NfL and GFAP in serum are associated with microstructural brain damage in progressive multiple sclerosis.

BACKGROUND: The potential of neurofilament light chain (NfL) and glial fibrillary acidic protein (GFAP) as biomarkers of disease activity and severity in progressive forms of multiple sclerosis (MS) is unclear. OBJECTIVE: To investigate the relationship between serum concentrations of NfL, GFAP, and magnetic resonance imaging (MRI) in progressive MS. METHODS: Serum concentrations of NfL and GFAP were measured in 32 healthy controls and 32 patients with progressive MS from whom clinical and MRI data including diffusion tensor imaging (DTI) were obtained during three years of follow-up. RESULTS: Serum concentrations of NfL and GFAP at follow-up were higher in progressive MS patients than in healthy controls and serum NfL correlated with the EDSS score. Decreasing fractional anisotropy (FA) in normal-appearing white matter (NAWM) correlated with worsening EDSS scores and higher serum NfL. Higher serum NfL and increasing T2 lesion volume correlated with worsening paced autitory serial addition test scores. In multivariable regression analyses with serum GFAP and NfL as independent factors and DTI measures of NAWM as dependent factors, we showed that high serum NfL at follow-up was independently associated with decreasing FA and increasing MD in NAWM. Moreover, we found that high serum GFAP was independently associated with decreasing MD in NAWM and with decreasing MD and increasing FA in cortical gray matter. CONCLUSION: Serum concentrations of NfL and GFAP are increased in progressive MS and are associated with distinct microstructural changes in NAWM and CGM.

Myasthenia and risk of cancer: a population-based case-control study.

BACKGROUND AND PURPOSE: To evaluate the association between having non-thymoma myasthenia and the risk of extra-thymic cancer in a population-based setting. METHODS: A nationwide case-control study was conducted in Denmark based on medical registries. The study included all cases with a first time diagnosis of cancer during 2000-2009. Each case was matched by birth year and gender with eight population controls using risk set sampling. Subjects with myasthenia were identified through a validated register-based algorithm. Conditional logistic regression was used to compute crude and adjusted odds ratios (ORs), with 95% confidence intervals (CIs), for cancer associated with a prior diagnosis of myasthenia. RESULTS: In all, 233 437 cases and 1 867 009 controls were identified. A total of 80 cases and 518 controls had a prior diagnosis of myasthenia. Myasthenia was not associated with an increased risk of overall cancer (OR 1.1; 95% CI 0.9-1.4). Adjusted ORs for major cancer sites were also close to unity, whereas an elevated risk of lymphomas was observed (OR 2.0; 95% CI 0.8-5.5). Early-onset myasthenia was associated with a slightly increased OR for overall cancer (1.5; 95% CI 1.0-2.3); however, this estimate was based on small numbers. CONCLUSIONS: Non-thymoma myasthenia was not associated with an increased risk of overall cancer. Larger studies are necessary to evaluate the association between myasthenia and risk of lymphoma and the potential effect modification by age of myasthenia onset in relation to cancer risk.

Risk of non-melanoma skin cancer in myasthenia patients treated with azathioprine.

BACKGROUND AND PURPOSE: The association between use of azathioprine and risk of non-melanoma skin cancer (NMSC) in patients with myasthenia was evaluated in a nationwide setting. Treatment of autoimmune myasthenia frequently involves long-term exposure to immunosuppressants, including azathioprine. Use of azathioprine increases the risk of NMSC in organ recipients and probably also in patients with other autoimmune disorders. No previous study has specifically investigated the risk of NMSC in myasthenia patients treated with azathioprine. METHODS: This is a case-control study based on Danish population-based registries. Cases were myasthenia patients with a first time diagnosis of NMSC during 2004-2009. Age- and sex-matched controls were selected amongst myasthenia patients with no history of cancer using incidence density sampling. Prior use of azathioprine in cases and controls was assessed through prescription records (1995-2009). Conditional logistic regression was used to calculate odds ratios (OR) with 95% confidence intervals (CI) for skin cancer associated with a high cumulative dose (≥150 g) or long-term use (≥5 years) of azathioprine, adjusted for confounders. RESULTS: Thirty NMSC cases and 360 matched controls were identified. Ever use of azathioprine was associated with a considerably increased risk of NMSC (OR 3.3, 95% CI 1.5-7.3) that was even more apparent in patients with high cumulative dose (OR 4.6, 95% CI 1.7-12.5) or long-term (OR 4.8; 95% CI 1.7-13.6) use of azathioprine. CONCLUSION: Azathioprine use in patients with myasthenia is associated with an increased risk of NMSC.

Use of azathioprine for non-thymoma myasthenia and risk of cancer: a nationwide case-control study in Denmark.

