M. Manfredi

The CC homozygosis of the -174G>C IL-6 polymorphism predicts a lower efficacy of rituximab therapy in rheumatoid arthritis

Identification of genetic biomarkers of response to biologics in rheumatoid arthritis (RA) is a relevant issue. Being IL-6 a key cytokine for B cell survival, the interleukin-6 (IL-6) -174G>C and the IL-6 receptor (IL-6R) D358A gene polymorphisms were investigated in 158 RA patients treated with rituximab (RTX). One hundred and twenty-eight (81.0%) were RF positive and 126 (79.7%) were anti-CCP positive. Response to therapy was evaluated at the end of the sixth month after the first RTX infusion, by using both the EULAR and the ACR criteria. The possible relationship with IL-6 serum levels was also studied. By univariate analysis, lack of response by the EULAR criteria was more prevalent in RA patients with the IL-6 -174 CC genotypes (39.1%), than in the GC/GG patients (18.5%) (OR 2.83; 95%CI=1.10-7.27; p=0.031). A good response was noticed in only one patient (4.3%) with the IL-6 -174 CC genotype, while it was present in 24.4% of GG/GC cases (p=0.06). By stepwise multivariate analysis (including RA duration, baseline DAS28, baseline HAQ, RF status, anti-CCP status and IL-6 genotype as covariates), the IL-6 -174CC genotype was selected as an independent predictor of no response to RTX by both EULAR and ACR≥50 criteria, while the IL-6R polymorphism resulted as not associated. No definite association between gene polymorphisms and IL-6 serum levels was noticed. Present results suggest a possible role for IL-6 genotyping to better plan treatment with RTX in RA, and larger studies are worthwhile.

Biomarkers of good EULAR response to the B cell depletion therapy in all seropositive rheumatoid arthritis patients: Clues for the pathogenesis

Objective To find out whether a high number of auto-antibodies can increase the probability of a “good-EULAR response” and to identify the possible biomarkers of response in seropositive rheumatoid arthritis (RA) patients undergoing the B cell depletion therapy (BCDT). Patients and Methods One hundred and thirty-eight patients with long standing RA (LSRA), 75% non or poorly responsive to one or more TNFα blockers, all seropositive for at least one autoantibody (AAB) (RF-IgM, RF-IgA, RF-IgG, anti-MCV, ACPA-IgG, ACPA-IgA, ACPA-IgM) received one full course of BCDT. The major outcomes (moderate or good-EULAR response) were assessed after 6 months of therapy. The IL6 and BAFF levels were also determined. Results At a 6-month follow-up, 33 (23.9%) of the RA patients achieved a good EULAR response. Having up to 5-AABs positivity increased the chances for treatment response. After a logistic regression analysis, however, only 4 baseline factors arose as associated with a good-EULAR response: no steroid therapy (ORu200a=u200a6.25), a lymphocyte count <1875/uL (ORu200a=u200a10.74), a RF-IgG level >52.1 IU/ml (ORu200a=u200a8.37) and BAFF levels <1011 pg/ml (ORu200a=u200a7.38). When all the AABs, except for RF-IgM and ACPA-IgG, were left in the analysis, the two final predictors were no-steroid therapy and low lymphocyte count. Discussion The number of AABs increased the chances of being a “good-EULAR” responder. The only predictors, however, at the baseline of a good response in this seropositive cohort of RA patients were 2 simple variables – no steroids and lymphocyte count – and two laboratory assays – IgG-RF and BAFF.

The 158VV Fcgamma receptor 3A genotype is associated with response to rituximab in rheumatoid arthritis: results of an Italian multicentre study

Objective The polymorphism 158V/F of Fc fragment of IgG (FCGR) type 3A may influence the response to rituximab (RTX) in rheumatoid arthritis (RA). We investigated the FCG3A polymorphism in a large cohort of RA patients treated with RTX, also by considering the possible loss of response from month +4 to +6 after RTX and the presence of established predictors of response. Methods The study analysed 212 RA patients. European League Against Rheumatism (EULAR) response was evaluated at months +4 and +6 after the first RTX infusion. The FCGR3A polymorphism was analysed by PCR followed by Sanger sequencing. Results The FCGR3A genotypes were associated with EULAR response (good or moderate) at month +6 (response in 34/38 (89.5%) VV vs 70/106 (66%) VF and in 51/77 (66.2%) FF patients; p=0.01), but not at month +4 (response in 32/37 (86.5%) VV vs 69/102 (67.6%) VF and 53/73 (72.6%) FF patients; p=0.09). Loss of response was observed only in VF and FF carriers ((VV vs VF vs FF: 0/37 (0%) vs 11/102 (10.8%) vs 12/73 (16.4%); p=0.02)). Probability of response at month +6 was very high when at least two of the three following items selected by multivariate analysis were present: positive rheumatoid factor and/or anticyclic citrullinated peptide antibodies, previous treatment with ≤1 anti-tumor necrosis factor (TNF) agent, and 158VV FCGR3A genotype (p<0.0001; OR 7.9, 95% CI 4.1 to 15.1). Conclusions The 158VV FCGR3A genotype was associated with response to RTX in a large cohort of RA patients. Patient genotyping may be helpful to plan RTX treatment, and may be integrated with clinical predictors.

