3′ untranslated region A>C (rs3212227) polymorphism of Interleukin 12B gene as a potential risk factor for Hodgkin’s lymphoma in Brazilian children and adolescents

Abstract

Interleukin 12 plays an important role in immunoregulation between the T helper 1/T helper 2 lymphocytes and in the antiviral and antitumor immune response. The aim of this study was to investigate the possible association between the interleukin 12B polymorphism rs3212227 and the risk to develop Hodgkin’s lymphoma in childhood and adolescents. A total of 100 patients with Hodgkin’s lymphoma and a group of 181 healthy controls were selected at random from a forensic laboratory of the University of Pernambuco. The AA genotype was detected in the controls (53.04%) and the AC genotype was found in the patients (54%). The AC genotype showed an association with the development of Hodgkin’s lymphoma (odds ratio = 2.091, 95% confidence interval = 1.240–3.523, p = 0.007). When AC + CC genotypes were analyzed together, an increase in risk of 1.9 times more chances for HL development could be observed (odds ratio = 1.923, 95% confidence interval = 1.166–3.170, p = 0.014). However, there was no association between the AC and CC genotypes of the interleukin 12B polymorphism with the clinical risk group (p = 0.992, p = 0.648, respectively). Our results suggest that the presence of the C allele may be contributing to the development of Hodgkin’s lymphoma in children and adolescents.

Keywords

Hodgkin’s lymphoma polymorphisms interleukin 12B

Introduction

Hodgkin’s lymphoma (HL) is a neoplasm that reaches the lymph nodes and lymphatic system and generally has a good prognosis in the pediatric population.^1–3 It is characterized by the large number of neoplastic cells called Hodgkin or Reed-Sternberg (HRS), and by infiltration of different types of immune system cells into the tumor microenvironment, including T and B cells.^4–6 Based on histological differences and cellular composition, classical Hodgkin lymphoma (CHL) is subdivided into four subtypes: nodular sclerosis classical Hodgkin lymphoma (NSCHL), mixed cellularity classical Hodgkin lymphoma (MCCHL), lymphocyte depletion classical Hodgkin lymphoma (LDCHL), and lymphocyte-rich classical Hodgkin lymphoma (LRCHL).^2,4–8 The clinical and pathological features of CHL result from an immune dysfunction characterized by deregulation of the expression of various cytokines and chemokines and an inadequate immune response due to the poor immunogenicity of HRS cells.^9,10 Among these cytokines are interferons (IFNs), which present a wide range of immunomodulatory activities, including the activation of natural killer (NK) cells, cytotoxic T cells, and the promotion of T helper 1 (Th1) type response.¹¹

NK cells represent a key component of the innate immune system against cancer. It was observed that patients with HL exhibit inactivated peripheral NK cells due to high serum levels of major histocompatibility complex (MHC) class I ligands (MICA A, MIC B, ULBP1-6) for the natural killer Group 2D (NKG2D) protein. NKG2D is the major NK cell receptor in the antitumor action, which belongs to the NK cell activator lectin superfamily (type C member).^12,13 According to the literature, inactivation of NK cells may be associated with NKG2D blockade by these ligands, and consequently may interfere on your antitumor function.^14–17 However, expression of NKG2D can be regulated by cytokines, such as Interleukin 12 (IL-12).^13,18–20

IL-12 is a multifunctional proinflammatory protein that acts as a key regulator of the Th1 response, through the stimulation of IFN-γ by T cells and NK.^21,22 It is produced by macrophages and dendritic cells, and plays an important role in the early stage of inflammation, but for a short period of time.^23,24 The induction to the cell to produce this cytokine is made through microbial stimuli, infections by intracellular bacteria, virus infections, and by induction of helper T cells already stimulated by these antigens.^23,24 It is the first cytokine released into an infected tissue being essential for the initiation and polarization of the immune response that is, the differentiation of Th1 and Th2 cells from non-polarized common (CD4+) precursor cells in addition to inhibiting apoptosis of Th1 cells.^21,23,25,26

The antitumor effects presented in animal models, and the various immunoregulatory activities of this interleukin, has aroused interest in this cytokine as a potential new agent to be developed in immunotherapy against cancer.^27–29

Single nucleotide polymorphisms (SNPs) have been identified at position +1188 3′ untraslated region (3′UTR) (A/C) of the IL-12B gene in several types of cancer, such as cancer of the cervix^24,30 and in HL in young adulthood.³¹

Considering the above, this study aimed to analyze the polymorphism at position +1188 3′UTR (A/C) of the IL-12B (rs3212227) as a possible risk factor for the development of HL in children and adolescents treated at a University Hospital in the Northeast of Brazil.

