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Original article
Analysis of associations between polymorphisms within genes coding for tumour necrosis factor (TNF)-alpha and TNF receptors and responsiveness to TNF-alpha blockers in patients with rheumatoid arthritis Jerzy Swierkot a,∗ , Katarzyna Bogunia-Kubik b , Beata Nowak c,d , Katarzyna Bialowas a , Lucyna Korman a , Katarzyna Gebura b , Katarzyna Kolossa e , Slawomir Jeka e , Piotr Wiland a a
Department of Rheumatology and Internal Medicine, Wroclaw Medical University, Borowska 213, 53114 Wroclaw, Poland Laboratory of Clinical Immunogenetics and Pharmacogenetics, L. Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland c Department of Pharmacology, Wroclaw Medical University, Wroclaw, Poland d Department of Rheumatology and Internal Medicine, Wroclaw University Hospital, Wroclaw, Poland e Clinical Department of Rheumatology and Connective Tissue Diseases, Hospital University Number 2 Jana Biziela Bydgoszcz, Wroclaw, Poland b
a r t i c l e
i n f o
Article history: Accepted 23 August 2014 Available online xxx
a b s t r a c t Introduction: Despite the fact that therapy with TNF-␣ inhibitors constitutes a breakthrough in rheumatoid arthritis management, no improvement is still achieved in approximately 30% of cases. The aim of the study was to evaluate whether single nucleotide polymorphisms (SNPs) within the TNF-␣ and TNF receptor encoding genes affect the efficacy of therapy with TNF-␣ inhibitors in patients with RA. Methods: Five SNPs within the TNF-␣ and TNF receptor encoding genes (TNFA: G-308A, G-238A, C-857T; TNFR1A G36A; TNFR1B T676G) were determined in 280 RA patients who had been treated with TNF-␣ inhibitors for at least 6 months or they stop therapy because of adverse events. The association between the relative change in DAS28 and SNP genotypes was tested by linear regression. Results: At week 24, low disease activity or remission was achieved by 45% of the patients. After 6 months remission of the disease or low disease activity were more frequently observed among patients homozygous for the TNFR1A 36A allele than among those who were GG homozygotes (52% vs. 34%, P = 0.04). At week 24 DAS28 was significantly lower in the subgroup of patients homozygous for the TNFA-857T variant compared to the C allele carriers (P = 0.045). The other polymorphisms were not found to be significantly associated with EULAR response at week 12 and 24 of the anti-TNF treatment. Conclusions: Homozygosity for the TNFR1A 36A allele and the TNFA-875T variant could act as a genetic factor associated with better response to anti-TNF treatment. © 2014 Société franc¸aise de rhumatologie. Published by Elsevier Masson SAS. All rights reserved.
1. Introduction Knowledge on rheumatoid arthritis (RA) aetiopathogenesis and immunological disorders has been recently expanded, which brought some modern therapeutic options. Basic medications used in RA therapy are still conventional synthetic diseasemodifying anti-rheumatic drugs (csDMARDs). A therapy with biological agents should be introduced in patients whose response to methotrexate (MTX) or other csDMARDs is insufficient and high
∗ Corresponding author. E-mail address: [email protected] (J. Swierkot).
activity of the disease persists. The first biological drugs registered several years ago for RA therapy were tumour necrosis factor alpha (TNF-␣) inhibitors. Considering the significant costs associated with biological therapy, individual countries and rheumatology associations define indications for its use in RA patients. Despite the fact that therapy with TNF-␣ inhibitors constitutes a breakthrough in RA management, no improvement is still achieved in approximately 30% of cases, and another 20% of patients discontinue the therapy because of side effects. There is an ongoing search for biochemical and clinical markers that would allow prediction of a good response to therapy with biologicals, including TNF-␣ inhibitors. Besides clinical factors, genetic predisposition may also be helpful in that prediction. A conviction of the importance of individualized
http://dx.doi.org/10.1016/j.jbspin.2014.08.006 1297-319X/© 2014 Société franc¸aise de rhumatologie. Published by Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Swierkot J, et al. Analysis of associations between polymorphisms within genes coding for tumour necrosis factor (TNF)-alpha and TNF receptors and responsiveness to TNF-alpha blockers in patients with rheumatoid arthritis. Joint Bone Spine (2014), http://dx.doi.org/10.1016/j.jbspin.2014.08.006
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2 Table 1 Patients’ characteristics. RA patients (n = )
280
Age (years) Females (%) Disease duration (years) Disease onset (years) Current smokers (%) RF+ (%) ACPA+ (%) DAS28 at baseline DAS28 at week 24 Anti-TNF drug Etanercept (%) Adalimumab (%) Infliximab (%) Certolizumab pegol (%) Glucocorticosteroids (%) Methotrexate (%)
50 ± 12.3 (range: 17–78) 79 18 ± 8.26 (range 1–44) 29.5 ± 12.1 (range 12–71) 18 65 84 6.53 ± 0.63 (range 5.14–8.69) 3.41 ± 1.10 (range 0.49–7.60) 52 34 9 5 92 (mean dose 6.6 mg prednisone daily) 92.5 (mean dose 22 mg weekly)
RA: rheumatoid arthritis; RF: rheumatoid factor; ACPA: anti-citrullinated protein antibodies; DAS 28: disease activity score 28; TNF: tumour necrosis factor.
pharmacotherapy becomes more and more common. Differences in genetic conditioning may influence the effect of a drug, both by a change in its pharmacokinetics and modification of its pharmacodynamic properties. Polymorphisms of genes coding TNF-␣ and its receptors may affect the efficacy of a drug and frequency of associated adverse effects. Based on published study results it is difficult to determine unequivocally which of the polymorphisms play the most important role and could be a good factor facilitating evaluation of efficacy and risk of adverse effects associated with therapy with TNF-␣ inhibitors. The aim of the study was to evaluate whether single nucleotide polymorphisms (SNPs) within the TNF-␣ (TNFA G-308A [rs1800629], TNFA G-238A [rs3615525], TNFA C-857T [rs1799724]) and TNF receptor (TNFR1A G36A [rs767455], TNFR1B T676G, Met196Arg [rs1061622]) encoding genes affect the efficacy of therapy with TNF-␣ inhibitors in patients with RA. 2. Methods 2.1. Characteristics of RA patients Three hundred and four RA patients of Caucasian origin who had been treated with recommended doses of TNF-␣ inhibitors (adalimumab [ADA], etanercept [ETA], infliximab [INF], certolizumab [CER]) for at least 6 months or who had stopped therapy because of adverse events were investigated. Patients enrolled in the study were men and women ≥ 18 years of age with active, adult-onset RA as defined by the 1987 revised criteria of the American College of Rheumatology (ACR) (the first patients were qualified for the study in January 2008). The mean disease duration was 18 (range: 1–44) years. All the patients provided written informed consent. The study was approved by the Wroclaw Medical University Ethics Committee. The patients’ characteristics are presented in Table 1. Twentyfour patients were excluded from the analysis due to irregular reporting to follow-up visits and incomplete data regarding activity of the disease and adverse effects. Two hundred and eighty patients, 221 females (79%) and 59 males (21%), were qualified for the final analysis. During the follow-up period, 11 patients (3.9%) discontinued therapy with TNF-␣ inhibitors, 5 (1.8%) because of side effects and 6 (2.1%) because of lack of improvement. The following inclusion criteria were accepted: consent to participate in the study; confirmed RA based on 1987 classification criteria of the American College of Rheumatology; active form of the disease–disease activity score based on erythrocyte sedimentation rate and an evaluation of 28 joints (DAS28) ≥ 5.1; failure of
treatment with at least 2 csDMARDs; over 18 years of age; women and men with reproductive potential had to use reliable contraception. There were the following exclusion criteria: pregnancy or breastfeeding; coexistence of other systemic diseases of connective tissue besides RA; clinically significant impairment of hepatic and renal function; alcohol abuse; infection with hepatotropic viruses; infections resistant to therapy; ongoing history of cancer if no cure was achieved; uncontrolled diabetes; patient unwilling or unable to cooperate. Patients were administered recommended doses of TNF-␣ inhibitors: 3 mg/kg body weight of INF, which was given as an intravenous infusion at weeks 0, 2, and 6, and every 8 weeks thereafter, subcutaneous injection of ADA at 40 mg every other week, subcutaneous injection of ETA at 50 mg every week and subcutaneous CER pegol 400 mg at week 0, 2, 4 and 200 mg every 2 weeks thereafter. The patients were allowed to continue treatment with csDMARDs, glucocorticoids, and/or non-steroidal anti-inflammatory drugs, if the treatment regimens were not modified for 4 weeks before the study. In total, 92.5% of patients were treated with a stable dose of methotrexate (mean dose: 22 mg weekly) and 92% patients were treated with glucocorticoids (mean dose: 6.6 mg daily of prednisone). Twenty-one patients (7.5%) were not treated with MTX. Among them, 11 (4%) were treated with biologic monotherapy (8 with ETA (5.5% of all ETA patients)) and 3 with ADA (3% of all ADA patients)) while remaining 10 patients (6 ETA, 4 ADA) were treated with another DMARD (5 leflunomide, 5 sulfasalazine). All patients treated with INF and CER were treated with MTX. To examine the response to anti-TNF therapy in RA, blood samples, laboratory data, and clinical data were collected at baseline (prior to anti-TNF therapy) and at 3 and 6 months after treatment. Clinical evaluation was based on medical history, number of painful and swollen joints, pain intensity assessed by the patient on a 100mm visual analogue scale (VAS) and laboratory tests (erythrocyte sedimentation rate [ESR], C-reactive protein [CRP]). The parameters allowed determination of improvement according to the criteria based on DAS28 suggested by the European League Against Rheumatism (EULAR). According to the EULAR definition, patients were classified as good, moderate, or non-responders, using the individual amount of change in the DAS28 (DAS28) and DAS28 values at 3 and 6 months. Moreover, in some analyses a comparison was made between non-responders versus patients in remission or low disease activity according to the EULAR response criteria. This stratification was due to the limited number of patients in each group. Safety of anti-TNF therapy was analysed on the basis of medical history, physical examination, and selected laboratory tests (including blood cell count, aspartate aminotransferase [AST], alanine aminotransferase [ALT], creatinine, urea levels, and urinalysis). The clinical and laboratory tests were completed before the start of the therapy and in months 3 and 6 of the follow-up. Outcomes were parameters of efficacy of TNF treatment and adverse events.