Anti-CarP antibodies as promising marker to measure joint damage and disease activity in patients with rheumatoid arthritis

Anti-citrullinated protein antibodies (ACPA) are important serological markers in the diagnosis of rheumatoid arthritis (RA) and are part of the recent diseasexa0classification criteria. However, there is a strong need for reliable markers for measuring and predicting joint damage and disease activity. Recently, antibodies directed against carbamylated antigens (anti-CarP antibodies) were identified. A total of 120 RA patients were tested for anti-CCP antibodies using different methods and for anti-CarP antibodies using carbamylated fetal calf serum according to the method described by Shi et al. Additionally, ACPA fine specificities (to three citrullinated peptides) were measured. Disease activity was assessed at baseline using the disease activity score 28 (DAS28) in 80 patients. For 40 RA patients, joint erosion score (JES) was established. The median JES was 14.1 with a standard deviation of 11.5. Anti-CarP antibodies were correlated with joint erosion score (ρxa0=xa00.34, 95xa0% CI 0.03–0.59; pxa0=xa00.0332). No correlation between ACPA and joint erosion score was observed. No individual marker correlated with DAS28. When one ACPA peptide was combined with anti-CarP antibodies in a score (ACPA peptide 1 divided by anti-CarP), a statistically relevant correlation was found (pxa0=xa00.0264). In thisxa0small cohort, the presence of anti-CarP antibodies, but not ACPA correlate with joint erosion score. Anti-CarP antibodies combined with ACPA fine specificities correlated with DAS28. Therefore, anti-CarP antibodies might represent a promising marker to predict joint damage and disease activity in RA patients.

Efficacy and safety of leflunomide or methotrexate plus subcutaneous tumour necrosis factor-alpha blocking agents in rheumatoid arthritis.

Several smaller retrospective case series have concluded that leflunomide (LEF) in combination with anti-TNF-α blocking agents appears to be effective and safe. Prospective case series and cohort studies have generally confirmed the efficacy of this combination. Overall, there is currently no evidence from controlled trials that an anti-TNF-α combination with LEF is as effective as an anti-TNF-α combination with methotrexate (MTX). We compared the effectiveness and safety of a therapeutic regimen associating subcutaneous anti-TNF-α agents, etanercept (ETN) and adalimumab (ADA), with leflunomide (LEF) or methotrexate (MTX), in a two year open-label study performed in clinical practice. We evaluated 96 patients with active rheumatoid arthritis undergoing therapy with ADA at the dose of 40 mg every other week, or with ETN at the dose of 50 mg/week for two years added to prednisolone (PDN) at the mean dose of 5.2±2.6 mg/day. Fifty-four of these patients were also undergoing therapy with MTX at the mean dose of 11.7±2.6 mg/week, while 42 patients were undergoing therapy with LEF at the daily dose of 20 mg. At 12 months, the analysis of variance showed an improvement of DAS28 in both groups (p<0.001), with a reduction in 33.3% of the patients in treatment with LEF and in 51.8% of the patients in treatment with MTX (p = 0.20). At 18 months, improvement was present in 33.3% of the patients in the LEF group and in 81.5% of the patients in the MTX group (p=0.001). This improvement seems to be independent of the anti-TNF-α agent, even if MTX produces the highest DAS28 reduction when used in association with ETN (p<0.078). We found no difference in drug discontinuation rates or in effectiveness measures between anti-TNFα+MTX and anti-TNFα+LEF. Our data showed a greater reduction of DAS28 in the MTX group and, in combination with ETN, better results after two years of therapy.