Materials and methods

Patients and controls

A cross-sectional study was performed comparing 100 children and adolescents (up to 19 years old) diagnosed with CHL at the Center of Pediatric Oncohematology—CEONHPE—University Oswaldo Cruz Hospital, with a group of 181 controls, comprising healthy individuals, aged 0–19 years, which were selected at random from a forensic laboratory of the University Center—Laboratory of Human Molecular Genetics of the Federal University of Pernambuco with at the same socio-geographic and ethnic origin as HL patients. Patients with a confirmed diagnosis for HIV and patients with previous treatment for HL and/or other types of neoplasms were excluded from this study.

Staging and classification of clinical risk group

Patients were stratified according to the Ann-Arbor classification system.³² In relation to the risk group, the patients were classified as favorable and unfavorable. Patients considered favorable were those presenting stage I, IIA, or IIIA, without symptoms B, with number of involved anatomic areas (IAA) < 4 and who are alive without disease. On the contrary, patients presenting stage IIB, IIIB, IV, with symptoms B, with a number of IAA ≥ 4 and who may have died or not, were classified as unfavorable.

Collection and biological samples

Ninety-one patients had clinical and biological characteristics available from medical records. The analyzed variables are described in Table 1. Biological samples consisted in peripheral blood obtained by venous puncture into vacuum tubes containing EDTA (ethylenediaminetetraacetic acid) collected from 100 alive HL patients.

Table 1.

Description of the clinical and biological characteristics of patients with HL.

Variables(measurable units)	Patients(n = 91)	%
Age at diagnosis
≤10 years	28	30.77
>10 years	63	69.23
Sex
Male	56	61.54
Female	35	38.46
Staging of Ann Arbor
I	8	8.8
II	32	35.16
III	37	40.66
IV	14	15.38
B symptoms
Yes	69	75.82
No	22	24.18
Histopathological diagnosis
Nodular sclerosis	56	61.54
Mixed cellularity	23	25.27
Lymphocyte depletion	0	0
Rich in lymphocytes	02	2.19
HL nodular lymphocytic predominance
Yes	10	11
No	81	89
Number of involved anatomic areas (IAA)
<4	55	60.44
≥4	36	39.56
Risk group
Favorable	23	25.27
Unfavorable	68	74.73

HL: Hodgkin’s lymphoma.

B symptoms: intermittent fever greater than 38°C for more than three consecutive days, nocturnal sweating, and loss of at least 10% of the body weight in the last 6 months.

DNA extraction from peripheral blood

DNA extraction from peripheral blood was performed by the “Mini Salting out” method.³³

Genotyping of the SNP rs3212227 in the IL-12B gene

Genotyping was determined using Polymerase Chain Reaction–Restriction Fragment Length Polymorphism (PCR-RFLP).³⁴

The reaction was performed in a final volume of 25 μL using approximately 200 ng of DNA, 2X Go Taq Colorless Master Mix (Promega), 2.0 mM. The primers used in the reaction were 5′-GATATCTTTGCTGTATTTGTATAGTT-3′ (sense) and 5′-AATATTTAAATAGCATGAAGGC-3′ (antisense), described by Chen et al.³⁵ At the PCR, the initial cycling was 95°C for 5 min, followed by 40 cycles of 95°C for 45 s, 53°C for 45 s, and 72°C for 45 s and a final extension of 72°C for 10 min. The PCR product was submitted to the electrophoresis on 2% agarose gel for 30 min at 100 V (Ultralum, Hamburg, Germany).

The 118 bp PCR product was digested by the enzyme TaqI (10 U/μL) for 4 h at 65°C. The digestion product was submitted to the electrophoresis on 4% agarose gel for 20 min at 200 V. Genotypes were determined as follows: AA with a fragment (118 bp); AC with three fragments (118 bp, 92 bp and 26 bp), and CC with two fragments (92 bp and 26 bp) as show in the Figure 1.

Figure 1

Genotypic representation of the IL-12B gene on 4% agarose gel from patients with HL. M = 50 bp molecular weight marker; 1–9 = children and adolescents with HL; AA = homozygote with 118 bp; AC = heterozygote with 118 bp, 92 bp, and 26 bp; CC = homozygous variable with 92 bp and 26 bp.

