Pilar Barrera

A clinical trial of CTLA-4 blockade with tremelimumab in patients with hepatocellular carcinoma and chronic hepatitis C

BACKGROUND & AIMS Tremelimumab is a monoclonal antibody that blocks cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), an inhibitory co-receptor that interferes with T cell activation and proliferation. The purpose of this pilot clinical trial was to test the antitumor and antiviral effect of tremelimumab in patients with hepatocellular carcinoma (HCC) and chronic hepatitis C virus (HCV) infection; and to study the safety of its administration to cirrhotic patients. METHODS Tremelimumab at a dose of 15 mg/kg IV every 90 days was administered until tumor progression or severe toxicity. Twenty patients were assessable for toxicity and viral response and 17 were assessable for tumor response. Most patients were in the advanced stage and 43% had an altered liver function (Child-Pugh class B). RESULTS A good safety profile was recorded and no patient needed steroids because of severe immune-mediated adverse events. Some patients had a transient albeit intense elevation of transaminases after the first dose, but not following subsequent cycles. Partial response rate was 17.6% and disease control rate was 76.4%. Time to progression was 6.48 months (95% CI 3.95-9.14). A significant drop in viral load was observed while new emerging variants of the hypervariable region 1 of HCV replaced the predominant variants present before therapy, particularly in those patients with a more prominent drop in viral load. This antiviral effect was associated with an enhanced specific anti-HCV immune response. CONCLUSIONS Tremelimumab safety profile and antitumor and antiviral activity, in patients with advanced HCC developed on HCV-induced liver cirrhosis, support further investigation.

Novel association in chromosome 4q27 region with rheumatoid arthritis and confirmation of type 1 diabetes point to a general risk locus for autoimmune diseases.

Recently, association of celiac disease with common single-nucleotide polymorphism (SNP) variants in an extensive linkage-disequilibrium block of 480 kb containing the KIAA1109, Tenr, IL2, and IL21 genes has been demonstrated in three independent populations (rs6822844P combined=1.3 x 10(-14)). The KIAA1109/Tenr/IL2/IL21 block corresponds to the Idd3 locus in the nonobese diabetic mouse model of type 1 diabetes (T1D). This block was recently found to be associated with T1D in a genomewide association study, although this finding lacks unequivocal confirmation. We therefore aimed to investigate whether the KIAA1109/Tenr/IL2/IL21 region is involved in susceptibility to multiple autoimmune diseases. We tested SNP rs6822844 for association with disease in 350 T1D-affected and 1,047 rheumatoid arthritis (RA)-affected Dutch patients and in 929 controls. We replicated the association with T1D (P=.0006; OR 0.64 [95% CI 0.50-0.83]), and revealed a similar novel association with RA (P=.0002; OR 0.72 [95% CI 0.61-0.86]). Our results replicate and extend the association found in the KIAA1109/Tenr/IL2/IL21 gene region with autoimmune diseases, implying that this locus is a general risk factor for multiple autoimmune diseases.

Synovial macrophage depletion with clodronate-containing liposomes in rheumatoid arthritis

OBJECTIVE To assess whether intraarticular (IA) administration of clodronate liposomes results in local macrophage depletion in patients with rheumatoid arthritis (RA). Primary goals were to address both the immunohistologic and potential toxic effects of this approach. Moreover, the correlation between immunohistologic findings and clinical assessments of disease activity and cartilage damage were assessed. METHODS An open study was conducted in consecutive RA patients who were scheduled for knee joint replacement in our department. Synovial biopsy tissue was obtained from the knee joint at 2 weeks before and at the time of surgery. This protocol was controlled for safety and immunohistologic concordance in 6 patients. One week before surgery, 10 patients received a single IA dose of clodronate liposomes. Staining of synovial tissue for cell markers (CD68, CD14, CD3, CD38) and adhesion molecules (vascular cell adhesion molecule 1 [VCAM-1], intercellular adhesion molecule 1 [ICAM-1]) was assessed by 2 blinded observers. Local and systemic parameters of disease activity were measured before each intervention. Cartilage damage was scored using standard radiologic techniques at baseline and during surgery. RESULTS A single IA dose of clodronate liposomes significantly reduced the number of CD68-positive cells (P = 0.005) and the expression of ICAM-1 and VCAM-1 in the synovial lining (P = 0.013 and P = 0.039, respectively). The intervention did not affect fibroblast-like synoviocytes, T cells, or plasma cells. No immunohistologic changes were observed in the control group. The procedure was well tolerated. The levels of ICAM-1 and VCAM-1 in the sublining layers correlated with the extent of macroscopic synovitis (P < 0.0005 and P < 0.005, respectively). The expression of ICAM-1 and CD14 in the sublining correlated with the levels of C-reactive protein (P < 0.0005 and P < 0.01, respectively). Cartilage destruction was correlated only with the expression of CD68 in the sublining (P = 0.02). CONCLUSION A single IA administration of clodronate liposomes leads to macrophage depletion and decreased expression of adhesion molecules in the synovial lining in patients with longstanding RA. The procedure is well tolerated, and its therapeutic potential is currently under investigation. The expression of adhesion molecules in the sublining layers reflects ongoing inflammation.

