The Association of Pre-diagnostic Inflammatory Markers and Adipokines and the Risk of Non-Hodgkin Lymphoma Development in Egypt

Abstract

Non-Hodgkin lymphoma (NHL) is an etiologically, clinically, and histologically heterogeneous group of lymphoproliferative disorders. Immune dysfunction is a well-known risk factor and dysregulation of cytokines may mediate disease progression. Obesity is one of the important relations connecting immune system abnormalities and lymphomagenesis. We conducted a study to find out the association between obesity as measured by body mass index (BMI), and risk of non-Hodgkin lymphoma (NHL) development by assessment the of inflammatory cytokines levels, (IL-6, IL-10, IFN-gamma and CRP) and adipokines levels (leptin and adiponectin). Also, to predict the effect of higher BMI on the incidence of NHL. The study included 180 NHL patients and 172 healthy controls. The inflammatory markers (IL-6, IL-10, IFN-γ & CRP) together with adiponectin were assessed by ELISA technique. IL-6, IL-10, CRP, IFN-γ and Adiponectin were statistically higher in cases than control. A positive significant difference of Leptin (p-value 0.001) was found with higher levels in patients with BMI (≥ 25 kg/m²) than in patients with < 25 kg/m². IL-6, IL-10, CRP, IFN-γ and Adiponectin could be implicated in lymphomagenesis in Egyptian NHL. The study results support the hypothesis that obesity has a major role in the development of NHL. An association between Leptin and NHL risk with higher levels in patients with BMI (≥ 25 kg/m²) was proved.

Introduction

Subclinical immune dysfunction is an established risk factor for NHL pathogenesis [1].Obesity is considered an important key factor connecting immune system abnormalities and lymphomagenesis. Obesity is considered a world wild health problem. It resulted due to disproportionate balance between the caloric intake and the energy exertion. Obesity cause severe morbidities, including cardiovascular diseases (CVDs), Type 2 diabetes mellitus (T2DM), and various forms of cancers. The key factor linking obesity and diseases is the excessive accumulation of visceral adipose tissue as well as its abnormal secretory function [2].

The adipose tissue is an endocrine organ.that secretes several hormones and inflammatory cytokines. Upon increasing the body mass index, the secretory profile of adipose tissue becomes disrupted and metabolic homeostasis is altered [2].

Among the hormones released from adipose tissue are the adipokines. Leptin controls the nutritional state of the human body and sends the feedback to the hypothalamus, resulting in specific neurotransmitters production [3]. Leptin stimulates the signal transduction pathways (JAK2/STAT, PI3K, and MAPK pathways). It enhances the secretion of inflammatory cytokine in macrophages, prevents cell apoptosis, and activates the invasiveness of malignant cells [4].

The development of different types of cancers is favored by the metabolic instability; dyslipidemia, hyperinsulinemia and chronic inflammation [5]. Altered JAK2/STAT pathway results in myeloproliferative neoplasms and T-cell ALL [6]. Disrupted PI3K pathway stimulates Leptin secretion which in turn inhibits cell apoptosis in NHL pathogenesis [7]. Adiponectin is an adipokine that has some anti-inflammatory properties [8]. Decreased adiponectin levels are associated with decreasedl apoptotic changes in malignant cells through suppression of the 5′-adenosine monophosphate-activated protein kinase/ mammalian target of rapamycin (AMPK/mTOR) signalling pathway [9].

The study aimed to examine the association between obesity as measured by body mass index (BMI) and risk of non-Hodgkin lymphoma (NHL) development by evaluating the role of inflammatory cytokines, (IL-6, IL-10, IFN-gamma and CRP) and adipokines (Leptin and Adiponectin) in Egyptian patients with B-NHL in comparison with healthy control.