Statistical analysis

Data analysis was performed using the BioEstat 5.0 program.³⁶ The Chi-square test (χ²) was used to verify if the observed genotypic frequencies, adjusted to their expected, were in agreement with the Hardy–Weinberg equilibrium hypothesis. Differences in allele and genotype frequencies of the polymorphism between the patients and controls were compared using the Williams G-test at a significance level of 5%. The odds ratio (OR) with 95% confidence intervals (CI) was used to verify the association between the allelic and genotype frequencies of the polymorphism, and to determine if they are involved at risk of developing HL. The associations were considered significant when p < 0.05.

Ethical considerations

The ethical aspects are in accordance with Resolution 466/2012 of the National Health Council, where bioethical principles were respected. The project was approved by the Ethics Committee of the Hospital Complex University Hospital Oswaldo Cruz (HUOC)/Pronto Cardiovascular Relief of Pernambuco (PROCAPE) (Certificate of Presentation for Appreciation Ethics [CAAE]: 2 43297515.6.0000.5192, Opinion Number: 1,191,857).

Results

Clinical and biological characteristics

The age at diagnosis ranged from 3 to 19 years (median 12.38 years), with 69.23% (63/91) of patients over 10 years. The male: female ratio (M:F) was 1.6:1, with 61.54% (56/91) of male and 38.46% (35/91) of female patients (Table 1).

The staging of the majority of the patients was II and III, representing 35.16% (32/91) and 40.66% (37/91), respectively. The presence of B symptoms (intermittent fever greater than 38°C for more than three consecutive days, nocturnal sweating, and loss of at least 10% of the body weight) was observed in 75.82% (69/91) in patients. When the group was classified according to IAA, 60.44% (55/91) of the patients had less than four. According to histopathology, CHL was responsible for 89% (81/91) of the cases, while 11% (10/91) were classified as nodular lymphocyte predominant Hodgkin lymphoma (NLPHL). The nodular sclerosis and mixed cellularity subtypes were observed in 61.54% (56/91) and 25.27% (23/91), respectively. The distribution of the clinical risk group revealed that 25.27% (23/91) of the patients were favorable and 74.73% (68/91) were unfavorable (Table 1).

Frequency of the +1188 3′UTR (A/C) polymorphism IL-12B gene

The studied populations are in Hardy–Weinberg equilibrium (p = 0.08 and p = 0.22), respectively. Analysis of genotype frequency distributions and the OR of the polymorphism IL-12B gene in children and adolescents with HL and controls can be seen in Table 2.

Table 2.

Distribution of genotype frequencies and odds ratio of the IL-12B gene A/C polymorphism in patients with Hodgkin’s lymphoma and controls.

GenotypeIL-12B	Patientsn = 100 (%)	Controlsn = 181 (%)	p*	OR	95% CI	p**
AA	37 (37)	96 (53.04)	0.020		–	–
AC	54 (54)	67 (37.02)		2.091	1.240–3.523	0.007
CC	9 (9)	18 (9.94)		1.297	0.535–3.144	0.730
AC + CC	63 (63)	85 (46.96)		1.923	1.166–3.170	0.014

OR: odds ratio; CI: confidence interval; IL-12B: Interleukin 12B.

p*: Williams G-test; p**: odds ratio value.

Analysis of the genotype frequency of the IL-12B gene polymorphism showed that the AA genotype was found in 53.04% (96/181) of the controls, and the AC genotype in 54% (54/100) of the patients. From the OR calculation, the AC genotype showed an increase of risk of two-fold for (p = 0.007) for the lymphoma condition. While the fusion of the AC + CC genotypes showed a frequency of 63% (63/100) with 1.9-fold increment of lymphoma risk (p = 0.014) (Table 2).

Clinical risk group

The IL-12B AC genotype was the most frequent in both the unfavorable group and the favorable disease group at treatment, with 55.88% (38/68) and 56.52% (13/23), respectively. The CC mutant genotype for the IL-12B gene was more frequent in the unfavorable patient group, with 10.29% (7/68) of the cases. However, there was no statistically significant association between the IL-12B gene polymorphism and the clinical risk group (Table 3).

Table 3.

Relationship between IL-12B gene genotypes of 91 HL patients and their response to treatment.

Polymorphism	Clinical risk group		p *	OR	95% CI	p **
Polymorphism	Favorablen = 23	Unfavorablen = 68	p *	OR	95% CI	p **
IL-12B
AA	09 (39.13)	23 (33.83)		–	–	–
AC	13 (56.52)	38 (55.88)	0.642	0.874	0.323–2.365	0.992
CC	01 (4.35)	7 (10.29)		0.365	0.039–3.403	0.648
AC + CC	14 (60.87)	45 (66.17)		0.795	0.299–2.111	0.835

IL-12B: Interleukin 12B; OR: odds ratio; CI: confidence interval; HL: Hodgkin’s lymphoma.

p*: Williams G-test; p**: odds ratio value.

