes, Leukemia, 16 (2002) 1437-1442. [40] S. Marino, F. Verzegnassi, P. Tamaro, G. Stocco, F. Bartoli, G. Decorti, M. Rabusin, Response to glucocorticoids and toxicity in childhood acute lymphoblastic leukemia: role of polymorphisms of genes involved in glucocorticoid response, Pediatric blood & cancer, 53 (2009) 984-991. [41] D. Franchimont, H. Martens, M.T. Hagelstein, E. Louis, W. Dewe, G.P. Chrousos, J. Belaiche, V. Geenen, Tumor necrosis factor alpha decreases, and interleukin-10 increases, the sensitivity of human monocytes to dexamethasone: potential regulation of the glucocorticoid receptor, The Journal of clinical endocrinology and metabolism, 84 (1999) 2834-2839. [42] B. Winkler, J. Taschik, I. Haubitz, M. Eyrich, P.G. Schlegel, V. Wiegering, TGFbeta and IL10 have an impact on risk group and prognosis in childhood ALL, Pediatric blood & cancer, 62 (2015) 72-79. [43] J.M. Horacek, T. Kupsa, M. Vasatova, L. Jebavy, P. Zak, Evaluation of serum levels of multiple cytokines and adhesion molecules in patients with newly diagnosed acute lymphoblastic leukemia using biochip array technology, Experimental oncology, 35 (2013) 229230. [44] M.B. Iravani M, Bahar B, Alimoghadam K, Moosavi A, Najar-Najafi S, Ardalan A and Ghavamzadeh A, Predictive significance of serum interleukin-10 for acute graft versus host disease prior to allogeneic bone marrow trasplantation, Acta Med Iran, 43 (2005) 6.

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Figure 1. IL-10 plasma levels in controls (CC) and ALL patients (a). Comparison of IL-10 concentration in high risk (HR) and low risk (LR) ALL groups (b) and between rs1800896 genotypes (c). Figure 2. IL-10 plasma levels in ALL patients, according to therapeutic regimen, at the time of sampling. Bars showing the mean plasma concentration of IL-10 regarding to the therapeutic regimen for the 61 ALL patients. S.E.M. as error bars. *p=0.01; ****p=0.0001.

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Figure 2

ACCEPTED MANUSCRIPT Table 1. Allele and genotype frequencies of the rs1800896 polymorphism in ALL patients and controls.

Genotypes

AA AG GG

23 31 13

34 46 20

33 32 10

AA AG + GG

23 44

34 66

33 42

Allele Carrier

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OR: Odds ratio; CI: confidence interval.

44 43 13

OR (CI 95%)

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A G

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Allele Frequency

N 77 57

1.4 (0.86 - 2.25)

44 56

p 0.18

IP

% 56 44

Control N % 98 65 52 35

SC R

ALL

1 (reference) 1.40 (0.67 - 2.90) 1.87 (0.70 - 4.98)

0.46 0.23

1.50 (0.76 - 2.97)

0.30

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Table 2. Risk association between IL-10 rs1800896 genotypes and ALL risk of relapse.

Genotypes % 35 65

N 2 9

GG

% 18 82

Low Risk High Risk

N 3 5

0.44

0.64 (0.12-3.29)

0.67

0.42 (0.07 - 2.37)

GA+AA % 38 62

N 8 21

% 28 72

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*Fisher’s exact test, p>0.05. OR: Odds ratio; CI: confidence interval.

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p value*

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OR (CI 95%)

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Groups