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Abstract

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Objective

To test whether the G-to-A polymorphism at position −308 in the promoter of the tumor necrosis factor α (TNFα) gene influences response to infliximab therapy in patients with rheumatoid arthritis (RA).

Methods

We genotyped 59 RA patients by polymerase chain reaction and subdivided them into two groups: those with the A/A or A/G genotype and those with the G/G genotype. We compared the groups' clinical responses to infliximab treatment after 22 weeks, using the Disease Activity Score in 28 joints (DAS28).

Results

We found that 42% of patients in the A/A and A/G group and 81% of patients in the G/G group had improvement of at least 1.2 in the DAS28 score (P = 0.0086). The average improvement in the DAS28 score was 1.24 in the A/A and A/G patients and 2.29 in the G/G patients (P = 0.029).

Conclusion

These data suggest that patients with a TNFα −308G/G genotype are better infliximab responders than are patients with A/A or A/G genotypes. TNFα −308 genotyping may be a useful tool for predicting response to infliximab treatment.

Anti–tumor necrosis factor α (anti-TNFα) therapy is a breakthrough in the management of rheumatoid arthritis (RA) (1). However, not all RA patients respond well to the standard infliximab regimen. Since anti-TNFα therapy is expensive and may have significant side effects, it would be extremely useful to be able to predict which patients will have improvement with anti-TNFα therapy and which patients will not. However, no clinical or biologic factor has yet been identified that would allow us to predict the response to this treatment.

The TNFα gene is located within the class III region of the major histocompatibility complex, between the HLA–B and the HLA–DRB1 genes. Individual variations in TNFα production have been observed in association with particular HLA–DR alleles (2). These findings suggest that polymorphism in the TNFα regulatory region might influence its production. Several polymorphisms have been identified in the vicinity of the TNFα gene. The influence of the G-to-A polymorphism at position −308 of the TNFα gene promoter on TNFα production, RA susceptibility, and RA severity has been studied extensively. Most groups have found that the rare allele TNFα −308A is associated with high TNFα production (3–6), but this finding is still somewhat controversial (7). In the present study, we investigated the relationship between TNFα −308 genotype and infliximab efficacy in 59 patients with RA treated with infliximab over a 22-week period.

PATIENTS AND METHODS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Patients.

Fifty-nine consecutive RA patients requiring infliximab therapy were included in this prospective study. All of them were enrolled in the Department of Rheumatology at La Conception Hospital in Marseille from September 2000 to January 2002. All patients fulfilled the American College of Rheumatology (formerly, the American Rheumatism Association) 1987 revised criteria for RA (8), and all had evidence of active disease, as indicated by a Disease Activity Score in 28 joints (DAS28) (9) of >3.2 despite methotrexate (MTX) treatment for at least 2 months. None of the patients had a history of chronic infectious disease, neoplasia, multiple sclerosis, or uncontrolled renal, hepatic, hematologic, or cardiac disease.

Treatment protocol.

All patients were given intravenous infusions of 3 mg/kg of infliximab at weeks 0, 2, 6, 14, and 22. MTX therapy was continued except when severe intolerance was known. No other disease-modifying drug was permitted. Patients were allowed to continue nonsteroidal antiinflammatory drugs and oral glucocorticoids.

Evaluation of clinical response.

Clinical response was evaluated before the first infusion (at week 0) and immediately before the fifth infusion (at week 22) using the following criteria: number of tender and swollen joints, patient's global assessment of disease status and assessment of pain (both on a visual analog scale), Health Assessment Questionnaire (HAQ) score (10), DAS28 score, erythrocyte sedimentation rate (ESR), and serum C-reactive protein (CRP) concentration.

Genotyping for TNFα polymorphism.

Genomic DNA was extracted from 10 ml of EDTA-anticoagulated blood using standard proteinase K digestion and salting out. Genotyping for biallelic polymorphism of TNFα at position −308 was performed by polymerase chain reaction (PCR) using sequence-specific primers (Cytokine Genotyping; One Lambda, Canoga Park, CA). Primer pairs were designed to match the −308G or −308A allele, resulting in amplification of the target sequence. An internal control primer for the human β-globin allele was included in every PCR reaction to assure integrity of the amplification.

Statistical analysis.

Patients who had the A/A or A/G genotype (at least 1 copy of the rare −308A allele) were grouped and compared with patients who had the G/G genotype, before initiation of infliximab treatment, according to the mean values for the following parameters: age, percentage of female patients, percentage of shared epitope (SE)–positive patients, duration of disease, rheumatoid factor (RF) seropositivity, glucocorticoid dosage, number of previous disease-modifying antirheumatic drugs (DMARDs), number of swollen and tender joints, duration of morning stiffness, patient's global assessment of disease status, DAS28 score, patient's assessment of pain, HAQ score, ESR, and serum CRP level. Comparison of responses for the 2 groups were evaluated by chi-square test and Student's 2-tailed t-test. Crude odds ratios (ORs; as estimates of relative risk) were calculated.