The Economic Burden of Biological Therapy in Rheumatoid Arthritis in Clinical Practice: Cost-Effectiveness Analysis of Sub-Cutaneous Anti-TNFα Treatment in Italian Patients

Rheumatoid arthritis (RA), with a prevalence of 0.46%, is found in about 272,004 patients in Italy. The socioeconomic cost of rheumatoid arthritis in Italy in 2002 has been estimated at €1,600 million. Cost-effectiveness evaluations have been based on the concept that, with treatment, patients will not progress to the next level(s) of disease severity or will take a longer time to progress, thus avoiding or delaying the high costs and low utility associated with more severe disease. Many cost-effective studies have been based on the variation of Health Assessment Questionnaire (HAQ) in clinical trials. The objective of this study is to perform a cost-effective analysis of 86 patients with rheumatoid arthritis in therapy with adalimumab 40 mg every other week and etanercept 50 mg/week for two years in a population of patients observed in clinical practice. The group of patients in therapy with adalimumab had also taken methotrexate, mean dose 12.4±2.5 mg/week (22 patients) or leflunomide 20 mg/day (16 patients). The group of patients in therapy with etanercept had also taken methotrexate, mean dose 11.7±2.6 mg/week (24 patients) or leflunomide 20 mg/day (24 patients). Incremental costs and QALYs (quality adjusted life years) gains are calculated compared with baseline, assuming that without biologic treatment patients would remain at the baseline level through the year. Conversion HAQ scores to utility were based on the Bansback algorithm. The results after two years showed: in the group methotrexate+adalimumab the QALY gained was 0.62±0.15 with a treatment cost of €26,517.62 and a QALY/cost of €42,521.13. In the group methotrexate+etanercept the QALY gained was 0.64±0.26 with a treatment cost of €25,020.96 and a QALY/cost of €39,171.76. The result of using etanercept in association with methotrexate is cost-effectiveness with a QALY gained under the acceptable threshold of €50,000. These are important data for discussion from an economic point of view when we choose a biologic therapy for rheumatoid arthritis in clinical practice.

The TTTT B lymphocyte stimulator promoter haplotype is associated with good response to rituximab therapy in seropositive rheumatoid arthritis resistant to tumor necrosis factor blockers

OBJECTIVEnTo investigate the polymorphisms in the promoter region of the B lymphocyte stimulator (BLyS) gene as markers of response to rituximab (RTX) in rheumatoid arthritis (RA).nnnMETHODSnThe study was first conducted in 152 Italian RA patients and then replicated in an additional 117 RA patients (73 Italian, 44 British). The European League Against Rheumatism response criteria were used to evaluate the response rate at months 4 and 6 after the first cycle of RTX, by means of the Disease Activity Score in 28 joints using the erythrocyte sedimentation rate; patients were classified according to the best response shown between months 4 and 6. BLyS promoter polymorphisms were analyzed by polymerase chain reaction followed by the analysis of the restriction fragments, BLyS promoter haplotypes were analyzed using the expectation-maximization algorithm, and BLyS serum levels were analyzed using enzyme-linked immunosorbent assay. Odds ratios (ORs) were calculated with 95% confidence intervals (95% CIs).nnnRESULTSnThe TTTT BLyS promoter haplotype appeared to be significantly associated with response to RTX only in the subset of seropositive patients (those positive for rheumatoid factor and/or anti-cyclic citrullinated peptide). The replication study confirmed that this association was limited to seropositive RA patients in whom treatment with anti-tumor necrosis factor (anti-TNF) agents had previously failed. In the whole series of seropositive patients in whom anti-TNF agents had previously failed, patients carrying the TTTT BLyS promoter haplotype were more prevalent in good responders (18 of 43 [41.9%]) than in moderate responders (20 of 83 [24.1%]) or in nonresponders (1 of 21 [4.8%]) (for good responders versus nonresponders, OR 14.4 [95% CI 1.77-117.39], P=0.0028). Furthermore, multivariate analysis selected the TTTT BLyS promoter haplotype as an independent marker of good response to RTX (for good responders versus nonresponders, OR 16.2 [95% CI 1.7-152.5], P=0.01; for good responders versus moderate responders and nonresponders combined, OR 3.1 [95% CI 1.2-7.8], P=0.02). The relationship between BLyS polymorphisms and BLyS serum levels remained unclear.nnnCONCLUSIONnBLyS promoter genotyping may be suitable for identifying seropositive RA patients who may have a good response to RTX after anti-TNF agents have failed.