Long term anti-tumour necrosis factor alpha monotherapy in rheumatoid arthritis: effect on radiological course and prognostic value of markers of cartilage turnover and endothelial activation.

Objectives: To investigate the effect of prolonged neutralisation of tumour necrosis factor α (TNFα) on the radiological course in rheumatoid arthritis (RA). To assess whether the radiological course can be predicted by clinical variables or biological markers of cartilage and synovium turnover and of endothelial activation. Patients and methods: Forty seven patients with active RA enrolled at our centre in monotherapy trials with adalimumab (D2E7), a fully human anti-TNFα monoclonal antibody, were studied for two years. Radiographs of hands and feet obtained at baseline and after one and two years were scored in chronological order by a single, blinded observer using the modified Sharp method. Radiological course was classified as stable or progressive using the smallest detectable difference as cut off point. The relation between radiological course and serum markers of cartilage and synovium turnover (metalloproteinases (MMP-1 and MMP-3), cartilage oligomeric matrix protein (COMP), human cartilage glycoprotein-39 (HC gp-39)), endothelial activation (soluble E-selectin and intercellular adhesion molecule (ICAM-1)), and integrated measures of disease activity were assessed using univariate and multivariate analysis. Results: Radiological evaluation was performed in 36 patients with paired sets of radiographs at baseline and two years. After two years a total of 15/36 (42%) presented no radiological progression. More patients with stable radiological course were still receiving anti-TNFα treatment after two years (13/15 (87%) v 11/21 (52%); p=0.03) and had lower baseline COMP and sICAM-1 levels (p=0.01 and 0.04, respectively) than those in the group with progressive disease. In a logistic regression model the combination of sustained TNF neutralisation and baseline COMP and sICAM-1 levels was predictive for radiological outcome (p=0.03). C reactive protein and disease activity score area under the curve were significantly correlated with changes in radiological scores after two years (r=0.40 and 0.37, p<0.05). Long term TNFα neutralisation decreased the levels of COMP, sICAM, MMPs, and HC gp-39, but not sE-selectin. Conclusion: The results suggest that long term monotherapy with anti-TNFα has a positive effect on radiological outcome and modulates cartilage and synovium turnover as measured by biological markers. Baseline serum sICAM-1 levels and COMP levels may be helpful to identify patients with progressive or non-progressive radiological outcome.

Successful treatment of dermatomyositis and polymyositis with anti-tumor-necrosis-factor-alpha: preliminary observations.

Tumor necrosis factor α neutralization seems a rational therapy for myositis because this proinflammatory cytokine has been implicated in the pathogenesis of this disorder. Until now, we have treated 2 patients with a chimeric anti-TNF-α monoclonal antibody (infliximab). Both patients demonstrated a marked and sustained subjective and objective improvement without the occurrence of any side effects. These preliminary results suggest that anti-TNF-α treatment with infliximab is a safe and rapidly effective therapy for myositis.