Discussion

In our study, we evaluated 100 children and adolescents diagnosed with HL treated at the University Hospital in Recife-Pernambuco from 0 to 19 years of age. We observed that HL frequency rates were higher in male patients (61.54%) older than 10 years at diagnosis, representing 69.23% of the cases. Spitz et al.²⁷ analyzed the epidemiological profile of 1109 patients with HL less than 20 years old and observed that the majority of patients were males aged 15–19 years.

According to the Brazilian National Cancer Institute (INCA),³⁷ there are two distinct points of increase in age distribution and the risk of developing HL: the first during adolescence and later in young adults. However, this profile is not observed in developing countries where the highest rates occur at younger ages.³⁷ Regarding B symptoms, we observed that 75.82% (69/91) of the patients presented B symptoms. In a meta-analysis, Dinand and Arya³⁸ found that 25%–30% of pediatric CHL cases in Western countries showed B symptoms. Sherief et al.³⁹ found that 39% of patients diagnosed with CHL exhibited symptoms. Thus, our results differ from those found by Dinand and Sherief, possibly because HL in childhood exhibits characteristics of epidemiological, clinical, and pathological aspects according to the various geographic areas, mainly according to the socio-economic level of the country.³⁸

In a retrospective analysis of pediatric HL cases in Brazil, Gualco et al.⁴⁰ observed that the Southeast region had a higher prevalence of cases of CHL and NLPHL with 91% and 9%, respectively, followed by the Northeast region with 84% of cases of CHL and 16% of NLPHL when compared with the other Brazilian regions. Our results show a similar frequency of number of cases in patients from Northeast Brazil. However, future studies are needed to understand this difference between these regions. Dinand and Arya³⁸ showed that in several countries, pediatric patients with HL had a predominance of mixed cellularity (MC) subtype. Oguz et al.⁴¹ found a predominance of 38.5% of MC subtype in pediatric HL patients. These results are inconsistent with those found in our study, where we found that 61.54% (56/91) of the cases belong to the nodular sclerosis (NS) subtype. However, our findings resemble those of Barros et al.⁴² and Monteiro et al.⁴³ who found a predominance of the NS subtype in pediatric patients with HL greater than 10 years of 69% and 50.8%, respectively. Although there is evidence that the NS subtype is detected in about 50% of HL cases in Brazil, it is not possible to determine the actual frequency of this histological subtype in our country. This diversity can be attributed to the age differences shown in the published studies, the scarcity of population studies divergences in the use of histological criteria for the diagnosis of HL subtype, and/or inequality in the socioeconomic level of the analyzed population, as well as to the small number of patients included in most studies published in the Brazilian regions.

Most HL treatment protocols consider stages I, IIA, and IIIA as a favorable risk group, and the others as an unfavorable prognosis.⁴⁴ Morais et al.⁴⁵ studied 69 pediatric HL patients and found that 31.9% were in the favorable disease group and 68.1% were in the unfavorable group. Similar results were observed in our study, in which 25.27% (23/91) of the cases presented favorable clinical stage and 74.73% (68/91) of the cases presented an unfavorable clinical stage.

The determination of the number of sites affected by HL as a predictor of the therapeutic response has been analyzed by some authors,^41,44 as justification that it would reflect the total tumor burden.^6,46,47 Vassilakopoulos et al.⁴⁸ observed that 32% of the patients with HL had an IAA number greater than or equal to five. Another study conducted by Morais et al.⁴⁵ found that 24.6% of pediatric patients with HL with more than five IAA, while 75.4% had less than five IAA. The findings found in these studies corroborate our results.

The identification of prognostic factors in HL is essential to facilitate the definition of risk groups, as well as the use of risk adjusted therapies,⁴⁹ since the use of exclusively clinical criteria for predicting response to treatment may fail until one-third of the patients.^47,50

IL-12 has become a promising candidate for tumor immunotherapy because of its ability to activate both NK cells and cytotoxic T lymphocytes.^49,50 Although potential anti-tumor effects of IL-12 are well established in the literature, this cytokine is considered incapable of directly inhibiting the development of cancer, although there may be some exceptions.⁵⁰ The association between polymorphism in the IL-12B gene and HL has also been reported.³¹ Cozen et al.³¹ observed a strong association between the IL-12p40 (1188 3′UTR) subunit polymorphism and the risk for HL in patients in young adulthood, providing further evidence for the role of IL-12 as a risk factor. These authors found that the presence of a variant allele was associated with a significant 2.8-fold increase in the risk of developing HL as compared with the control group.