Tailored first-line biologic therapy in patients with rheumatoid arthritis, spondyloarthritis, and psoriatic arthritis

OBJECTIVEnA multidisciplinary expert panel, the Italian board for the TAilored BIOlogic therapy (ITABIO), was constituted to formulate evidence-based decisional statements for the first-line tailored biologic therapy in patient with rheumatoid arthritis (RA), spondyloarthritis (SpA), and psoriatic arthritis (PsA).nnnMETHODSnSystematic review of the literature to identify English-language articles on the variables influencing the first-line biologic choice, including the efficacy and safety of the drug, the route of administration, the availability of response predictor biomarkers, the need of monotherapy, the patient socio-economic status, lifestyle, cultural level, personality, fertility and childbearing potential in women, the presence of comorbidities, the host-related risk factors for infection and latent tuberculosis infection (LTBI) reactivation, the cardiovascular (CV) risk, and costs.nnnRESULTSnSome variables, including the patients preference, the indication for anti-TNF monotherapy in potential childbearing women, and the intravenous route with dose titration in obese subjects resulted valid for all the three rheumatic conditions. Further, evidence of a better cost-effectiveness profile for etanercept (ETN) and biosimilar infliximab (IFX) in RA was found. Any biologic may be employed in absence of choice driving factors in RA. Otherwise, a high infection risk or LTBI positivity drive the choice toward abatacept (ABA), tocilizumab (TCZ), or ETN. TCZ should be the first choice if monotherapy is required. High rheumatoid factor (RF) and anti-citrullinated protein antibodies (ACPA) titers should drive the choice toward TCZ or ABA, while in patients at high CVD risk anti-TNF choice, with preference for ETN, seems appropriate. Presence of anterior uveitis or inflammatory bowel disease drives the choice to monoclonal antibody anti-TNFs (MoAb anti-TNFs). In PsA, ustekinumab (UTK), and to a lesser extent ETN, represents the first choice in patients at high infection and TB risk. Anti-TNFs or UTK choice is guided by skin or articular disease severity, enthesitis, and dactylitis, whereas ETN should be preferred if metabolic syndrome or high CV risk complicate PsA.nnnCONCLUSIONnTaking in account of multiple choice driving variables, first-line biologic therapy may be optimized in patients with RA, SpA, and PsA.

Determinants of Risk Infection During Therapy with Anti TNF-Alpha Blocking Agents in Rheumatoid Arthritis

The use of TNF-alpha antagonists (infliximab, etanercept, adalimumab) has changed the course of many rheumatic diseases including rheumatoid arthritis (RA). Since their approval, some questions regarding their safety including infections have been observed. The aim of the study was to evaluate the changes in cytokines levels and cells subsets in patients with RA during anti TNF blocking agents treatment and the possible effect on infections’ development. We evaluated in 89 RA patients [39 treated with etanercept (ETN), 29 with adalimumab (ADA) and 21 with infliximab (IFN)] at baseline and after 6 months the following parameters: procalcitonin, ESR, CRP, cytokines as TNF, IL-6, IL-10, IL-8 and the TNF/IL-10 ratio, and peripheral mononuclear cells as CD3+, CD3+/CD4+, CD3+/CD8+, CD19+, CD3- /CD16+/56+, CD14+HLADR+, CD20+, CD19+/CD38+. Peripheral mononuclear cells were detected by flow cytometric system Cytomics FC500 and cytokines circulating levels by a quantitative sandwich enzyme immunoassay technique (Human IL-8 Instant ELISAe Bioscience, Human IL-6 Instant ELISA e Bioscience, Human IL-10 Instant ELISAe Bioscience and Human TNF-a Quantikine immunoassay RD system). A lower reduction of CD14+HLADR+ in ADA group 54.6±10.4% vs ETA 48.4±15.7% vs INF 40.7±16.5%, p<0.039 was found. No differences in all three groups on peripheral mononuclear cells CD3+, CD3+/CD4+, CD3+/CD8+, CD19+, CD 20+, CD19+/CD38+, CD3-/CD16+/56+, and cytokine circulating levels were found. The number of infections at 6 months was: 10.3% in ADA group, 12.8% in ETN group and 19.04% in IFN group. A correlation was found between the reduction in CD14+HLADR+ cells and IFN treatment. Our data showed that the level of CD14+HLADR+ cells was reduced during therapy with IFN. ADA and ETN don’t reduce lymphocyte populations and their subsets such as CD14+HLADR+ cells that play an important role host defence.

The clinical impact of Anti-DFS70 antibodies in undifferentiated connective tissue disease: case reports and a review of the literature

Anti-nuclear antibody (ANA) positivity suggests CTD but can also lead to a diagnosis of UCTD when a patient does not fulfill the CTD diagnostic criteria. An anti-dense fine speckled (DFS) immunofluorescence (IIF) pattern can be observed when using an ANA test on HEp-2 cells and is due to the presence of antibodies to the nuclear DFS70 antigen that has rarely found in CTD. Serological testing for anti-DFS70 antibodies could therefore play a very interesting negative predictive role in stratifying patients on the basis of the evolution of UCTD to CTD. We described two patients ANAxa0and anti-DFS70 positive in which the use of new method allowing the immunoadsorption of anti-DFS70 antibodies has permitted to exclude the incorrect diagnosis of CTD.