Modulation of lipoprotein plasma concentrations during long-term anti-TNF therapy in patients with active rheumatoid arthritis

Objective: Durable blockade of tumour necrosis factor-alpha (TNF-α) in patients with rheumatoid arthritis (RA) suppresses disease activity and its progression. Cardiovascular diseases are 1.5–2-fold more frequent in RA patients than in the general population. Although TNF-α has well-established effects on lipid metabolism, the long-term effects of TNF-α blockade on lipid pattern are still unclear. In the present study, we investigated the effects of 1-year therapy with anti-TNF on the lipid profile of RA patients. Methods: Disease activity (DAS28) and plasma lipoproteins concentrations (total, HDL and LDL-cholesterol, triglycerides, ApoA, ApoB) were assessed in 55 RA patients and 55 controls. The whole RA group was followed up for 6 months, and 31 of the patients were followed up for 1 year. Results: In RA patients, DAS28 decreased after 2 weeks from the start of therapy (p<0.001) and remained low during the entire study duration. Short-term effects of anti-TNF on plasma lipid concentrations seemed beneficial and anti-atherogenic. However, these changes did not persist: plasma concentrations of total and LDL-cholesterol and the atherogenic index increased after 6 months and 1 year from the start of therapy. During therapy, the changes in disease activity and inflammatory status were inversely correlated with changes in plasma total and HDL cholesterol levels and positively correlated with the variation of atherogenic index. Conclusion: We conclude that one-year therapy with infliximab is likely to lead to a more pro-atherogenic pattern of the plasma lipids concentrations. However, the overall impact of these changes on the cardiovascular risk is more complex, considering the strong anti-inflammatory effects of anti-TNF drugs.

Differential association of the PTPN22 coding variant with autoimmune diseases in a Dutch population.

Protein tyrosine phosphatase PTPN22 is involved in the negative regulation of T-cell responsiveness. Recently, the association of a coding variant of the PTPN22 gene-R620W(1858C>T) with a number of autoimmune diseases has been described. Therefore, we tested the association of PTPN22 1858*T allele in Dutch early onset type 1 diabetes (T1D) and rheumatoid arthritis (RA) patients, as well as celiac disease (CD) patients, for which no previous study of PTPN22 has been reported. The PTPN22 variant was strongly associated with T1D in cases vs controls (P=2 × 10−7, OR=2.3, 95% CI=1.7–3.1) as well as in a transmission disequilibrium test in nuclear trios (P=9 × 10−9, OR=3.3, CI=2.1–5.0), RA (case/control: P=0.003, OR=1.8 CI =1.2–2.6), but not CD, in spite of a trend of increased homozygosity (P=0.05) and early age at onset (P=0.01). PTPN22 is not generally associated with T-cell mediated autoimmune diseases, although it might play a role in the CD patients with early clinical manifestation.

Effects of treatment with a fully human anti-tumour necrosis factor alpha monoclonal antibody on the local and systemic homeostasis of interleukin 1 and TNFalpha in patients with rheumatoid arthritis.

OBJECTIVES To study the short term effects of a single dose of D2E7, a fully human anti-tumour necrosis factor (TNFα) monoclonal antibody (mAb), on the local and systemic homeostasis of interleukin 1β (IL1β) and TNFα in patients with rheumatoid arthritis (RA). METHODS All patients with RA enrolled in a phase I, single dose, placebo controlled study with D2E7 in our centre were studied. Systemic cytokine levels, acute phase reactants, and leucocyte counts were studied at days 0, 1, and 14 after the first administration of anti-TNF mAb (n=39) or placebo (n=11). The cellularity and the expression of IL1 and TNFα in synovial tissue were studied in knee biopsy specimens obtained at baseline and at day 14 in 25 consenting patients. RESULTS A single dose of anti-TNF mAb induced a rapid clinical improvement, a decrease in acute phase reaction, and increased lymphocyte counts in patients with active RA. The protein levels of IL1β in the circulation were low and remained unchanged, but the systemic levels of IL1β mRNA (p=0.002) and the concentrations of IL1 receptor antagonist (IL1ra) and IL6 (p=0.0001) had already dropped within 24 hours and this persisted up to day 14. Systemic levels of TNFα mRNA were low and remained unchanged, though total TNFα (free and bound) in the circulation increased after D2E7, probably reflecting the presence of TNF-antiTNF mAb complexes (p<0.005, at days 1 and 14). Both TNF receptors dropped below baseline levels at day 14 (p<0.005). Despite clinical improvement of arthritis, no consistent immunohistological changes were seen two weeks after anti-TNF administration. Endothelial staining for IL1β tended to decrease in treated patients (p=0.06) but not in responders. The staining for IL1β and TNFα in sublining layers and vessels was mutually correlated (rs =0.47 and 0.58 respectively, p<0.0005) and the microscopic scores for inflammation correlated with sublining TNFα and IL1β scores (rs =0.65 and 0.54 respectively, p<0.0001), though none of these showed significant changes during the study. CONCLUSIONS Blocking TNFα in RA results in down regulation of IL1β mRNA at the systemic level and in reduction of the endogenous antagonists for IL1 and TNF and of other cytokines related to the acute phase response, such as IL6, within days. At the synovial level, anti-TNF treatment does not modulate IL1β and TNFα in the short term. The synovial expression of these cytokines does not reflect clinical response to TNF neutralisation.