Methodology

The present case–control study included 180 B-Cell Non-Hodgkin lymphoma (NHL) patients & 172 age & sex matching healthy controls. Patients were selected from Clinical Hematology unit, Kasr Al-Aini Hospital, Faculty of Medicine, Cairo University. The study was approved by the Research Ethical Committee of Internal Medicine department as well as Clinical & Chemical Pathology department, Faculty of Medicine, Cairo University. Informed consents were obtained from the participants prior to enrollment in the study.

Patients were Egyptians ≥ 18 years old with newly diagnosed or relapsed B-cell NHL. Diagnosis of NHL subtypes was confirmed by laboratory and/or histopathological assays.

They were subjected to Full history taking and thorough clinical examination, assessment of lymph node, organomegaly and the body mass index (BMI: kg/m²), identification of Ann Arbor Clinical staging for NHL.

Also, Pan CT was done for radiological staging confirmation, excisional biopsy of the involved lymph node or tissue biopsy of the mass for examination and immunophenotyping, bone marrow examination to assess cellularity and infiltration as well as CD20 gating for immunohistochemistry.

Blood samples were withdrawn for routine laboratory workup (Complete blood count, liver and kidney functions, LDH), and Specific marker assessment; IL-6, IL-10, IFN-γ, CRP, Leptin & Adiponectin.

Quantitative Detection of; Human IL-6 PicoKine™ ELISA Kit (Cat. Num.: EK0410), (Range: 4.69-300 pg/ml), Human IL-10 PicoKine™ ELISA Kit (Cat. Num.: EK0416), (Range: 3.4-250 pg/ml), Human IFN Gamma PicoKine™ ELISA Kit (Cat. Num.: EK0373), (Range: 15.6-1000 pg/ml), Human Leptin PicoKine™ ELISA Kit (Cat. Num.: EK0437), (Range: 62.5-4000 pg/ml), Human Adiponectin PicoKine™ ELISA Kit (Cat. Num.: 94,566), (Range: 1.56-100 ng/ml) was assayed by Sandwich ELISA kit (BOSTER BIOLOGICAL TECHNOLOGY Co., Ltd. Pleasanton, CA).

Statistical Analysis

Data were coded and entered using the statistical package SPSS (Statistical Package for the Social Sciences) version 23. Data was summarized using mean, standard deviation, median, minimum and maximum in quantitative data and using frequency (count) and relative frequency (percentage) for categorical data. Comparisons between quantitative variables were done using the non-parametric Kruskal–Wallis and Mann–Whitney tests. For comparing categorical data, Chi square (χ2) test was performed. Exact test was used instead when the expected frequency is less than 5. P-values less than 0.05 were considered as statistically significant.

Results

The study comprised 116 (64%) males and 64 (36%) females. Their age ranged between 22 and 79 years with median 55 years. There were 20 (11%) patients with BMI < 18.5, 44 (24.5%) patients with BMI 18.5–24.9, 44 (24.5%) patients with BMI 25–29.9 and 72 (40%) patients with BMI ≥ 30.

There was 96 newly diagnosed cases as well as 84 chronic cases; of them; 120 patients (66.7%) with diffuse large B-cell lymphoma (DLBCL), 30 patients (16.7%) having mucosa-associated lymphoid tissue—B variant (MALT), 25 patients (13.9%) having follicular lymphoma (FL), 3 patients (1.6%) with small lymphocytic lymphoma and 2 patients (1.1%) with MZL (Table 1).

Table 1.

The demographic data of the patients

Item	Number (180)
Gender
Male	116
Female	64
Age
< 60	68
≥ 60	112
BMI
< 18.5	20
18.5–24.9	44
25–29.9	44
> 30	72
Splenomegaly	60
Hepatomegaly	36
Disease status
Newly diagnosed	96
Chronic	84
Ann Arbor staging
I/II	56
III/IV	124
B symptoms
Absent	120
Present	60
Performance status
< 2	125
2 ≤	55
Number of extra nodal sites
< 2	88
2 ≤	92
Histopathological subtypes
DLBCL	120
MALT	30
FL	25
Small lymphocytic lymphoma	3
MZL	2
Fate
CR	96
Relapse	19
No available data	65

