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Non-Hodgkin lymphoma (NHL) has been associated with immunological defects, chronic inflammatory and autoimmune conditions. Given the link between immune dysfunction and NHL, genetic variants in toll-like receptors (TLRs) have been regarded as potential predictive factors of susceptibility to NHL. Adequate anti-tumoral responses are known to depend on TLR9 function, such that the use of its synthetic ligand is being targeted as a therapeutic strategy. We investigated the association between the functional rs5743836 polymorphism in the TLR9 promoter and risk for B-cell NHL and its major subtypes in three independent case–control association studies from Portugal (1160 controls, 797 patients), Italy (468 controls, 494 patients) and the US (972 controls, 868 patients). We found that the rs5743836 polymorphism was significantly overtransmitted in both Portuguese (odds ratio (OR), 1.85; P = 7.3E–9) and Italian (OR, 1.84; P = 6.0E–5) and not in the US cohort of NHL patients. Moreover, the increased transcriptional activity of TLR9 in mononuclear cells from patients harboring rs5743836 further supports a functional effect of this polymorphism on NHL susceptibility in a population-dependent manner.
Non-Hodgkin lymphoma (NHL) includes a heterogeneous group of malignant lymphoproliferative diseases whose incidence has substantially increased over the past decades in Western countries.1 Individuals with specific immune deficiencies associated with immune-suppressive therapy after transplantation, HIV infection and congenital conditions are among those with higher incidence rates of lymphoid malignancies. Additionally, clinical and experimental data consistently associated several autoimmune and chronic inflammatory disorders with increased risk of NHL.2,3
Toll-like receptors (TLRs) are widely studied members of the pattern recognition receptor family with a major role in activation and homeostasis of the immune system upon pathogen recognition.4 Among the TLRs that bind nucleic acids, TLR9 recognizes unmethylated CpG DNA motifs (present at a much higher frequency in the genomes of prokaryotes than of eukaryotes) as a ‘danger signal’ that activates the innate immune system. In humans, this receptor is expressed in plasmacytoid dendritic cells and B lymphocytes, known to have a diverse TLR repertoire, where TLR9 predominates.5 A role for TLR9 on viral, fungal, mycobacterial and Helicobacter pylori infections has been described.6–10 TLR9 (and TLR7) has been also involved in the recognition of self-nucleic acids, which is believed to have an important role in the pathogenesis of auto-immune diseases, particularly in systemic lupus erythematosus11 and psoriasis.12
In addition to self-recognition, inappropriate TLR9 activation leading to disease may also involve mechanisms of transcriptional deregulation of the TLR9 gene. Consistently, cells from different lymphoma types (mantle cell, B-cell small lymphocytic, follicular and diffuse large B-cell) have been reported to overexpress TLR9 and to show an increased proliferation upon CpG stimulation, although with a heterogeneous pattern when comparing either different lymphomas or different patients.5,13 More specifically, the highly prevalent rs5743836 polymorphism in the TLR9 gene (1237T>C), has been demonstrated to predispose to Hodgkin lymphoma, as well as to several autoimmune and chronic inflammatory diseases, including asthma and Crohn’s disease.14–18
Given the substantial weight of evidence for immune dysfunction as the underlying basis of lymphomagenesis, a genetic variant unbalancing the regulation and expression of TLR9-associated mechanisms may have a major impact upon host anti-tumoral responses and therefore, an important role in NHL pathogenesis. Epidemiological and genetic studies have shown that inflammatory and infectious conditions, particularly bacterial and viral infections, are associated with increased NHL risk,3,19–22 suggesting that chronic immune stimulation has a role in NHL development.3,21,23–26
Given the role of TLR9 in bacterial and viral recognition, and the link of the TLR9 rs5743836 polymorphism with Hodgkin lymphoma, we hypothesized that rs5743836 could also influence NHL risk. We therefore analyzed whether this polymorphism was associated with susceptibility to B-cell-derived NHL in a large-scale, case–control study.
All cases were histologically confirmed and coded according to the World Health Organization (WHO) classification. Genotype frequencies of Portuguese control individuals (n = 1160) for rs5743836 were in Hardy–Weinberg equilibrium (P>0.05). A comparison of genotype distribution between the NHL patients (n = 797, including cases of follicular lymphoma (FL) n = 240, 30.1%; diffuse large B-cell lymphoma (DLBCL) n = 200, 25.1%, marginal zone lymphoma n = 125, 15.7%, chronic lymphoid leukemia/small lymphocytic lymphoma, (CLL/SLL) n = 85, 10.7%, mantle cell lymphoma n = 82, 10.3%; B-lymphoblastic lymphoma, n = 55, 6.9%, and Burkitt lymphoma, n = 10; 1.2%) and controls revealed a positive association between the presence of rs5743836 and risk of NHL (Table 1), using the dominant (P = 7.3E–9, odds ratio (OR) = 1.85, 95% confidence interval (CI), (1.50–2.27)) or the additive (P = 1.1E–7, OR = 1.66) genetic models. Accordingly, significant associations were also observed for the major NHL subtypes, such as FL (P = 2.9E–4; OR = 1.77; 95% CI, 1.30–2.42), DLBCL (P = 7.2E–6; OR = 2.07; 95% CI, 1.50–2.85) and CLL/SLL (P = 7.06E–6; OR = 2.76; 95% CI, 1.74–4.35), thus pointing to a broad impact of rs5743836 in B-cell NHL, independently of its subtypes.