2.2. Polymorphism study DNA was extracted from peripheral blood taken on EDTA using Maxwell 16 Blood DNA Purification Kit (Promega Corp., Madison, WI, USA) following the recommendation of the manufacturer. Genetic polymorphisms within the TNFA promoter (rs1800629, G308A; rs3615525, G-238A; rs1799724, C-857T), TNFR1A (rs767455, G36A) and TNFR1B (rs1061622, T676G) were analysed using a polymerase chain reaction (PCR) amplification employing LightSNiP assays designed by TIB MOLBIOL (GmbH, Berlin, Germany). PCR
Please cite this article in press as: Swierkot J, et al. Analysis of associations between polymorphisms within genes coding for tumour necrosis factor (TNF)-alpha and TNF receptors and responsiveness to TNF-alpha blockers in patients with rheumatoid arthritis. Joint Bone Spine (2014), http://dx.doi.org/10.1016/j.jbspin.2014.08.006
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amplifications and analysis of the typing results were performed using a Roche LightCycler 480 instrument.
Table 2 Distribution of TNFA and TNFR1A, TNRF1B alleles and genotypes in RA patients. Gene/Polymorphism
2.3. Statistical analysis The association between the relative change in DAS28 and SNP genotypes was tested by linear regression. In addition, Chi2 test was applied to compare genotypes in non-responders versus patients in remission or low disease activity according to the EULAR response criteria. Statistical analysis was performed with STATISTICA (data analysis software system), version 10 (StatSoft, Inc. 2011). 3. Results 3.1. Response to treatment Clinical data of 280 Caucasians patients with RA treated with TNF-␣ inhibitors were analysed. Among them, 52% were treated with ETA, 34% with ADA, 9% with INF and 5% with CER (Table 1). Mean DAS28 at the onset of biologic treatment was 6.53 ± 0.63 (range 5.14–8.69). No difference in DAS28 values (ETA–6.51 ± 0.66, ADA–6.54 ± 0.63, INF–6.56 ± 0.51, CER–6.70 ±0.40; P = 0.1390) was detected between subgroups treated with different biologics. There was no difference between patients carrying the various alleles or genotypes of the SNPs studied. Mean DAS28 after 24 weeks of treatment was 3.41 ± 1.10 (range 0.49–7.6). After 24 weeks DAS28 was significantly higher in the subgroup treated with INF (P = 0.0045). There was no difference in disease activity as assessed by DAS28 between other subgroups (ETA–3.29, ADA–3.43, INF–4.13, CER–3.22). A moderate EULAR response was achieved in 69% of patients, while a good EULAR response was achieved in 23% of patients at 3 months. At week 24, low disease activity or remission was achieved by 45% of the patients (ETA–54.8%, ADA–37.9%, CER–42.9%, INF–17.4%). No response according to EULAR or loss of response was observed in 24.4%. We have further analyzed the ACPA (+) patients and compared with the ACPA (–) group. Remission after 24 weeks was achieved by 50.4% ACPA (+) vs. 33% of ACPA (–) (P = 0.0237) although there was no statistically significant difference in disease activity at baseline. 3.2. Genotyping study Distributions of the TNFA (G-238A; G-308A; C-857T), TNFR1A (G36A) and TNFR1B (T676G) alleles and genotypes in patients with RA are presented in Table 2. These distributions were consistent with data from public databases for Caucasians (http://www.ncbi.nlm.nih.gov/projects/ SNP). None of the polymorphism studied was found to be associated with predisposition to RA. 3.3. Association of the TNFR1A and TNFA polymorphisms with response to TNF-˛ inhibitors Among the SNPs studied, only the TNFR1A (rs767455, G36A) and one of the TNFA (rs1799724, C-857T) promoter polymorphisms were found to be associated with response to anti-TNF therapy. It appeared that significantly more patients with the homozygous TNFR1A36 AA genotype achieved a good EULAR response at month 3 compared to patients carrying the G allele (P = 0.011). After 6 months remission of the disease or low disease activity were also more frequently observed among patients homozygous for the TNFR1A 36A allele than among those who were GG homozygotes (52% vs. 34%, P = 0.04, Table 3). Analyzing the data for the ACPA (+) patients only we obtained similar results as for the whole population. In the ACPA (+) group, remission of the disease or low disease activity were also more frequently observed among
3
RA patients Genotype/allele distribution n
TNFA G-238A (rs3615525) GG GA AA G A TNFA G-308A (rs1800629) GG GA AA G A TNFA C-857T (rs1799724) CC CT TT C T TNFR1A G36A (rs767455) GG GA AA G A TNFR1B T676G (rs1061622) TT TG GG T G
Allelic frequencies
[%]
(N = 278) 265 6 7 271 13 (N = 278)
95.3 2.2 2.5 97.5 4.7
G A
0.964 0.036
189 85 4 274 89 (N = 279)
68.0 30.6 1.4 97.5 32.0
G A
0.833 0.167
193 80 6 273 86 (N = 276)
69.2 28.7 2.1 97.8 30.8
C T
0.835 0.165
58 145 73 203 218 (N = 278)
21.0 52.5 26.4 73.5 79.0
G A
0.482 0.517
168 97 13 265 120
60.4 34.9 4.7 95.3 40.2
T G
0.779 0.221
N: number of patients studied for a given SNP; n: number of patients with a particular allele/genotype; [%]: percentage of patients with a particular allele/genotype.
patients homozygous for the TNFR1A 36A allele than among those who were GG homozygotes (58% vs. 38%, P = 0.0384). This additionally confirms the beneficial effect of the TNF1A 36AA homozygosity. The difference in the remission rate was not secondary to the differences in distribution of the alleles in subgroups treated with different biologics. Moreover, failure of therapy, identified as the lack of at least moderate improvement according to EULAR, was observed in 33% of patients with the TNFR1A GG genotype and in 19% of patients with the A allele (AA and GA genotypes) (P = 0.02). At week 24, DAS28 was significantly lower in the subgroup of patients homozygous for the TNFA -857T variant (DAS28–2.05) compared to the C allele carriers (DAS28–3.41) (P = 0.012), although the remission rate was not higher in this subgroup. The other polymorphisms were not found to be significantly associated with EULAR response at week 12 and 24 of the anti-TNF treatment. No significant association was found between the polymorphisms studied and the occurrence of adverse side effects. 4. Discussion Major genetic effects on the anti-TNF response might be expected to arise from variation within genes implicated in the TNF-␣ pathway. Candidate gene studies until now have had limited success and no single gene influencing the anti-TNF response in RA has been definitively identified [1]. Pharmacogenetic studies of the influence of the TNFA gene polymorphisms on response to
Please cite this article in press as: Swierkot J, et al. Analysis of associations between polymorphisms within genes coding for tumour necrosis factor (TNF)-alpha and TNF receptors and responsiveness to TNF-alpha blockers in patients with rheumatoid arthritis. Joint Bone Spine (2014), http://dx.doi.org/10.1016/j.jbspin.2014.08.006
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Table 3 TNFA, TNFR1A and TNFR1B in RA patients characterized with different response to therapy with TNF- inhibitors at week 24 after treatment initiation. Gene/Polymorphism
Geno-type
EULAR remission/low disease activity at week 24 [n]
Non-responders at week 24 [n]
TNFA G-308A (rs1800629) N = 278
AG
36 (42%)
21 (25%)
GG
AA
TNFA G-238A (rs3615525) N = 278
GG
AA
AG
TNFA C-857T (rs1799724) N = 279
CT
CC
TT
TNFR1A G36A (rs767455) N = 276
ETA 22
INF 0
CER 2
ADA 5
ETA 9
INF 6
CER 1
91 (49%) ADA 30
ETA 55
INF 2
CER 4
35 (19%) ADA 10
ETA 12
INF 11
CER 2
2 (50%) ADA 1
ETA 1
INF 0
CER 0
2 (50%) ADA 2
ETA 0
INF 0
CER 0
122 (46%)
ETA 70
INF 5
CER 5
ADA 16
ETA 23
INF 14
CER 4
3 (43%) ADA 0
ETA 3
INF 0
CER 0
2 (29%) ADA 1
ETA 1
INF 0
CER 0
5 (83%) ADA 0
ETA 5
INF 0
CER 0
1 (17%) ADA 0
ETA 0
INF 1
CER 0
38 (47.5%)
ETA 25
INF 2
CER 2
ADA 3
ETA 3
INF 8
CER 1
89 (46%) ADA 31
ETA 52
INF 3
CER 3
44 (23%) ADA 16
ETA 19
INF 2
CER 7
3 (50%) ADA 2
ETA 1
INF 0
CER 0
1 (17%) ADA 0
ETA 1
INF 0
CER 0
ETA 10
INF 3
CER 1
ETA 12
INF 8
CER 1
ETA 4
INF 5
CER 1
GA
ADA 5 69 (48%) ADA 23
GT
GG
15 (19%)
ADA 9
20 (34%)a
TT
57 (22%)
ADA 42
GG
AA
TNFR1B T676G (rs1061622) N = 278
ADA 12
38 (52%)a ADA 13
19 (33%)b
ETA 14
INF 0
CER 1
ETA 41
INF 2
CER 3
ADA 5 28 (19%)b ADA 7
ETA 20
INF 3
CER 2
13 (18%)b ADA 3
80 (48%)
40 (24%)
ADA 30
ETA 46
INF 1
CER 3
ADA 12
ETA 19
INF 7
CER 2
43 (44%) ADA 9
ETA 29
INF 3
CER 2
18 (19%) ADA 5
ETA 4
INF 9
CER 0
7 (54%) ADA 3
ETA 3
INF 1
CER 0
2 (15%) ADA 1
ETA 1
INF 0
CER 0
N: number of patients studied for a given SNP (the % of patients with remission/low disease activity + non-responders is not 100% because there were also patients with moderate response to treatment); n: number of patients with a given genotype; % percentage of patients with a given genotype; ADA: adalimumab; ETA: etanercept; INF: infliximab; CER: certolizumab. a P = 0.04 (TNFR1A: AA vs. GG). b P = 0.02 (GG vs. AA + GA).