There was a highly significant relationship (p-value 0.001) regarding the inflammatory markers between NHL patients and their corresponding controls, IL-6 (31.09 ± 3.48) in patients versus IL-6 (3.77 ± 0.40) in controls, IL-10 (18.72 ± 2.20) in patients versus IL-10 (3.89 ± 0.29) in controls, IFN-γ (7.08 ± 0.31) in patients versus IFN-γ (3.71 ± 0.17) in controls and CRP (35.26 ± 3.19) in patients versus CRP (3.46 ± 0.82) in controls (Table 2).

Table 2.

Inflammatory and adiposity markers levels between between B-NHL cases and healthy control

	Inflammatory markers				Adiposity markers
	IL6 (pg/ml)	IL10 (pg/ml)	IFN-γ (pg/ml)	CRP (mg/l)	Lep (pg/ml)	APN (ng/ml)
Cases	31.09 ± 3.48	18.72 ± 2.20	7.08 ± 0.31	35.26 ± 3.19	4182.30 ± 246.95	468.92 ± 6.47
Control	3.77 ± 0.40	3.89 ± 0.29	3.71 ± 0.17	3.46 ± 0.82	4782.00 ± 193.65	412.27 ± 13.36
p-value	0.001	0.001	0.001	0.001	0.061	0.002

IL6 interleukin-6, IL10 interleukin-10, IFN-γ interferon-γ, CRP C-reactive protein

p-value <0.05 is statistically significant

Regarding the adiposity markers, there was a highly significant relationship (p-value 0.002) between patient’s adiponectin (468.92 ± 6.47) and controls adiponectin (412.27 ± 13.36). There was an insignificant relationship (p-value 0.002) between patient’s leptin (4182.30 ± 246.95) and controls leptin (4782.00 ± 193.65) (Table 2).

Regarding sex, there was no significant differences between males and females neither in the inflammatory markers nor in Adiponectin. But leptin was found higher in females compared to males with significant p-value 0.001 (data not shown).

Regarding (BMI) as an indicator of obesity, IL-6 and leptin were found higher in obese patients (≥ 25 kg/m²) compared to non-obese (<25 kg/m²) with significant p-value 0.001 (data not shown).

The different pathological type of lymphoma and the disease status did not affect neither the level of the inflammatory markers nor the adipokines markers.

Regarding the Ann Arbor staging, there was no significant differences between different staging grades neither in the inflammatory markers nor in Leptin. But APN was significantly higher in stage IV NHL patients with p-value 0.015 (data not shown).

Discussion

NHL is one of the most commonly diagnosed cancers in the Western world. Over the past few decades the incidence of NHL has increased dramatically. Factors affecting an individual’s risk of developing non-Hodgkin lymphoma have been extensively studied. These factors include immune disorders, medicines, infections, lifestyle, genetics, race, family history, and occupational factors [10]. Immune dysfunction, obesity & chronic inflammation are thought to underlie the development of NHL. Several studies were designed to explore the association between obesity and cancer, but the exact mechanisms are still unclear [11].

Basically, the focus is on three hormonal systems that include insulin and insulin-like growth factor (IGF) axis, sex steroids and adipokines. Obesity-related inflammatory cytokines, genetic background, obesity-related hypoxia, and oxidative stress were also noted as possible causes that may have a role in the occurrence and development of NHL [12].

The present study included 180 B-NHL patients and 172 healthy control, the inflammatory markers (Il-6, Il-10, INF-gamma & CRP) and adipokines (Leptin & Adiponectin) were assayed in B-NHL patients and compared to healthy control regarding their levels as well as their correlation to BMI to judge their impact in the development of B-NHL in Egyptians.