To validate our findings, two independent Caucasian populations were used: an Italian cohort (467 NHL cases, including cases of CLL/SLL, n = 242, 49.0%; FL, n = 150, 30.4%; DLBCL, n = 55, 11.1%; mantle cell lymphoma, n = 22, 4.5%; marginal zone lymphoma, n = 18, 3.6% and mucosa-associated lymphoid tissue lymphoma, n = 7, 1.4%, and 468 controls) and a US population of European ancestry (821 NHL cases, including cases of CLL/SLL, n = 201, 24.5%; FL, n = 194, 23.6%; DLBCL, n = 255, 31.1%; mantle cell lymphoma, n = 31, 3.8%; marginal zone lymphoma, n = 80, 9.7%, other B-NHL cases, n = 60, 7.3%, and 1001 controls).27 In both populations, rs5743836 frequencies for controls were in Hardy–Weinberg equilibrium (P>0.05). Whereas the association with overall NHL (with either the dominant (P = 6.0E–5; OR = 1.84; 95% CI, 1.36–2.48) or the additive (P = 1.1E–4; OR = 1.60) genetic models) or its major subtypes FL (P = 1.9E–2; OR = 1.66; 95% CI, 1.08–2.53), DLBCL (P = 1.7E–2; OR = 2.07; 95% CI, 1.13–3.81) and CLL/ SLL (P = 3.3E–4; OR = 1.91; 95% CI, 1.34–2.73) was confirmed in the Italian cohort, no association between NHL susceptibility and the presence of rs5743836 was found in the US cohort (Table 1).
There are several possible reasons for the discrepant results obtained for the association of rs5743836 with NHL. It is noteworthy that several other studies have found minor allele control frequencies of rs5743836, similar to those assessed in our European populations.8,28,29 This suggests that, despite the fact that the populations enrolled in our study shared the same population bottleneck expanding out of Europe, the decreased minor allele frequency detected in the Portuguese and Italian cohorts likely reflects a greater genetic drift in Europe.30 On the other hand, familial aggregation and twin studies for lymphoproliferative disorders have ruled out the role of highly penetrant genes in affecting risk of the majority of NHL cases.31 It is therefore likely that the additive effects of genetic variants and interactions with environmental and infectious agents may have a significant role in the differential relevance of the rs5743836 polymorphism in the risk for NHL. This hypothesis is in agreement with epidemiological and genetic studies, showing that inflammatory and infectious conditions are associated with increased NHL risk, suggesting that chronic immune stimulation has a role in NHL development.3,20–22
The rs5743836 polymorphism in TLR9 has been consistently associated with increased transcriptional activity.9,32,33 Indeed, we have also found that unstimulated mononuclear cells isolated from both healthy controls and NHL patients bearing the rs5743836 polymorphism display increased TLR9 mRNA expression (Figure 1), thereby supporting the notion that rs5743836 triggers an enhanced TLR9 function. Therefore, it is possible to predict that the complex immunological response of various cell types to CpG, involving both direct and indirect effects of TLR9, is likely deregulated in individuals carrying the rs5743836 polymorphism. In fact, it has been shown that increased B-cell proliferation upon CpG stimulation is a common feature of different NHL subtypes, even though the proliferation pattern varies among NHL subtypes and individual patients.13 Signal transduction initiated by TLR9 activation results in nuclear translocation of nuclear factor-κB34 that may enhance lymphocyte neoplastic transformation by promoting proliferation and survival of mutated cells.35 In this regard, it may be worthwhile to investigate whether the rs5743836 polymorphism displays a preferential association with lymphoma subtypes with constitutive activation of nuclear factor-κB pathways. The neoplastic process leading to the development of NHL may be usurping impairments in TLR signaling pathways to advance cancer progression, which suggests that targeting these pathways may open novel therapeutic avenues. CpG molecules are now regarded as highly promising for cancer therapy, mostly due to their direct effect on TLR9 activation on immune cell subpopulations that have an important role in anti-tumor immunity, including B cells.36 However, therapeutic applications of these CpG should be individually tailored, as individual genetic variations may affect the outcome of the TLR9 signaling pathway.
In summary, our results suggest a potential role for the host genetic background in B-cell NHL susceptibility. Altogether, and despite the fact that B-cell lymphomas are known for their ability to trigger disparate responses to TLR9 agonists,13 the genetic associations found herein were not, for the most part, subtype-specific. This suggests that altered TLR9 signaling pathways may underlie shared mechanisms of susceptibility to B-cell NHL, particularly to the subtypes where the findings were stronger, such as CLL/SLL. As a matter of fact, apoptosis of human CLL B-cells has been found to be intimately related with TLR9 activation,37 thereby suggesting that genetic variation of this receptor may also impact the outcome of TLR9 agonist therapy. However, deeper insights into the pathogenetic mechanisms underlying TLR9-mediated mechanisms in NHL are needed. Ultimately, functional studies dissecting the role of the rs5743836 may allow the identification of potential therapeutic targets.
AC, NSO, MTC and AJA were financially supported by a fellowship from Fundação para a Ciência e Tecnologia, Portugal. MS is a Ciência 2007 fellow. This study was supported by Fundação para a Ciência e Tecnologia, Portugal (PIC/IC/83313/2007) and by Fundação Calouste Gulbenkian, Serviço de Saúde e Desenvolvimento Humano, Portugal (Grant Number:Proc/60666-MM/734). CFS, PB and LC were supported by National Institutes of Health (NIH) grants CA122663 and CA104682, and PB also by NIH grants CA45614 and CA89745. We are grateful to Paulo Vieira, Cecília Leão, Manuel T Silva, Nuno Sousa, Jorge Correia-Pinto, Joana Palha, Margarida Correia-Neves, Margarida Lima and Matthew Berry for all their input throughout these studies and critical reading of the manuscript. We are grateful to the patients who joint this study, as well as to all members of the Life and Health Sciences Research Institute and School of Health Sciences, University of Minho, who contributed in any way to the development of this work.
Conflict of interest
The authors declare no conflict of interest.