TNF blocker treatment have given conflicting results. Many explanations may account for such discrepancies as the differences in: the number of subjects, study design, criteria used in qualifying for biological treatment, response criteria chosen as primary endpoint and duration of follow-up, concomitant medication and the
ethnic origin of the patients [2–4]. Most studies of the TNFA promoter have focused on separate analysis of selected SNPs, mainly on the TNFA G-308A polymorphism. In some studies, the authors have suggested that patients carrying the -308A allele express higher levels of TNF-␣ [5] and that subjects with a high response to TNF
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blockers could also have high TNF-␣ bioactivity [6] or high synovial levels of this cytokine at baseline [7]. Poorer responders might be those who produce low levels of TNF-␣. Two small studies reported an association of the TNFA-308GG genotype with better response to anti-TNF treatment with INF [8] or ETA [3]. In another study involving 88 patients with rheumatic diseases, the TNFA -308GG genotype was found to be associated with a better response to TNF blockers at week 24 [4]. No association of the TNFA G-308A polymorphism with response to ETA was found in a study of 70 Asian patients [9]. Also, in three other studies involving larger sample sizes of patients treated with ETA (n = 151) [2], with INF (n = 198) [10] and with ADA (n = 381) [11], no association with the TNFA G-308A polymorphism was observed. Also, in our present study we did not observe an association between the response to anti-TNF therapy and this SNP. Five further meta-analyses showed inconsistent results of the impact of this polymorphism on the efficacy of treatment with TNF-␣ blockers. Three of them suggested that patients with RA who carried the rare A allele showed a worse response to anti-TNF therapy [12,13]. This was not confirmed in two larger meta-analyses. The results of 13 studies were included in the meta-analysis carried out by Lee et al. [15]. In this analysis, studies with a small number of subjects (< 100) showed that the odds ratio for the A allele carrier state was significantly lower among responders, but studies with a higher number of subjects (> or = 100) found no association between the TNFA G-308A polymorphism and responsiveness to TNF-␣ blockers. Furthermore, a larger meta-analysis on a total of 2576 patients also did not show any association between this SNP and the response to anti-TNF treatment [16]. Probably the data on the impact on the TNFA G-308A SNP are not conclusive because there are differences in study design, and many of them are limited by the small patient numbers and the low frequency of the TNF–308AA genotype, which is particularly rare. Zeng et al. suggested that it is possible that a poor response among carriers of the A variant might not be specific to anti-TNF-␣ agents, but could merely reflect a more aggressive manifestation of the disease in these patients and/or their poorer response to any therapeutic regimen [14]. The importance of the other TNFA polymorphisms was evaluated in subsequent studies. Eleven SNPs in the TNFA promoter region were analysed in 70 representatives of the Asian population [9]. In the study by Kange et al., patients homozygous for the TNFA857 C allele were poor responders to ETA. In our study, at week 24 DAS28 was significantly lower in the patients carrying the TNFA857TT homozygous genotype, which could indicate a more effective treatment. However, no statistically significant differences were observed for this SNP when analysing the effectiveness of response according to EULAR. As for the other polymorphisms located within the TNFA promoter analysed in our present study, TNFA G-238A showed no association with TNF-␣ inhibitors related efficacy. Some previous investigations have also failed to detect any significant association between the TNF receptor-associated factor (TRAF) polymorphism and treatment response [17]. There have been attempts to identify certain combinations of polymorphic variants that predispose to more effective treatment. Limited practical use of this type of results is caused by a small number of patients who have a particular combination of alleles. Nevertheless, haplotype reconstruction of the TNF locus revealed that the GGC haplotype (-238G/-308G/-857 C) in a homozygous form was significantly associated with a lower ACR50 response to ADA at 12 weeks [11], although none of the SNPs studied was correlated with the response to treatment in separate analysis. Similar analysis was performed in our group of RA patients. However, we did not find any association with treatment response and the presence of the GGC haplotype. The effectiveness of the treatment with TNF-␣ blockers may also be affected by the polymorphisms in genes encoding receptors for
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TNF-␣. Aggarwal et al. [18] showed that TNF-␣ and TNF- initiate their effects on cell function by binding to common cell surface receptors–TNFRI (TNFR1A, TNFRSF1A) and TNFII (TNF1B, TNFRSFAB). Soluble TNFRI and TNFRII levels were found to be higher in RA patients than healthy subjects [19]. In 105 patients with RA investigated after 3 and 12 months of treatment with ADA, ETA, INF, carriers of the TNFR1B 676TG genotype were characterized by a significantly lower ACR response than those with the 676TT genotype [20]. Two other relatively small studies found a lower degree of response to anti-TNF-␣ treatment in patients carrying the G allele [21,22]. However, these results were not confirmed by the further studies–conducted by Toonen [23] and our present one. In the study of Morales-Lara et al., evaluating the effect of the TNFR1A (G36A) polymorphisms on the efficacy of TNF inhibitor therapy, an association between the TNFR1A 36 AA genotype and a poorer response at 3 months was identified. A percentage of 39.3 of the AA positive patients and only 19% of those with the G allele were characterized by a less effective response (P = 0.04). However, the A allele showed only a trend to significance when compared with the G allele (RA with non-EULAR response: A 30.1% vs. G 19.5%; P = 0.07) [24]. This study was conducted on a Spanish population and included 145 (90 rheumatoid arthritis and 55 psoriatic arthritis) treated with anti-TNF inhibitors. Our study included a greater number of patients, was related to other populations and there were differences in the percentage of subsequent use of TNF blockers. These differences may be responsible for other results. In our study, more patients with the homozygous TNFR1A 36 AA genotype achieved a good EULAR response and remission at 3 months compared to patients carrying the G allele. Interestingly, in the Spanish study in patients with PsA the results were similar to those in our study–the TNFR1A polymorphism in PsA was associated with a better response at 3 months (AA 88% vs. AG/GG 58.9%; P = 0.04). SNPs of TNFR1A (rs767455) and TNFR1B (rs1061624 and rs3397) were also evaluated in 80 Japanese patients with Crohn’s disease [25]. The minor allele of rs767455 SNP was associated with a lack of efficacy of INF compared to the major genotype. This finding was confirmed by Pierik et al. [26]. In contrast, a previous study conducted in a Caucasian population concluded that the TNFR1A and TNFR1B polymorphisms could be thoroughly excluded as pharmacogenomic markers for a treatment response to INF in Crohn’s disease [8]. There have been a number of studies that aimed to evaluate the effect of polymorphisms in genes that control the activity of cytokines on the effectiveness of anti-TNF therapy. Their results showed no difference in response to treatment with respect to the sIL-6R A358 C polymorphism, and the polymorphism in the promoter region of the IL-10 gene (G − 1082A) [27,28]. Previous investigations of RA susceptibility markers in determining the response to anti-TNF drugs have found that neither the presence of the HLA–DRB1 shared epitope nor the PTPN22 locus was correlated with the response to biologics treatment [29]. Among genes encoding proteins involved in TNF signalling, the TNFAIP3, TRAF6, REL and PTPRC genes represent attractive candidate loci for the analysis of response to TNF antagonists [1]. There are conflicting results regarding the impact of age, sex, disease activity, and disease duration on the effectiveness of antiTNF therapy. It has been suggested that gender is probably not a predictor of response, but disease activity and poor functional ability at baseline could be [30]. In our study, all RA patients exhibited high disease activity at baseline. Concomitant treatment with methotrexate improved the response to anti-TNF therapies and 92% of our patients were treated with such a combination of drugs. Clinical factors explained only a minority of the variation in response to anti-TNF therapy. There is an increasing need for an individualised treatment strategy for patients with RA. Considering the fact that commonly, the first months of the