Circulating soluble tumor necrosis factor receptors, interleukin‐2 receptors, tumor necrosis factor α, and interleukin‐6 levels in rheumatoid arthritis.

Objective. To assess whether circulating concentrations of soluble tumor necrosis factor receptors (sTNFR; p55 and p75), soluble interleukin-2 receptors (sIL-2R), tumor necrosis factor α (TNFα), and interleukin-6 (IL-6) reflect clinical response and whether changes are dependent on the drug used in rheumatoid arthritis (RA) patients taking methotrexate (MTX) or azathioprine (AZA). Methods. These cytokines and soluble receptors were assessed in 20 control subjects and serially for up to 48 weeks in 61 RA patients, by bioassay (IL-6) and immunoassays (sTNFR, sIL-2R, TNFα, and IL-6). Results. Concentrations of p55 and p75, sIL-2R, and TNFα (but not IL-6) were significantly higher in RA patients than in controls. Significant decreases in sIL-2R and p55 concentrations were associated with clinical improvement and were observed in patients treated with MTX, but not AZA. Both treatments induced decreases in IL-6 concentrations, but circulating AZA (or its metabolites) appears to interfere with the measurement of IL-6 bioactivity. TNFα and p75 levels did not show significant changes. Conclusion. Measurement of circulating sIL-2R, p55, and IL-6 may be useful in the evaluation of RA disease activity and response to therapy. Interference by circulating levels of drugs must be ruled out when bioassays are used to evaluate cytokine levels.

Common and different genetic background for rheumatoid arthritis and coeliac disease

Recent genome-wide association studies (GWAS) have revealed genetic risk factors in autoimmune and inflammatory disorders. Several of the associated genes and underlying pathways are shared by various autoimmune diseases. Rheumatoid arthritis (RA) and coeliac disease (CD) are two autoimmune disorders which have commonalities in their pathogenesis. We aimed to replicate known RA loci in a Dutch RA population, and to investigate whether the effect of known RA and CD risk factors generalize across the two diseases. We selected all loci associated to either RA or CD in a GWAS and confirmed in an independent cohort, with a combined P-value cut-off P < 5 x 10(-6). We genotyped 11 RA and 11 CD loci in 1368 RA patients, 795 CD patients and 1683 Dutch controls. We combined our results in a meta-analysis with UK GWAS on RA (1860 cases; 2938 controls) and CD (767 cases; 1422 controls). In the Dutch RA cohort, the PTPN22 and IL2/IL21 variants showed convincing association (P = 3.4 x 10(-12) and P = 2.8 x 10(-4), respectively). Association of RA with the known CD risk variant in the SH2B3 was also observed, predominantly in the subgroup of rheumatoid factor-positive RA patients (P = 0.0055). In a meta-analysis of Dutch and UK data sets, shared association with six loci (TNFAIP3, IL2/IL21, SH2B3, LPP, MMEL1/TNFRSF14 and PFKFB3/PRKCQ) was observed in both RA and CD cohorts. We confirmed two known loci and identified four novel ones for shared CD-RA genetic risk. Most of the shared loci further emphasize a role for adaptive and innate immunity in these diseases.