In our study, the genotype variant AC of the IL-12B gene polymorphism was detected in 54% (54/100) of the patients with HL, and showed an increase of risk in two times more chances (p = 0.007) for the development of HL. When we analyzed the sum of the AC and CC genotypes, we observed a frequency of 63% (63/100) of the cases and an increase in risk in 1.9 times more chances (p = 0.014) for the susceptibility to HL. This fact may be associated with a greater expression of the NK receptor inhibitor that impairs the antitumor function of this cell and possibly increases the risk of development of this cancer, not acting effectively on the neutralization of neoplastic cells. In addition, two other factors may also be associated with IL-12B expression and the development of carcinogenesis.

The first factor is based on the inflammatory response by expressing IL-12B in the presence of the SNP. The polymorphism studied is located at position +1188 3′UTR (A/C). Therefore, the presence of a SNP in this region may lead to post-transcriptional alterations in IL-12B gene expression that prevents the ribosome from identifying the stop codon and continues the polypeptide chain synthesis in an uncontrolled manner, leading to high levels of this interleukin. Because it is a pro-inflammatory cytokine, its high production is related to a chronic inflammatory response, increasing the production of macrophages and IFNs that causes cytotoxic effects in the host cells, leading to cell proliferation and differentiation, favoring carcinogenesis.⁵¹

The second is based on the inflammatory response by expressing IL-12B in the presence of Epstein–Barr Virus (EBV). When EBV latent membrane protein 1 (LMP-1) infects host B cells, a chronic inflammatory response is triggered. High levels of this interleukin will be produced in response to the infection and consequently, macrophages and IFNs are also synthesized, stimulating host cell cytotoxicity, favoring carcinogenesis.⁵² During the development of this study, we had difficulty in relation to the rescue of the paraffin samples, due to the fact that they came from a laboratory outsourced by the Child Cancer Support Group. Thus, access to the information of these patients was very limited, which made it impossible to perform the analysis of EBV.

In a meta-analysis with 18 case-control studies conducted by Zhou et al.,³⁰ it was observed that the polymorphism of rs3212227 (3′UTR A → C) IL-12B may be a potential cancer risk biomarker among Asians, especially for cancer of the cervix and nasopharynx. Sima et al.⁵³ also demonstrated that the interaction of IL-12A polymorphism (rs2243115) and IL-12B (rs3212227) with increased of risk of 2.62 for the glioma development.

Liu et al.²³ observed that only one of the variants of IL-12 (IL-12A rs568408) was associated with the risk of hepatonuclear carcinoma (HCC) in the Chinese population, showing that there was no significant association of IL-12B rs3212227 A/C and risk of HCC.

This study was performed by spontaneous demand, with an average frequency of six patients per year. This fact justifies the small number of samples analyzed in this study. Another fact that reinforces the limitation of sample size is the difficulty in achieving the release of peripheral blood samples from the under-age patients, due to the fear of their mothers in authorizing the collection. Another circumstance may also be related to the same limitation, is that the majority of patients already treated, don’t return to annual periodic medical appointments. It is also attributed the difficulty of access to some medical records and absence of some information necessary for the analysis of the studied variables.

Although HL is considered a rare malignant type with bimodal age distribution, this type of neoplasia can affect any age group.³⁷ In Brazil, the guidelines for the early treatment of cancer in children and adolescents are generally established for patients up to the age of 19 years. However, this criterion may vary according to each hospital.³⁷

In this work, we decided not to stratify patients according to self-declared ethnicity due to the potential bias of this approach already reported for the Brazilian population.⁵⁴

Based on the results of this study, we suggest that there is an association between the heterozygous AC genotype and the risk for development of HL. This association was also observed when we analyzed the AC and CC genotypes together. In view of this, it is possible that the presence of the C allele may be contributing to the development of HL in the children and adolescents studied. Thus, the identification of this polymorphism may help in the stratification of patients with HL according to the risk for the disease. This is the first study to analyze this type of association in children and adolescents with HL in Brazil. However, other studies in other populations are important to investigate this association, since the antitumor mechanisms of this interleukin are not yet fully understood.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Bárbara de Oliveira Silva

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