Please cite this article in press as: Swierkot J, et al. Analysis of associations between polymorphisms within genes coding for tumour necrosis factor (TNF)-alpha and TNF receptors and responsiveness to TNF-alpha blockers in patients with rheumatoid arthritis. Joint Bone Spine (2014), http://dx.doi.org/10.1016/j.jbspin.2014.08.006
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disease are associated with possible permanent articular damage and organ-based complications, immediate introduction of correct therapy is highly important. In our opinion, for the effectiveness of treatment with biological agents many genes are likely to be responsible, probably each with a small effect size. Patients with unfavourable genotypes could be treated with biologic agents with different mechanisms of action, or would require a more frequent rheumatological control, evaluating efficacy and safety of their therapy [31]. We hope that in the future, a physician will be able to select a correct medication for each individual patient, considering their current condition, previously used drugs, results of clinical and observational examination, concurrent diseases and–possibly–also genetic predispositions. The results of our present study imply that the TNFR1A gene polymorphism can affect the responsiveness to TNF-␣ blockers in Caucasians patients with rheumatoid arthritis. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgements This work was supported by a grant from the National Centre of Science (Poland)–grant no. 2011/01/B/NZ5/05367. References [1] Plant D, Prajapati R, Hyrich KL, et al. Biologics in Rheumatoid Arthritis Genetics and Genomics Study Syndicate. Barton A: replication of association of the PTPRC gene with response to anti-tumor necrosis factor therapy in a large UK cohort. Arthritis Rheum 2012;64:665–70. [2] Criswell LA, Lum RF, Turner KN, et al. The influence of genetic variation in the HLA-DRB1 and LTA-TNF regions on the response to treatment of early rheumatoid arthritis with methotrexate or etanercept. Arthritis Rheum 2004;50:2750–6. [3] Fonseca JE, Carvalho T, Cruz M, et al. Polymorphism at position -308 of the tumour necrosis factor alpha gene and rheumatoid arthritis pharmacogenetics. Ann Rheum Dis 2005;64:793–4. [4] Seitz M, Wirthmuller U, Moller B, et al. The -308 tumour necrosis factor-alpha gene polymorphism predicts therapeutic response to TNF{alpha}-blockers in rheumatoid arthritis and spondyloarthritis patients. Rheumatology (Oxford) 2007;46:93–6. [5] Bouma G, Crusius JB, Oudkerk Pool M, et al. Secretion of tumour necrosis factor alpha and lymphotoxin alpha in relation to polymorphisms in the TNF genes and HLA-DR alleles. Relevance for inflammatory bowel disease. Scand J Immunol 1996;43:456–63. [6] Marotte H, Maslinski W, Miossec P. Circulating tumour necrosis factor-alpha bioactivity in rheumatoid arthritis patients treated with infliximab: link to clinical response. Arthritis Res Ther 2005;7:149–55. [7] Wijbrandts CA, Dijkgraaf MG, Kraan MC, et al. The clinical response to infliximab in rheumatoid arthritis is in part dependent on pretreatment tumour necrosis factor alpha expression in the synovium. Ann Rheum Dis 2008;67:1139–44. [8] Mascheretti S, Hampe J, Kühbacher T, et al. Pharmacogenetic investigation of the TNF/TNF-receptor system in patients with chronic active Crohn’s disease treated with infliximab. Pharmacogenomics J 2002;2:127–36. [9] Kang CP, Lee KW, Yoo DH, et al. The influence of a polymorphism at position -857 of the tumour necrosis factor alpha gene on clinical response to etanercept therapy in rheumatoid arthritis. Rheumatology (Oxford) 2005;44: 547–52. [10] Marotte H, Pallot-Prades B, Grange L, et al. The shared epitope is a marker of severity associated with selection for, but not with response to, infliximab in a large rheumatoid arthritis population. Ann Rheum Dis 2006;65:342–7.
[11] Miceli-Richard C, Comets E, Verstuyft C, et al. A single tumour necrosis factor haplotype influences the response to adalimumab in rheumatoid arthritis. Ann Rheum Dis 2008;67:478–84. [12] Lee YH, Rho YH, Choi SJ, et al. Association of TNF-alpha -308G/A polymorphism with responsiveness to TNF-alpha-blockers in rheumatoid arthritis: a metaanalysis. Rheumatol Int 2006;27:157–61. [13] O’Rielly DD, Roslin NM, Beyene J, et al. TNFalpha- 308 G/A polymorphism and responsiveness to TNF-alpha blockade therapy in moderate to severe rheumatoid arthritis: a systematic review and meta-analysis. Pharmacogenomics J 2009;9:161–7. [14] Zeng Z, Duan Z, Zhang T, et al. Association between tumor necrosis factor-␣ (TNF-␣) promoter -308 G/A and response to TNF-␣ blockers in rheumatoid arthritis: a meta-analysis. Mod Rheumatol 2013;23:489–95. [15] Lee YH, Ji JD, Bae SC, et al. Associations between tumor necrosis factor-a (TNF-a) -308 and -238 G/A polymorphisms and shared epitope status and responsiveness to TNF-a blockers in rheumatoid arthritis: a metaanalysis update. J Rheumatol 2010;37:740–6. [16] Pavy S, Toone EJM, Miceli-Richard C. Tumour necrosis factor a-308G? A polymorphism is not associated with response to TNF-a blockers in Caucasian patients with rheumatoid arthritis: systematic review and meta-analysis. Ann Rheum Dis 2010;69:1022–8. [17] Tan R, Gibbons L, Potter C, et al. Investigation of rheumatoid arthritis susceptibility genes identifies association of AFF3 and CD226 variants with response to anti-tumour necrosis factor treatment. Ann Rheum Dis 2010;69:1029–35. [18] Aggarwal BB, Eessalu TE, Hass PE. Characterization of receptors for human tumour necrosis factor and their regulation by gamma-interferon. Nature 1985;318:665–7. [19] Valle Y, Ledezma-Lozano IY, Torres-Carrillo N, et al. Circulating TNFRI and TNFRII levels correlated with the disease activity score (DAS28) in rheumatoid arthritis. Scand J Rheumatol 2009;38:332–5. [20] Ongaro A, De Mattei M, Pellati A, et al. Can tumor necrosis factor receptor II gene 676T > G polymorphism predict the response grading to anti-TNFalpha therapy in rheumatoid arthritis? Rheumatol Int 2008;28:901–8. [21] Fabris M, Di PE, D’Elia A, et al. Tumor necrosis factor-alpha gene polymorphism in severe and mild-moderate rheumatoid arthritis. J Rheumatol 2002;29:29–33. [22] Chatzikyriakidou A, Georgiou I, Voulgari PV, et al. Combined tumour necrosis factor-alpha and tumour necrosis factor receptor genotypes could predict rheumatoid arthritis patients’ response to anti-TNF-alpha therapy and explain controversies of studies based on a single polymorphism. Rheumatology (Oxford) 2007;46:1034–5. [23] Toonen EJ, Coenen MJ, Kievit W, et al. The tumour necrosis factor receptor superfamily member 1b 676T > G polymorphism in relation to response to infliximab and adalimumab treatment and disease severity in rheumatoid arthritis. Ann Rheum Dis 2008;67:1174–7. ˜ [24] Morales-Lara MJ, Canete JD, Torres-Moreno D, et al. Effects of polymorphisms in TRAILR1 and TNFR1A on the response to anti-TNF therapies in patients with rheumatoid and psoriatic arthritis. Joint Bone Spine 2012;79: 591–6. [25] Matsukura H, Ikeda S, Yoshimura N, et al. Genetic polymorphisms of tumour necrosis factor receptor superfamily 1A and 1B affect responses to infliximab in Japanese patients with Crohn’s disease. Aliment Pharmacol Ther 2008;27:765–70. [26] Pierik M, Vermeire S, Steen KV, et al. Tumour necrosis factor-a receptor 1 and 2 polymorphisms in inflammatory bowel disease and their association with response to infliximab. Aliment Pharmacol Ther 2004;20: 303–10. [27] Hassan B, Maxwell JR, Hyrich KL, et al. Genotype at the sIL-6R A358 C polymorphism does not influence response to anti-TNF therapy in patients with rheumatoid arthritis. Rheumatology (Oxford) 2010;49:43–7. [28] de Paz B, Alperi-López M, Ballina-García FJ, et al. Interleukin 10 and tumor necrosis factor-alpha genotypes in rheumatoid arthritis–association with clinical response to glucocorticoids. J Rheumatol 2010;37: 503–11. [29] Potter C, Hyrich KL, Tracey A, et al. Association of rheumatoid factor and anticyclic citrullinated peptide positivity, but not carriage of shared epitope or PTPN22 susceptibility variants, with anti-tumour necrosis factor response in rheumatoid arthritis. Ann Rheum Dis 2009;68:69–74. [30] Emery P, Dörner T. Optimising treatment in rheumatoid arthritis: a review of potential biological markers of response. Ann Rheum Dis 2011;70: 2063–70. [31] Liu C, Batliwalla F, Li W, et al. Genome-wide association scan identifies candidate polymorphisms associated with differential response to anti-TNF treatment in rheumatoid arthritis. Mol Med 2008;14:575–81.