RESULTS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Baseline characteristics of patients (Table 1).

The baseline characteristics of the 59 patients are shown in Table 1. The majority of patients (78%) were women. Their mean ± SD age was 56.0 ± 12.6 years, and their mean ± SD disease duration was 13.2 ± 9 years. More than two-thirds of the patients (69.5%) were RF positive, and 74.6% were SE positive. A mean of 3.7 DMARDs had been used before infliximab treatment. At study entry, most patients had active disease despite MTX treatment, as indicated by a mean DAS28 score of >5. The distribution of TNFα −308 genotypes was as follows: −308G/G, 76.3%; −308A/G, 22.0%. One patient had the rare −308A/A genotype. Patients were divided into genotype group A/A and A/G (presence of at least 1 rare −308A allele; 23.7% of patients) and genotype group G/G (76.3% of patients). With the exception of HLA–DR3 positivity, baseline characteristics were similar in the two groups. Six patients (2 from group A/A and A/G, 4 from group G/G; P not significant) were excluded from the study: 2 because of infection, 1 because of cardiac sudden death, 3 because of noncompliance.

Table 1. Baseline characteristics of the study patients*
 All patients (n = 59)A/A and A/G genotypes (n = 14)G/G genotype (n = 45)P
  • *

    Except where indicated otherwise, values are the mean ± SD. SE = shared epitope; RF = rheumatoid factor; DMARDs = disease-modifying antirheumatic drugs; VAS = visual analog scale; DAS28 = Disease Activity Score in 28 joints; HAQ = Health Assessment Questionnaire; ESR = erythrocyte sedimentation rate; CRP = C-reactive protein. See Patients and Methods for description of groups.

Demographics
 Age, years56.0 ± 12.656.7 ± 15.955.8 ± 11.40.85
 Women, no. (%)46 (78)9 (64.3)37 (82.2)0.16
Disease status
 SE positive, no. (%)44 (74.6)9 (64.3)35 (77.8)0.31
 Disease duration, years13.2 ± 916 ± 11.512.1 ± 8.30.19
 RF seropositive, no. (%)41 (69.5)10 (71.4)31 (68.9)0.42
 HLA–DR3 positive, no. (%)9 (15.3)7 (50)2 (4.4)3.5 × 10−5
Drug treatment
 Glucocorticoids, mg/day12.5 ± 1111.6 ± 6.712.1 ± 12.00.71
 Previous DMARDs3.73 ± 1.54.2 ± 1.73.6 ± 1.40.17
Clinical assessment
 Swollen and tender joints10.4 ± 5.79 ± 4.810.9 ± 6.10.3
 Duration of morning stiffness, minutes120 ± 9092 ± 84128 ± 1030.23
 Patient's global assessment of disease status (0–10 VAS)6.9 ± 1.97.36 ± 1.86.8 ± 1.90.35
 DAS28 score5.7 ± 15.54 ± 0.95.8 ± 1.10.40
 Patient's assessment of pain (0–10 VAS)6.4 ± 1.96.86 ± 1.86.3 ± 1.80.35
 HAQ score1.9 ± 0.761.9 ± 0.61.9 ± 0.80.98
Biologic assessment
 ESR, mm/hour30.5 ± 23.325 ± 15.231.7 ± 25.90.29
 Serum CRP level, mg/dl29.0 ± 37.522.8 ± 2032.2 ± 43.30.46

Infliximab response and TNFα genotypes (Tables 2 and 3, Figure 1).

Good responders were defined, according to the European League Against Rheumatism consensus statement, as patients whose DAS28 score improved by at least 1.2 at week 22 compared with their DAS28 score before the first infusion (11). Similarly, nonresponders were defined as patients whose DAS28 score improved by <1.2 (11). Most patients (71.7%) were good responders (80.5% in group G/G, 41.7% in group A/A and A/G) (Table 2). The percentage of good responders was significantly higher in group G/G than in group A/A and A/G (OR 1.93, P = 0.0086). Comparison of the variation of DAS28 scores in group A/A and A/G with those in group G/G between weeks 0 and 22 showed a higher mean DAS28 improvement in group G/G (2.29) than in group A/A and A/G (1.24) (P = 0.029) (Table 3 and Figure 1).

Table 2. Correlation between infliximab response and polymorphism at position −308 of the tumor necrosis factor α gene*
 All patients (n = 53)A/A and A/G genotypes (n = 12)G/G genotype (n = 41)
  • *

    See Patients and Methods for description of groups.

  • Defined as improvement of at least 1.2 in the Disease Activity Score in 28 joints between the score before the first infliximab infusion and the score at week 22.

Responders, no. (%)38 (71.7)5 (41.7)33 (80.5)
Nonresponders, no. (%)15 (28.3)7 (58.3)8 (19.5)
Odds ratio 0.51.93
Table 3. Improvement in DAS28 scores with infliximab therapy and correlation with polymorphism at position −308 of the tumor necrosis factor α gene*
 A/A and A/G genotypes (n = 12)G/G genotype (n = 41)P
  • *

    Values are the mean ± SD. DAS28 = Disease Activity Score in 28 joints; NS = not significant. See Patients and Methods for description of groups.