The hypothesis that prediagnostic levels of adipokines have a role in the development of NHL was previously discussed by Conroy et al. [13] who tried to trace the association of diet, lifestyle, and genetic factors with the incidence of cancer in a prospective cohort study of 67,594 participants. They recruited only 272 patient’s samples that were withdrawn before NHL diagnosis in their nested case–control analysis study. They concluded that, in a period of 2.7 years from sample withdrawal to NHL diagnosis, IL-10, IL-8, TNF-α and leptin were significantly correlated to NHL incidence. On the other hand, IFN-g, IL-1b, IL-2, IL-4, IL-5, IL-6, CRP and adiponectin were not associated with NHL incidence [13].

There was a significant elevation of serum level of IL-6 in studied cases (p-value 0.001). In agreement with Hadi et al. [14] who found a significant difference in the serum IL-6 levels between NHL patients and controls (p-value 0.0001). Lymphoma cells themselves and reactive lymphocytes in lymphomatous nodes can produce IL-6 [15]. On the contrary, Purudue et al. [16] and Conroy et al. [13] didn’t find significant association between IL-6 and NHL risk.

Regarding IL-10, there was a significant elevation of serum level of IL-10 in studied cases (p-value 0.001). This was in aggreement with Guney et al. [17], Hadi et al. [14] who found a significant difference in the serum IL-10 levels between NHL patients and controls (p-value 0.001, p-value 0.038) respectively. Also, Conroy et al. [14] reported high IL-10 as a risk factor for NHL. The role of IL-10 in NHL pathogenesis is unclear. It is a critical mediator of Th1/Th2 balance, apoptosis potential, and regulation of inflammation. It may promote lymphomagenesis through the proliferation of B-cells and, thereby increase the likelihood of DNA-modifications, such as chromosomal translocations and oncogene mutations [18]. Voorzanger et al. [19] reported that elevated IL-10 levels observed among NHL patients appeared to be caused by increased expression from lymphoma cells and stimulated lymphoma proliferation, suggesting that IL-10 is an autocrine lymphoma growth factor.

There was a significant elevation of serum level of IFN-γ in studied cases (p-value 0.001). Our findings came in agreement with Chen et al. [20] who suggested that the IFN-γ transduction pathway could play a role in the relationship between BMI and risk of NHL. On the other side Saberi et al. [21] found low expression of Th1 cytokines, including IFN-γ, in NHL cases compared with controls.

There was a significant elevation of serum level of CRP in studied cases (p-value 0.001). this positive significant relationship came in agreement with Shevappa et al. [22] research who had explored the association between the dietary inflammatory index and NHL risk and suggested a positive association between CRP levels and NHL risk. Conversely, previous studies failed to prove a positive relation between CRP levels and risk of NHL development [13, 16].

The positive significant association between the inflammatory markers and NHL incidence might be due to the up-regulation of proinflammatory and antiapoptotic signals, via the nuclear transcription factor (NF)-κB pathway, thus promoting lymphomagenesis. Also alterations in pro inflammatory cytokines played an important role in the pathogenesis of NHL [23].

In this study, Leptin was apparently higher in controls rather than in NHL patient (p-value 0.61). Conroy et al. [13] studied Leptin as one of the cytokines related to NHL development found inverse association between leptin and NHL with 50% reduction in NHL risk. He suggested that, through its immunomodulatory effect, Leptin may enhance the immune response, resulting in a more effective immunosurveillance by promoting NK-cell cytotoxicity and a switch to Th1-cell immune response [24]. A previous hypothesis suggested that Leptin may mediate the positive obesity-NHL association [25]. Another study supported the role of leptin in NHL etiology [26]. The negative relationship between Leptin and NHL in the working study may be explained as some cases had low BMI.

Adiponectin was positively associated to NHL risk in the current study. There is a controversy in the literatures regarding serum adiponectin as a predictive marker of hematological malignancies from “lymphoid” origin [27].

Petridou et al. [28] studied Adiponectin serum levels in NHL patient and healthy controls and found elevated serum adiponectin level which was associated with childhood NHL and poor prognosis, suggesting that Adiponectin may represent not only a potential clinically significant diagnostic and prognostic marker but also a molecule that may be implicated in NHL pathogenesis. Also, an observational study evaluated Adiponectin in newly diagnosed lymphoproliferative disorders. It proved significantly higher Adiponectin level In NHL patients than in CLL and controls groups [29].