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Available online at
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Original article
Analysis of associations between polymorphisms within genes coding for tumour necrosis factor (TNF)-alpha and TNF receptors and responsiveness to TNF-alpha blockers in patients with rheumatoid arthritis Jerzy Swierkot a,∗ , Katarzyna Bogunia-Kubik b , Beata Nowak c,d , Katarzyna Bialowas a , Lucyna Korman a , Katarzyna Gebura b , Katarzyna Kolossa e , Slawomir Jeka e , Piotr Wiland a a
Department of Rheumatology and Internal Medicine, Wroclaw Medical University, Borowska 213, 53114 Wroclaw, Poland Laboratory of Clinical Immunogenetics and Pharmacogenetics, L. Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland c Department of Pharmacology, Wroclaw Medical University, Wroclaw, Poland d Department of Rheumatology and Internal Medicine, Wroclaw University Hospital, Wroclaw, Poland e Clinical Department of Rheumatology and Connective Tissue Diseases, Hospital University Number 2 Jana Biziela Bydgoszcz, Wroclaw, Poland b
a r t i c l e
i n f o
Article history: Accepted 23 August 2014 Available online xxx
a b s t r a c t Introduction: Despite the fact that therapy with TNF-␣ inhibitors constitutes a breakthrough in rheumatoid arthritis management, no improvement is still achieved in approximately 30% of cases. The aim of the study was to evaluate whether single nucleotide polymorphisms (SNPs) within the TNF-␣ and TNF receptor encoding genes affect the efficacy of therapy with TNF-␣ inhibitors in patients with RA. Methods: Five SNPs within the TNF-␣ and TNF receptor encoding genes (TNFA: G-308A, G-238A, C-857T; TNFR1A G36A; TNFR1B T676G) were determined in 280 RA patients who had been treated with TNF-␣ inhibitors for at least 6 months or they stop therapy because of adverse events. The association between the relative change in DAS28 and SNP genotypes was tested by linear regression. Results: At week 24, low disease activity or remission was achieved by 45% of the patients. After 6 months remission of the disease or low disease activity were more frequently observed among patients homozygous for the TNFR1A 36A allele than among those who were GG homozygotes (52% vs. 34%, P = 0.04). At week 24 DAS28 was significantly lower in the subgroup of patients homozygous for the TNFA-857T variant compared to the C allele carriers (P = 0.045). The other polymorphisms were not found to be significantly associated with EULAR response at week 12 and 24 of the anti-TNF treatment. Conclusions: Homozygosity for the TNFR1A 36A allele and the TNFA-875T variant could act as a genetic factor associated with better response to anti-TNF treatment. © 2014 Société franc¸aise de rhumatologie. Published by Elsevier Masson SAS. All rights reserved.
1. Introduction Knowledge on rheumatoid arthritis (RA) aetiopathogenesis and immunological disorders has been recently expanded, which brought some modern therapeutic options. Basic medications used in RA therapy are still conventional synthetic diseasemodifying anti-rheumatic drugs (csDMARDs). A therapy with biological agents should be introduced in patients whose response to methotrexate (MTX) or other csDMARDs is insufficient and high
∗ Corresponding author. E-mail address: [email protected] (J. Swierkot).
activity of the disease persists. The first biological drugs registered several years ago for RA therapy were tumour necrosis factor alpha (TNF-␣) inhibitors. Considering the significant costs associated with biological therapy, individual countries and rheumatology associations define indications for its use in RA patients. Despite the fact that therapy with TNF-␣ inhibitors constitutes a breakthrough in RA management, no improvement is still achieved in approximately 30% of cases, and another 20% of patients discontinue the therapy because of side effects. There is an ongoing search for biochemical and clinical markers that would allow prediction of a good response to therapy with biologicals, including TNF-␣ inhibitors. Besides clinical factors, genetic predisposition may also be helpful in that prediction. A conviction of the importance of individualized
http://dx.doi.org/10.1016/j.jbspin.2014.08.006 1297-319X/© 2014 Société franc¸aise de rhumatologie. Published by Elsevier Masson SAS. All rights reserved.
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2 Table 1 Patients’ characteristics. RA patients (n = )
280
Age (years) Females (%) Disease duration (years) Disease onset (years) Current smokers (%) RF+ (%) ACPA+ (%) DAS28 at baseline DAS28 at week 24 Anti-TNF drug Etanercept (%) Adalimumab (%) Infliximab (%) Certolizumab pegol (%) Glucocorticosteroids (%) Methotrexate (%)
50 ± 12.3 (range: 17–78) 79 18 ± 8.26 (range 1–44) 29.5 ± 12.1 (range 12–71) 18 65 84 6.53 ± 0.63 (range 5.14–8.69) 3.41 ± 1.10 (range 0.49–7.60) 52 34 9 5 92 (mean dose 6.6 mg prednisone daily) 92.5 (mean dose 22 mg weekly)
RA: rheumatoid arthritis; RF: rheumatoid factor; ACPA: anti-citrullinated protein antibodies; DAS 28: disease activity score 28; TNF: tumour necrosis factor.
pharmacotherapy becomes more and more common. Differences in genetic conditioning may influence the effect of a drug, both by a change in its pharmacokinetics and modification of its pharmacodynamic properties. Polymorphisms of genes coding TNF-␣ and its receptors may affect the efficacy of a drug and frequency of associated adverse effects. Based on published study results it is difficult to determine unequivocally which of the polymorphisms play the most important role and could be a good factor facilitating evaluation of efficacy and risk of adverse effects associated with therapy with TNF-␣ inhibitors. The aim of the study was to evaluate whether single nucleotide polymorphisms (SNPs) within the TNF-␣ (TNFA G-308A [rs1800629], TNFA G-238A [rs3615525], TNFA C-857T [rs1799724]) and TNF receptor (TNFR1A G36A [rs767455], TNFR1B T676G, Met196Arg [rs1061622]) encoding genes affect the efficacy of therapy with TNF-␣ inhibitors in patients with RA. 2. Methods 2.1. Characteristics of RA patients Three hundred and four RA patients of Caucasian origin who had been treated with recommended doses of TNF-␣ inhibitors (adalimumab [ADA], etanercept [ETA], infliximab [INF], certolizumab [CER]) for at least 6 months or who had stopped therapy because of adverse events were investigated. Patients enrolled in the study were men and women ≥ 18 years of age with active, adult-onset RA as defined by the 1987 revised criteria of the American College of Rheumatology (ACR) (the first patients were qualified for the study in January 2008). The mean disease duration was 18 (range: 1–44) years. All the patients provided written informed consent. The study was approved by the Wroclaw Medical University Ethics Committee. The patients’ characteristics are presented in Table 1. Twentyfour patients were excluded from the analysis due to irregular reporting to follow-up visits and incomplete data regarding activity of the disease and adverse effects. Two hundred and eighty patients, 221 females (79%) and 59 males (21%), were qualified for the final analysis. During the follow-up period, 11 patients (3.9%) discontinued therapy with TNF-␣ inhibitors, 5 (1.8%) because of side effects and 6 (2.1%) because of lack of improvement. The following inclusion criteria were accepted: consent to participate in the study; confirmed RA based on 1987 classification criteria of the American College of Rheumatology; active form of the disease–disease activity score based on erythrocyte sedimentation rate and an evaluation of 28 joints (DAS28) ≥ 5.1; failure of
treatment with at least 2 csDMARDs; over 18 years of age; women and men with reproductive potential had to use reliable contraception. There were the following exclusion criteria: pregnancy or breastfeeding; coexistence of other systemic diseases of connective tissue besides RA; clinically significant impairment of hepatic and renal function; alcohol abuse; infection with hepatotropic viruses; infections resistant to therapy; ongoing history of cancer if no cure was achieved; uncontrolled diabetes; patient unwilling or unable to cooperate. Patients were administered recommended doses of TNF-␣ inhibitors: 3 mg/kg body weight of INF, which was given as an intravenous infusion at weeks 0, 2, and 6, and every 8 weeks thereafter, subcutaneous injection of ADA at 40 mg every other week, subcutaneous injection of ETA at 50 mg every week and subcutaneous CER pegol 400 mg at week 0, 2, 4 and 200 mg every 2 weeks thereafter. The patients were allowed to continue treatment with csDMARDs, glucocorticoids, and/or non-steroidal anti-inflammatory drugs, if the treatment regimens were not modified for 4 weeks before the study. In total, 92.5% of patients were treated with a stable dose of methotrexate (mean dose: 22 mg weekly) and 92% patients were treated with glucocorticoids (mean dose: 6.6 mg daily of prednisone). Twenty-one patients (7.5%) were not treated with MTX. Among them, 11 (4%) were treated with biologic monotherapy (8 with ETA (5.5% of all ETA patients)) and 3 with ADA (3% of all ADA patients)) while remaining 10 patients (6 ETA, 4 ADA) were treated with another DMARD (5 leflunomide, 5 sulfasalazine). All patients treated with INF and CER were treated with MTX. To examine the response to anti-TNF therapy in RA, blood samples, laboratory data, and clinical data were collected at baseline (prior to anti-TNF therapy) and at 3 and 6 months after treatment. Clinical evaluation was based on medical history, number of painful and swollen joints, pain intensity assessed by the patient on a 100mm visual analogue scale (VAS) and laboratory tests (erythrocyte sedimentation rate [ESR], C-reactive protein [CRP]). The parameters allowed determination of improvement according to the criteria based on DAS28 suggested by the European League Against Rheumatism (EULAR). According to the EULAR definition, patients were classified as good, moderate, or non-responders, using the individual amount of change in the DAS28 (DAS28) and DAS28 values at 3 and 6 months. Moreover, in some analyses a comparison was made between non-responders versus patients in remission or low disease activity according to the EULAR response criteria. This stratification was due to the limited number of patients in each group. Safety of anti-TNF therapy was analysed on the basis of medical history, physical examination, and selected laboratory tests (including blood cell count, aspartate aminotransferase [AST], alanine aminotransferase [ALT], creatinine, urea levels, and urinalysis). The clinical and laboratory tests were completed before the start of the therapy and in months 3 and 6 of the follow-up. Outcomes were parameters of efficacy of TNF treatment and adverse events.