BACKGROUND AND PURPOSE: To evaluate the association between the use of azathioprine and risk of cancer in patients with non-thymoma myasthenia gravis (MG) in a nationwide setting. METHODS: Case-control study based on population-based registries. Cases were patients with MG with a first time diagnosis of cancer (except non-melanoma skin cancer) registered during 2000-2009, and controls were patients with MG with no history of cancer. Prior use of azathioprine in cases and controls was assessed through prescription records (1995-2009). We used unconditional logistic regression to calculate odds ratios (ORs) with 95% confidence intervals (CIs) for cancer associated with a high cumulative dose [≥ 1000 defined daily doses (DDD)] or long-term use (≥ 5 years) of azathioprine, compared with never use of the drug and adjusted for potential confounders. RESULTS: We identified 89 cases and 873 controls. The prevalence of ever use of azathioprine was similar among cases (39.3%) and controls (39.4%). We observed a slightly elevated OR for cancer overall associated with long-term use of azathioprine (1.22; 95% CI: 0.62-2.40, P = 0.56). The highest ORs were observed for use of 2000 DDD or more of azathioprine; however, these risk estimates were based on small numbers. CONCLUSIONS: Use of azathioprine in patients with non-thymoma MG may be associated with a slightly increased risk of cancer overall. Larger studies are necessary to address the risk of site-specific cancers.

Late-onset myasthenia not on the increase: a nationwide register study in Denmark, 1996-2009.

BACKGROUND: An increase in late-onset myasthenia gravis (MG) has been reported. There are few large population-based studies over longer periods of time reflecting recent developments in MG incidence. METHODS: We identified a nationwide cohort of patients with incident myasthenia in Denmark in 1996-2009. We used a validated algorithm to track subjects based on a combination of diagnosis and prescription (pyridostigmine) data from nationwide registers. Patients with myasthenia were classified into early onset (<50 years old) and late onset (50+ years). We calculated incidence rates (IRs) and corresponding 95% confidence intervals. RESULTS: We identified 693 patients (362 women) with incident MG in the study period corresponding to an IR of 9.2 per million person-years (8.5-9.9). Overall, 207 (29.9%) were classified as early-onset and 486 (70.1%) as late-onset MG. Women predominated in the early-onset group (70.5%), but not in the late-onset group (44.4%). The incidence rate of early-onset MG was 4.2 (3.6-4.8) and late-onset MG 18.9 (17.3-20.7) per million person-years and it did not vary over time in the study period (P-values for trend 0.54 and 0.15, respectively). CONCLUSION: Late-onset MG comprised a large proportion of all incident cases in Denmark, was more common in men than women, and occurred with a stable incidence in the 14-year study period. Therefore, we speculate whether previous reports of a rise in late-onset MG reflect a non-biological phenomenon, that is, a gradual improvement in the diagnosis of MG in this age group in previous years.

Correlation between anti-interferon-ß binding and neutralizing antibodies in interferon-ß-treated multiple sclerosis patients.

BACKGROUND AND PURPOSE: Measurements of binding antibodies (BAbs), neutralizing antibodies (NAbs) and MX1 mRNA expression are used to analyse the immunological reactions in patients with MS treated with IFN-ß. The correlations between these are yet not fully understood. METHODS: We measured BAbs and NAbs to IFN-ß in 110 serum samples from 83 patients with MS treated with IFN-ß, and in a subgroup, antibody titre was compared with corresponding expressions of MX1 mRNA. The methods used were capture ELISA assay, luciferase reporter gene assay and mRNA RT-PCR for MX1 gene expression. RESULTS: There were significant correlations between binding, neutralizing and MX1 results. Cut-off values are suggested for the definition of samples of BAbs and NAbs as negative, positive and grey zones. Naturally occurring groups of low and high antibody titres were identified by the correlation between BAbs and NAbs, probably as a result of an immunological maturation process of antibodies. The low-titre group had lower correlations between BAbs and NAbs than the high-titre group. CONCLUSIONS: High correlation is demonstrated between the results obtained by the three methods, and we suggest the possibility of using ELISA measurements of BAbs to identify patients with high titres of anti-IFN-ß antibodies that block the biological response to IFN-ß. Ιn patients with low titres, we suggest to supplement ELISA with measurement of MX1 mRNA to establish whether the bioavailability of IFN-ß is preserved.

Identification of new sensitive biomarkers for the in vivo response to interferon-beta treatment in multiple sclerosis using DNA-array evaluation.

OBJECTIVE: Neutralizing antibodies (NAbs) occur in a proportion of multiple sclerosis (MS) patients treated with interferon (IFN)-beta. NAbs impair the effect of treatment. The biological effect of IFN-beta can be measured as the induction of the myxovirus resistance protein A (MxA) molecule. However, other markers could be more sensitive for evaluating the response to IFN-beta. We used DNA array analysis to identify genes that are strongly induced in blood cells by IFN-beta, and measured their expression in MS patients with different NAb levels. METHODS: Gene expression was studied on DNA arrays in untreated patients, in NAb negative patients, and in MS patients with varying NAb levels 9-12 h and 36-48 h after IFN-beta administration. The expression of selected genes was measured by real-time PCR. NAb levels were assessed by a cytopathic effect assay. RESULTS: Several hundred genes were induced 9-12 h after an injection of IFN-beta. The molecules CXCL10, CCL2 and IFI27 were among the most strongly induced. Gene induction was generally much less pronounced after 36-48 h, but IFI27 remained strongly induced. The strong induction of these molecules and MxA was confirmed by real-time PCR. Induction of MxA, CCL2, CXCL10 and IFI27 was reduced in patients with low NAb levels and lost in patients with intermediate/high NAb levels. CONCLUSION: We identify IFI27, CCL2 and CXCL10 as sensitive biomarkers for the response to IFN-beta. The expression of these markers adequately reflects bioactivity of IFN-ss as documented by the decreased induction in low NAb-positive patients and the lost induction in patients with moderate/high NAb levels.