  • By Student's 2-tailed t-test.

DAS28 scores
 Week 05.60 ± 0.945.77 ± 1.09NS
 Week 224.36 ± 1.163.48 ± 1.420.05
Improvement in DAS28 scores1.24 ± 1.742.29 ± 1.330.029
thumbnail image

Figure 1. Improvement in Disease Activity Score in 28 joints (DAS28) with infliximab treatment between week 0 and week 22 in 12 patients with tumor necrosis factor α (TNFα) −308 A/A or A/G genotypes and 41 patients with the TNFα −308G/G genotype. Thick bars show the mean DAS28 scores; thin bars show the median DAS28 scores.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

In this study, we followed up 59 patients with RA who were being treated with infliximab, and we tested whether the clinical response at 22 weeks (8 weeks after the fourth infusion of infliximab) could be predicted by analysis of the polymorphism at position −308 of the TNFα gene promoter. We found that patients with the −308G/G genotype were twice as likely to have a positive response as patients with the −308A/G or −308A/A genotype. We believe that this is the first report of a TNFα gene polymorphism predicting response to anti-TNF treatment in RA. A similar study in Crohn's disease did not find any influence of −308 TNFα gene polymorphism on response to infliximab (12).

The most straightforward interpretation of these data is that in A/A and A/G patients, standard infliximab treatment is overwhelmed by the high production of TNFα associated with −308 A/G or A/A genotypes. However, this was not demonstrated in this study. One way to confirm that this interpretation of our data is correct would be to show that higher doses of infliximab lead to better responses in A/A and A/G patients.

It could be argued that poor outcome with infliximab only indicates that the A/G or A/A genotypes are associated with particular severity in RA patients. However, we did not observe any difference in disease severity between patients in group A/A and A/G (poor infliximab responders) and those in group G/G (good infliximab responders) before treatment with infliximab (Table 1). The lack of influence of −308 TNFα gene polymorphism on the severity of RA has already been reported by others (13, 14).

In addition, since the TNFα gene is in linkage disequilibrium with HLA–DR, the difference in response to treatment with infliximab could just reflect more frequent expression of the HLA–DR SE in A/A and A/G patients. However, this is not the case; more G/G patients than A/A and A/G patients expressed the SE (Table 1), a finding consistent with the fact that the TNFα −308A allele is associated with HLA–DR3, an SE-negative allele.

Finally, genes other than TNFα on the HLA–DR3 haplotype (which is linked with the TNFα −308A allele) might be responsible for the poor response in patients in the A/A and A/G group. Indeed, the central portion of the HLA–A1;B8;DR3 haplotype may contain one or more risk factors for RA that are independent of the SE (15). A high-producer TNFα gene is likely to be one of them, but such a gene may not be the only risk factor in this region, which is rich in immune system genes (15).

Thus, we have observed that RA patients with TNFα −308 genotypes A/A or A/G show a poor response to infliximab treatment at 22 weeks. We therefore believe that TNFα genotyping in RA patients may be a useful tool for predicting efficacy of treatment with infliximab.

REFERENCES

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES
  • 1
    Maini R, St Clair EW, Breedveld F, Furst D, Kalden J, Weisman M, et al, the ATTRACT Study Group. Infliximab (chimeric anti-tumour necrosis factor alpha monoclonal antibody) versus placebo in rheumatoid arthritis patients receiving concomitant methotrexate: a randomised phase III trial. Lancet 1999; 354: 19329.
  • 2
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  • 3
    Braun N, Michel U, Ernst B, Metzner R, Bitsch A, Weber F, et al. Gene polymorphism at position −308 of the tumor-necrosis-factor-alpha (TNF-alpha) in multiple sclerosis and it's influence on the regulation of TNF-alpha production. Neurosci Lett 1996; 215: 758.
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  • 12
    Louis E, Vermeire S, Rutgeerts P, De Vos M, van Gossum A, Pescatore P, et al. A positive response to infliximab in Crohn disease: association with a higher systemic inflammation before treatment but not with −308 TNF gene polymorphism. Scand J Gastroenterol 2002; 37: 81824.
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    Lacki J, Moser R, Korczowska I, Mackiewicz S, Muller W. TNF-alpha gene polymorphism does not affect the clinical and radiological outcome of rheumatoid arthritis. Rheumatol Int 2000; 19: 13740.
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    Brinkman B, Huizinga H, Kurban S, van der Velde E, Schreuder G, Hazes J, et al. Tumor necrosis factor α gene polymorphisms in rheumatoid arthritis: association with susceptibility to or severity of disease? Br J Rheumatol 1997; 36: 51621.
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    Jawaheer D, Li W, Graham R, Chen W, Damle A, Xiao X, et al. Dissecting the genetic complexity of the association between human leukocyte antigens and rheumatoid arthritis. Am J Hum Genet 2002; 71: 58594.