2.2. Polymorphism study DNA was extracted from peripheral blood taken on EDTA using Maxwell 16 Blood DNA Purification Kit (Promega Corp., Madison, WI, USA) following the recommendation of the manufacturer. Genetic polymorphisms within the TNFA promoter (rs1800629, G308A; rs3615525, G-238A; rs1799724, C-857T), TNFR1A (rs767455, G36A) and TNFR1B (rs1061622, T676G) were analysed using a polymerase chain reaction (PCR) amplification employing LightSNiP assays designed by TIB MOLBIOL (GmbH, Berlin, Germany). PCR
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amplifications and analysis of the typing results were performed using a Roche LightCycler 480 instrument.
Table 2 Distribution of TNFA and TNFR1A, TNRF1B alleles and genotypes in RA patients. Gene/Polymorphism
2.3. Statistical analysis The association between the relative change in DAS28 and SNP genotypes was tested by linear regression. In addition, Chi2 test was applied to compare genotypes in non-responders versus patients in remission or low disease activity according to the EULAR response criteria. Statistical analysis was performed with STATISTICA (data analysis software system), version 10 (StatSoft, Inc. 2011). 3. Results 3.1. Response to treatment Clinical data of 280 Caucasians patients with RA treated with TNF-␣ inhibitors were analysed. Among them, 52% were treated with ETA, 34% with ADA, 9% with INF and 5% with CER (Table 1). Mean DAS28 at the onset of biologic treatment was 6.53 ± 0.63 (range 5.14–8.69). No difference in DAS28 values (ETA–6.51 ± 0.66, ADA–6.54 ± 0.63, INF–6.56 ± 0.51, CER–6.70 ±0.40; P = 0.1390) was detected between subgroups treated with different biologics. There was no difference between patients carrying the various alleles or genotypes of the SNPs studied. Mean DAS28 after 24 weeks of treatment was 3.41 ± 1.10 (range 0.49–7.6). After 24 weeks DAS28 was significantly higher in the subgroup treated with INF (P = 0.0045). There was no difference in disease activity as assessed by DAS28 between other subgroups (ETA–3.29, ADA–3.43, INF–4.13, CER–3.22). A moderate EULAR response was achieved in 69% of patients, while a good EULAR response was achieved in 23% of patients at 3 months. At week 24, low disease activity or remission was achieved by 45% of the patients (ETA–54.8%, ADA–37.9%, CER–42.9%, INF–17.4%). No response according to EULAR or loss of response was observed in 24.4%. We have further analyzed the ACPA (+) patients and compared with the ACPA (–) group. Remission after 24 weeks was achieved by 50.4% ACPA (+) vs. 33% of ACPA (–) (P = 0.0237) although there was no statistically significant difference in disease activity at baseline. 3.2. Genotyping study Distributions of the TNFA (G-238A; G-308A; C-857T), TNFR1A (G36A) and TNFR1B (T676G) alleles and genotypes in patients with RA are presented in Table 2. These distributions were consistent with data from public databases for Caucasians (http://www.ncbi.nlm.nih.gov/projects/ SNP). None of the polymorphism studied was found to be associated with predisposition to RA. 3.3. Association of the TNFR1A and TNFA polymorphisms with response to TNF-˛ inhibitors Among the SNPs studied, only the TNFR1A (rs767455, G36A) and one of the TNFA (rs1799724, C-857T) promoter polymorphisms were found to be associated with response to anti-TNF therapy. It appeared that significantly more patients with the homozygous TNFR1A36 AA genotype achieved a good EULAR response at month 3 compared to patients carrying the G allele (P = 0.011). After 6 months remission of the disease or low disease activity were also more frequently observed among patients homozygous for the TNFR1A 36A allele than among those who were GG homozygotes (52% vs. 34%, P = 0.04, Table 3). Analyzing the data for the ACPA (+) patients only we obtained similar results as for the whole population. In the ACPA (+) group, remission of the disease or low disease activity were also more frequently observed among
3
RA patients Genotype/allele distribution n
TNFA G-238A (rs3615525) GG GA AA G A TNFA G-308A (rs1800629) GG GA AA G A TNFA C-857T (rs1799724) CC CT TT C T TNFR1A G36A (rs767455) GG GA AA G A TNFR1B T676G (rs1061622) TT TG GG T G
Allelic frequencies
[%]
(N = 278) 265 6 7 271 13 (N = 278)
95.3 2.2 2.5 97.5 4.7
G A
0.964 0.036
189 85 4 274 89 (N = 279)
68.0 30.6 1.4 97.5 32.0
G A
0.833 0.167
193 80 6 273 86 (N = 276)
69.2 28.7 2.1 97.8 30.8
C T
0.835 0.165
58 145 73 203 218 (N = 278)
21.0 52.5 26.4 73.5 79.0
G A
0.482 0.517
168 97 13 265 120
60.4 34.9 4.7 95.3 40.2
T G
0.779 0.221
N: number of patients studied for a given SNP; n: number of patients with a particular allele/genotype; [%]: percentage of patients with a particular allele/genotype.
patients homozygous for the TNFR1A 36A allele than among those who were GG homozygotes (58% vs. 38%, P = 0.0384). This additionally confirms the beneficial effect of the TNF1A 36AA homozygosity. The difference in the remission rate was not secondary to the differences in distribution of the alleles in subgroups treated with different biologics. Moreover, failure of therapy, identified as the lack of at least moderate improvement according to EULAR, was observed in 33% of patients with the TNFR1A GG genotype and in 19% of patients with the A allele (AA and GA genotypes) (P = 0.02). At week 24, DAS28 was significantly lower in the subgroup of patients homozygous for the TNFA -857T variant (DAS28–2.05) compared to the C allele carriers (DAS28–3.41) (P = 0.012), although the remission rate was not higher in this subgroup. The other polymorphisms were not found to be significantly associated with EULAR response at week 12 and 24 of the anti-TNF treatment. No significant association was found between the polymorphisms studied and the occurrence of adverse side effects. 4. Discussion Major genetic effects on the anti-TNF response might be expected to arise from variation within genes implicated in the TNF-␣ pathway. Candidate gene studies until now have had limited success and no single gene influencing the anti-TNF response in RA has been definitively identified [1]. Pharmacogenetic studies of the influence of the TNFA gene polymorphisms on response to
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Table 3 TNFA, TNFR1A and TNFR1B in RA patients characterized with different response to therapy with TNF- inhibitors at week 24 after treatment initiation. Gene/Polymorphism
Geno-type
EULAR remission/low disease activity at week 24 [n]
Non-responders at week 24 [n]
TNFA G-308A (rs1800629) N = 278
AG
36 (42%)
21 (25%)
GG
AA
TNFA G-238A (rs3615525) N = 278
GG
AA
AG
TNFA C-857T (rs1799724) N = 279
CT
CC
TT
TNFR1A G36A (rs767455) N = 276
ETA 22
INF 0
CER 2
ADA 5
ETA 9
INF 6
CER 1
91 (49%) ADA 30
ETA 55
INF 2
CER 4
35 (19%) ADA 10
ETA 12
INF 11
CER 2
2 (50%) ADA 1
ETA 1
INF 0
CER 0
2 (50%) ADA 2
ETA 0
INF 0
CER 0
122 (46%)
ETA 70
INF 5
CER 5
ADA 16
ETA 23
INF 14
CER 4
3 (43%) ADA 0
ETA 3
INF 0
CER 0
2 (29%) ADA 1
ETA 1
INF 0
CER 0
5 (83%) ADA 0
ETA 5
INF 0
CER 0
1 (17%) ADA 0
ETA 0
INF 1
CER 0
38 (47.5%)
ETA 25
INF 2
CER 2
ADA 3
ETA 3
INF 8
CER 1
89 (46%) ADA 31
ETA 52
INF 3
CER 3
44 (23%) ADA 16
ETA 19
INF 2
CER 7
3 (50%) ADA 2
ETA 1
INF 0
CER 0
1 (17%) ADA 0
ETA 1
INF 0
CER 0
ETA 10
INF 3
CER 1
ETA 12
INF 8
CER 1
ETA 4
INF 5
CER 1
GA
ADA 5 69 (48%) ADA 23
GT
GG
15 (19%)
ADA 9
20 (34%)a
TT
57 (22%)
ADA 42
GG
AA
TNFR1B T676G (rs1061622) N = 278
ADA 12
38 (52%)a ADA 13
19 (33%)b
ETA 14
INF 0
CER 1
ETA 41
INF 2
CER 3
ADA 5 28 (19%)b ADA 7
ETA 20
INF 3
CER 2
13 (18%)b ADA 3
80 (48%)
40 (24%)
ADA 30
ETA 46
INF 1
CER 3
ADA 12
ETA 19
INF 7
CER 2
43 (44%) ADA 9
ETA 29
INF 3
CER 2
18 (19%) ADA 5
ETA 4
INF 9
CER 0
7 (54%) ADA 3
ETA 3
INF 1
CER 0
2 (15%) ADA 1
ETA 1
INF 0
CER 0
N: number of patients studied for a given SNP (the % of patients with remission/low disease activity + non-responders is not 100% because there were also patients with moderate response to treatment); n: number of patients with a given genotype; % percentage of patients with a given genotype; ADA: adalimumab; ETA: etanercept; INF: infliximab; CER: certolizumab. a P = 0.04 (TNFR1A: AA vs. GG). b P = 0.02 (GG vs. AA + GA).