Efficacy of natalizumab in multiple sclerosis patients with high disease activity: a Danish nationwide study.

INTRODUCTION: Previous studies of natalizumab (Tysabri) in relapsing multiple sclerosis (MS) patients have included patients with moderate disease activity. We studied a patient population with high disease activity. PATIENTS AND METHODS: We analyzed data from 234 consecutive, natalizumab-treated patients, followed for at least 3 months. Three groups of patients were eligible for natalizumab therapy: patients with two or more documented relapses or sustained increase of 2 EDSS points on disease modifying therapy (DMT) in the previous year; patients switching from mitoxantrone; and patients with very active MS as de novo therapy. RESULTS: During a median observation time of 11.3 months (range 3.0-21.5) the annualized relapse rate decreased to 0.68 from a pre-treatment rate of 2.53 (73% reduction). We assessed the annualized relapse rate in three subgroups: (i) 0.83 in 14 (6.0%) de novo treated patients; (ii) 0.71 in 175 (74.8%) patients with >or=2 relapses or sustained increase in EDSS of >or=2 points on a first-line DMT; and (iii) 0.56 in 45 (19.2%) patients switching from mitoxantrone. Nine anaphylactoid reactions, two severe, were reported. Out of 215 patients 7 (3%) were persistently positive for antibodies to natalizumab. CONCLUSIONS: Tysabri appears to be effective in MS patients with high disease activity, but the relapse rate was higher than in the pivotal study after the first treatment year. This is likely to reflect differences in disease activity before the initiation of natalizumab treatment.

Treatment with azathioprine and cyclic methylprednisolone has little or no effect on bioactivity in anti-interferon beta antibody-positive patients with multiple sclerosis.

BACKGROUND: It is unknown whether immunosuppression of patients who have developed interferon-beta (IFN-beta) neutralizing antibodies (NAbs) hastens disappearance of NAbs in the blood. OBJECTIVE: We wanted to test whether immunosuppression with cyclic methylprednisolone (MP) in combination with azathioprine (AZA) for 6 months accelerates recovery of IFN-beta bioactivity in patients with multiple sclerosis (MS) with abolished in-vivo myxovirus resistance protein A (MxA) mRNA response to IFN-beta. METHODS: We included 13 patients with MS with NAbs and a low IFN-beta bioavailability detected by the MxA-mRNA response in a descriptive, non-randomized trial. Another 14 NAb-positive patients with a low MxA-mRNA response served as controls. The primary outcome was the fraction of patients who regained an MxA-mRNA response to IFN-beta. NAbs were measured by means of a clinically validated cytopathic effect assay and a new reporter gene assay. The in-vivo MxA-mRNA response was measured by real-time polymerase chain reaction. RESULTS: A total of 11 patients in the treatment group completed the trial. In all, two of these 11 patients regained an in-vivo MxA-mRNA response as compared to one of 14 patients in the control group. CONCLUSION: Treatment with AZA and cyclic MP for 6 months has little or no effect on IFN-beta bioactivity in NAb-positive patients with MS.

Native and transformed alpha2-macroglobulin in plasma from patients with multiple sclerosis.

Multiple sclerosis (MS) is an inflammatory demyelinating disease with unknown etiology. Various proteinases have been observed in increased levels in the central nervous system of patients with MS, which may contribute to the release of immunogenic myelin components. alpha2-Macroglobulin (alpha2M) inhibits a broad spectrum of proteinases sterically, undergoing major conformational changes induced by the proteinases themselves. Moreover, alpha2M acts as a carrier of several cytokines in the systemic circulation. By use of radial immunodiffusion, we determined the total alpha2M levels in plasma from 28 MS patients and 15 control subjects [14 patients with other neurologic diseases (OND) and one healthy individual]. No significant differences in total alpha2M concentration were observed between the MS patients and the control subjects. A comparison of the degree of alpha2M transformation in MS patients with different disease courses and controls was performed, using monoclonal antibodies (mAbs) specific for binding to native and transformed alpha2M, respectively. The fractions of transformed alpha2M were significantly increased in patients with secondary or primary progressive disease course compared with the controls. No significant differences were obtained using a native-specific mAb. At least a major proportion of alpha2M from the MS patients was able to change conformation from its native to its transformed state, as demonstrated by a shift in mAb reactivity, following methylamine treatment of the plasma samples. In conclusion, the results indicate that plasma alpha2M may be inactivated at a higher degree in patients with chronic progressive MS compared with patients with OND. This may influence the levels of proteinases and cytokines in the systemic circulation and may furthermore have diagnostic implications.