On the other side, Dalamaga et al. [27] and Conroy et al. [13] found no significant association between adipokines and risk of B-cell chronic lymphocytic leukemia (B-CLL) and B-NHL.

Wei et al. [30] tried to correlate between circulating Adiponectin levels and cancer development. He reporteded that hypoadiponectinemia has been associated with risk of many types of cancers.

The high prevelence of HCV in Egypt commonly deteriorates the liver functions by cirrhosis and virus-related hepatic malignancies. The affected liver improperly degrade the Adiponectin, leading to higher elevation in its levels among the Egyptian NHL patients.

Different variables affecting cytokines in B-NHL patients were studied. Female gender affected Leptin level significantly (p-value 0.001). Hellström et al. [31] observed higher serum leptin levels in women and confirmed that plasma leptin is related to BMI. Another study conducted by Kelly et al. [32] who, evaluated the association of BMI during early adulthood with risk of NHL in correlation to gender. There was a significant evidence of gender difference in serum Leptin levels, with higher levels in female. But, Conroy et al., mentioned no substantial evidence of sex modification on Leptin levels [13].

NHL subtype stratification revealed significant high IL-6 levels (p-value 0.006) in follicular lymphoma. This comes in agreement with Conroy et al. [13] who mentioned that IL-6 was associated with an elevated risk for follicular lymphoma specifically.

Regarding BMI, patients were sub-grouped into; group A consisted of 16 patients with BMI < 25 kg/m² and Group B consisted of 29 patients with BMI ≥ 25 kg/m². Levels of inflammatory & adiposity markers in both groups were studied.

The inverse association that was found between Leptin and NHL risk was slightly strengthened (p-value was 0.61 and became 0.001) with higher levels in the second group with BMI ≥ 25 kg/m². This finding is supported by Conroy et al., who reported that BMI significantly modified the association of Leptin with NHL [13].

Additionally, unexpected significant increase in IL-6 levels in non-obese patients was found (p-value 0.001). CRP, Il-10, IFN-γ & Adiponectin were not statistically correlated to BMI.

There is mounting evidence that obesity is associated with an increased risk for the lymphoma development. Patel et al. [33] stated that obese individuals (BMI ≥ 30 kg/m²) had 23% higher risk of NHL incidence compared to those with normal weight (BMI 18.5- < 25 kg/m²). Also, a prospective analysis studied the relation between body size and risk of NHL during childhood, adolescence, and adulthood. It proved that larger body size in in all age groups predicts increased risk of NHL, particularly of DLBCL and follicular lymphoma [34].

Additionally, a meta-analysis observed the influence of obesity on FL incidence, has shown a significant direct association between obesity and increased risk for the development of FL [35].

Obesity-related inflammation is thought to result from the proinflammatory cytokines and chemokines that are produced by adipocytes and macrophages in adipose tissue [36]. With weight gain, the numbers of adipocytes and macrophages increase as adipose tissue expands, increasing production of cytokines such as, Leptin, IL6, as well as chemokines and acute phase proteins [36].

In this study, the stage of the disease did not affect the levels of inflammatory or adiposity markers. Only, late stage of NHL (stage VI) was associated with higher level of Adiponectin (p-value 0.014). No significant difference between denovo and relapse diseases regarding inflammatory markers as well as adiposity markers.

In conclusion, the study findings support the hypothesis that obesity has a major role in the development of NHL. Elevated levels of Leptin are associated with higher risk of NHL incidence in increased BMI (≥ 25 kg/m²).

Further studies including larger number of patients with repeated markers measurements over a longer period of time are required to clarify the biological mechanism that underlies the observed associations between obesity and lymphoma as well as to prove further important associations between these immune system molecules and NHL pathogenesis.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.