TNF blocker treatment have given conflicting results. Many explanations may account for such discrepancies as the differences in: the number of subjects, study design, criteria used in qualifying for biological treatment, response criteria chosen as primary endpoint and duration of follow-up, concomitant medication and the
ethnic origin of the patients [2–4]. Most studies of the TNFA promoter have focused on separate analysis of selected SNPs, mainly on the TNFA G-308A polymorphism. In some studies, the authors have suggested that patients carrying the -308A allele express higher levels of TNF-␣ [5] and that subjects with a high response to TNF
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blockers could also have high TNF-␣ bioactivity [6] or high synovial levels of this cytokine at baseline [7]. Poorer responders might be those who produce low levels of TNF-␣. Two small studies reported an association of the TNFA-308GG genotype with better response to anti-TNF treatment with INF [8] or ETA [3]. In another study involving 88 patients with rheumatic diseases, the TNFA -308GG genotype was found to be associated with a better response to TNF blockers at week 24 [4]. No association of the TNFA G-308A polymorphism with response to ETA was found in a study of 70 Asian patients [9]. Also, in three other studies involving larger sample sizes of patients treated with ETA (n = 151) [2], with INF (n = 198) [10] and with ADA (n = 381) [11], no association with the TNFA G-308A polymorphism was observed. Also, in our present study we did not observe an association between the response to anti-TNF therapy and this SNP. Five further meta-analyses showed inconsistent results of the impact of this polymorphism on the efficacy of treatment with TNF-␣ blockers. Three of them suggested that patients with RA who carried the rare A allele showed a worse response to anti-TNF therapy [12,13]. This was not confirmed in two larger meta-analyses. The results of 13 studies were included in the meta-analysis carried out by Lee et al. [15]. In this analysis, studies with a small number of subjects (< 100) showed that the odds ratio for the A allele carrier state was significantly lower among responders, but studies with a higher number of subjects (> or = 100) found no association between the TNFA G-308A polymorphism and responsiveness to TNF-␣ blockers. Furthermore, a larger meta-analysis on a total of 2576 patients also did not show any association between this SNP and the response to anti-TNF treatment [16]. Probably the data on the impact on the TNFA G-308A SNP are not conclusive because there are differences in study design, and many of them are limited by the small patient numbers and the low frequency of the TNF–308AA genotype, which is particularly rare. Zeng et al. suggested that it is possible that a poor response among carriers of the A variant might not be specific to anti-TNF-␣ agents, but could merely reflect a more aggressive manifestation of the disease in these patients and/or their poorer response to any therapeutic regimen [14]. The importance of the other TNFA polymorphisms was evaluated in subsequent studies. Eleven SNPs in the TNFA promoter region were analysed in 70 representatives of the Asian population [9]. In the study by Kange et al., patients homozygous for the TNFA857 C allele were poor responders to ETA. In our study, at week 24 DAS28 was significantly lower in the patients carrying the TNFA857TT homozygous genotype, which could indicate a more effective treatment. However, no statistically significant differences were observed for this SNP when analysing the effectiveness of response according to EULAR. As for the other polymorphisms located within the TNFA promoter analysed in our present study, TNFA G-238A showed no association with TNF-␣ inhibitors related efficacy. Some previous investigations have also failed to detect any significant association between the TNF receptor-associated factor (TRAF) polymorphism and treatment response [17]. There have been attempts to identify certain combinations of polymorphic variants that predispose to more effective treatment. Limited practical use of this type of results is caused by a small number of patients who have a particular combination of alleles. Nevertheless, haplotype reconstruction of the TNF locus revealed that the GGC haplotype (-238G/-308G/-857 C) in a homozygous form was significantly associated with a lower ACR50 response to ADA at 12 weeks [11], although none of the SNPs studied was correlated with the response to treatment in separate analysis. Similar analysis was performed in our group of RA patients. However, we did not find any association with treatment response and the presence of the GGC haplotype. The effectiveness of the treatment with TNF-␣ blockers may also be affected by the polymorphisms in genes encoding receptors for
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TNF-␣. Aggarwal et al. [18] showed that TNF-␣ and TNF- initiate their effects on cell function by binding to common cell surface receptors–TNFRI (TNFR1A, TNFRSF1A) and TNFII (TNF1B, TNFRSFAB). Soluble TNFRI and TNFRII levels were found to be higher in RA patients than healthy subjects [19]. In 105 patients with RA investigated after 3 and 12 months of treatment with ADA, ETA, INF, carriers of the TNFR1B 676TG genotype were characterized by a significantly lower ACR response than those with the 676TT genotype [20]. Two other relatively small studies found a lower degree of response to anti-TNF-␣ treatment in patients carrying the G allele [21,22]. However, these results were not confirmed by the further studies–conducted by Toonen [23] and our present one. In the study of Morales-Lara et al., evaluating the effect of the TNFR1A (G36A) polymorphisms on the efficacy of TNF inhibitor therapy, an association between the TNFR1A 36 AA genotype and a poorer response at 3 months was identified. A percentage of 39.3 of the AA positive patients and only 19% of those with the G allele were characterized by a less effective response (P = 0.04). However, the A allele showed only a trend to significance when compared with the G allele (RA with non-EULAR response: A 30.1% vs. G 19.5%; P = 0.07) [24]. This study was conducted on a Spanish population and included 145 (90 rheumatoid arthritis and 55 psoriatic arthritis) treated with anti-TNF inhibitors. Our study included a greater number of patients, was related to other populations and there were differences in the percentage of subsequent use of TNF blockers. These differences may be responsible for other results. In our study, more patients with the homozygous TNFR1A 36 AA genotype achieved a good EULAR response and remission at 3 months compared to patients carrying the G allele. Interestingly, in the Spanish study in patients with PsA the results were similar to those in our study–the TNFR1A polymorphism in PsA was associated with a better response at 3 months (AA 88% vs. AG/GG 58.9%; P = 0.04). SNPs of TNFR1A (rs767455) and TNFR1B (rs1061624 and rs3397) were also evaluated in 80 Japanese patients with Crohn’s disease [25]. The minor allele of rs767455 SNP was associated with a lack of efficacy of INF compared to the major genotype. This finding was confirmed by Pierik et al. [26]. In contrast, a previous study conducted in a Caucasian population concluded that the TNFR1A and TNFR1B polymorphisms could be thoroughly excluded as pharmacogenomic markers for a treatment response to INF in Crohn’s disease [8]. There have been a number of studies that aimed to evaluate the effect of polymorphisms in genes that control the activity of cytokines on the effectiveness of anti-TNF therapy. Their results showed no difference in response to treatment with respect to the sIL-6R A358 C polymorphism, and the polymorphism in the promoter region of the IL-10 gene (G − 1082A) [27,28]. Previous investigations of RA susceptibility markers in determining the response to anti-TNF drugs have found that neither the presence of the HLA–DRB1 shared epitope nor the PTPN22 locus was correlated with the response to biologics treatment [29]. Among genes encoding proteins involved in TNF signalling, the TNFAIP3, TRAF6, REL and PTPRC genes represent attractive candidate loci for the analysis of response to TNF antagonists [1]. There are conflicting results regarding the impact of age, sex, disease activity, and disease duration on the effectiveness of antiTNF therapy. It has been suggested that gender is probably not a predictor of response, but disease activity and poor functional ability at baseline could be [30]. In our study, all RA patients exhibited high disease activity at baseline. Concomitant treatment with methotrexate improved the response to anti-TNF therapies and 92% of our patients were treated with such a combination of drugs. Clinical factors explained only a minority of the variation in response to anti-TNF therapy. There is an increasing need for an individualised treatment strategy for patients with RA. Considering the fact that commonly, the first months of the
Please cite this article in press as: Swierkot J, et al. Analysis of associations between polymorphisms within genes coding for tumour necrosis factor (TNF)-alpha and TNF receptors and responsiveness to TNF-alpha blockers in patients with rheumatoid arthritis. Joint Bone Spine (2014), http://dx.doi.org/10.1016/j.jbspin.2014.08.006
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disease are associated with possible permanent articular damage and organ-based complications, immediate introduction of correct therapy is highly important. In our opinion, for the effectiveness of treatment with biological agents many genes are likely to be responsible, probably each with a small effect size. Patients with unfavourable genotypes could be treated with biologic agents with different mechanisms of action, or would require a more frequent rheumatological control, evaluating efficacy and safety of their therapy [31]. We hope that in the future, a physician will be able to select a correct medication for each individual patient, considering their current condition, previously used drugs, results of clinical and observational examination, concurrent diseases and–possibly–also genetic predispositions. The results of our present study imply that the TNFR1A gene polymorphism can affect the responsiveness to TNF-␣ blockers in Caucasians patients with rheumatoid arthritis. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgements This work was supported by a grant from the National Centre of Science (Poland)–grant no. 2011/01/B/NZ5/05367. References [1] Plant D, Prajapati R, Hyrich KL, et al. Biologics in Rheumatoid Arthritis Genetics and Genomics Study Syndicate. Barton A: replication of association of the PTPRC gene with response to anti-tumor necrosis factor therapy in a large UK cohort. Arthritis Rheum 2012;64:665–70. [2] Criswell LA, Lum RF, Turner KN, et al. The influence of genetic variation in the HLA-DRB1 and LTA-TNF regions on the response to treatment of early rheumatoid arthritis with methotrexate or etanercept. Arthritis Rheum 2004;50:2750–6. [3] Fonseca JE, Carvalho T, Cruz M, et al. Polymorphism at position -308 of the tumour necrosis factor alpha gene and rheumatoid arthritis pharmacogenetics. Ann Rheum Dis 2005;64:793–4. [4] Seitz M, Wirthmuller U, Moller B, et al. The -308 tumour necrosis factor-alpha gene polymorphism predicts therapeutic response to TNF{alpha}-blockers in rheumatoid arthritis and spondyloarthritis patients. Rheumatology (Oxford) 2007;46:93–6. [5] Bouma G, Crusius JB, Oudkerk Pool M, et al. Secretion of tumour necrosis factor alpha and lymphotoxin alpha in relation to polymorphisms in the TNF genes and HLA-DR alleles. Relevance for inflammatory bowel disease. Scand J Immunol 1996;43:456–63. [6] Marotte H, Maslinski W, Miossec P. Circulating tumour necrosis factor-alpha bioactivity in rheumatoid arthritis patients treated with infliximab: link to clinical response. Arthritis Res Ther 2005;7:149–55. [7] Wijbrandts CA, Dijkgraaf MG, Kraan MC, et al. The clinical response to infliximab in rheumatoid arthritis is in part dependent on pretreatment tumour necrosis factor alpha expression in the synovium. Ann Rheum Dis 2008;67:1139–44. [8] Mascheretti S, Hampe J, Kühbacher T, et al. Pharmacogenetic investigation of the TNF/TNF-receptor system in patients with chronic active Crohn’s disease treated with infliximab. Pharmacogenomics J 2002;2:127–36. [9] Kang CP, Lee KW, Yoo DH, et al. The influence of a polymorphism at position -857 of the tumour necrosis factor alpha gene on clinical response to etanercept therapy in rheumatoid arthritis. Rheumatology (Oxford) 2005;44: 547–52. [10] Marotte H, Pallot-Prades B, Grange L, et al. The shared epitope is a marker of severity associated with selection for, but not with response to, infliximab in a large rheumatoid arthritis population. Ann Rheum Dis 2006;65:342–7.
[11] Miceli-Richard C, Comets E, Verstuyft C, et al. A single tumour necrosis factor haplotype influences the response to adalimumab in rheumatoid arthritis. Ann Rheum Dis 2008;67:478–84. [12] Lee YH, Rho YH, Choi SJ, et al. Association of TNF-alpha -308G/A polymorphism with responsiveness to TNF-alpha-blockers in rheumatoid arthritis: a metaanalysis. Rheumatol Int 2006;27:157–61. [13] O’Rielly DD, Roslin NM, Beyene J, et al. TNFalpha- 308 G/A polymorphism and responsiveness to TNF-alpha blockade therapy in moderate to severe rheumatoid arthritis: a systematic review and meta-analysis. Pharmacogenomics J 2009;9:161–7. [14] Zeng Z, Duan Z, Zhang T, et al. Association between tumor necrosis factor-␣ (TNF-␣) promoter -308 G/A and response to TNF-␣ blockers in rheumatoid arthritis: a meta-analysis. Mod Rheumatol 2013;23:489–95. [15] Lee YH, Ji JD, Bae SC, et al. Associations between tumor necrosis factor-a (TNF-a) -308 and -238 G/A polymorphisms and shared epitope status and responsiveness to TNF-a blockers in rheumatoid arthritis: a metaanalysis update. J Rheumatol 2010;37:740–6. [16] Pavy S, Toone EJM, Miceli-Richard C. Tumour necrosis factor a-308G? A polymorphism is not associated with response to TNF-a blockers in Caucasian patients with rheumatoid arthritis: systematic review and meta-analysis. Ann Rheum Dis 2010;69:1022–8. [17] Tan R, Gibbons L, Potter C, et al. Investigation of rheumatoid arthritis susceptibility genes identifies association of AFF3 and CD226 variants with response to anti-tumour necrosis factor treatment. Ann Rheum Dis 2010;69:1029–35. [18] Aggarwal BB, Eessalu TE, Hass PE. Characterization of receptors for human tumour necrosis factor and their regulation by gamma-interferon. Nature 1985;318:665–7. [19] Valle Y, Ledezma-Lozano IY, Torres-Carrillo N, et al. Circulating TNFRI and TNFRII levels correlated with the disease activity score (DAS28) in rheumatoid arthritis. Scand J Rheumatol 2009;38:332–5. [20] Ongaro A, De Mattei M, Pellati A, et al. Can tumor necrosis factor receptor II gene 676T > G polymorphism predict the response grading to anti-TNFalpha therapy in rheumatoid arthritis? Rheumatol Int 2008;28:901–8. [21] Fabris M, Di PE, D’Elia A, et al. Tumor necrosis factor-alpha gene polymorphism in severe and mild-moderate rheumatoid arthritis. J Rheumatol 2002;29:29–33. [22] Chatzikyriakidou A, Georgiou I, Voulgari PV, et al. Combined tumour necrosis factor-alpha and tumour necrosis factor receptor genotypes could predict rheumatoid arthritis patients’ response to anti-TNF-alpha therapy and explain controversies of studies based on a single polymorphism. Rheumatology (Oxford) 2007;46:1034–5. [23] Toonen EJ, Coenen MJ, Kievit W, et al. The tumour necrosis factor receptor superfamily member 1b 676T > G polymorphism in relation to response to infliximab and adalimumab treatment and disease severity in rheumatoid arthritis. Ann Rheum Dis 2008;67:1174–7. ˜ [24] Morales-Lara MJ, Canete JD, Torres-Moreno D, et al. Effects of polymorphisms in TRAILR1 and TNFR1A on the response to anti-TNF therapies in patients with rheumatoid and psoriatic arthritis. Joint Bone Spine 2012;79: 591–6. [25] Matsukura H, Ikeda S, Yoshimura N, et al. Genetic polymorphisms of tumour necrosis factor receptor superfamily 1A and 1B affect responses to infliximab in Japanese patients with Crohn’s disease. Aliment Pharmacol Ther 2008;27:765–70. [26] Pierik M, Vermeire S, Steen KV, et al. Tumour necrosis factor-a receptor 1 and 2 polymorphisms in inflammatory bowel disease and their association with response to infliximab. Aliment Pharmacol Ther 2004;20: 303–10. [27] Hassan B, Maxwell JR, Hyrich KL, et al. Genotype at the sIL-6R A358 C polymorphism does not influence response to anti-TNF therapy in patients with rheumatoid arthritis. Rheumatology (Oxford) 2010;49:43–7. [28] de Paz B, Alperi-López M, Ballina-García FJ, et al. Interleukin 10 and tumor necrosis factor-alpha genotypes in rheumatoid arthritis–association with clinical response to glucocorticoids. J Rheumatol 2010;37: 503–11. [29] Potter C, Hyrich KL, Tracey A, et al. Association of rheumatoid factor and anticyclic citrullinated peptide positivity, but not carriage of shared epitope or PTPN22 susceptibility variants, with anti-tumour necrosis factor response in rheumatoid arthritis. Ann Rheum Dis 2009;68:69–74. [30] Emery P, Dörner T. Optimising treatment in rheumatoid arthritis: a review of potential biological markers of response. Ann Rheum Dis 2011;70: 2063–70. [31] Liu C, Batliwalla F, Li W, et al. Genome-wide association scan identifies candidate polymorphisms associated with differential response to anti-TNF treatment in rheumatoid arthritis. Mol Med 2008;14:575–81.
Please cite this article in press as: Swierkot J, et al. Analysis of associations between polymorphisms within genes coding for tumour necrosis factor (TNF)-alpha and TNF receptors and responsiveness to TNF-alpha blockers in patients with rheumatoid arthritis. Joint Bone Spine (2014), http://dx.doi.org/10.1016/j.jbspin.2014.08.006
