Drug Efficacy Monitoring in Pharmacotherapy of Multiple Sclerosis With Biological Agents.

Multiple sclerosis is a heterogenous disease. Although several EMA-approved disease-modifying treatments including biopharmaceuticals are available, their efficacy is limited, and a certain percentage of patients are always nonresponsive. Drug efficacy monitoring is an important tool to identify these nonresponsive patients early on. Currently, detection of antidrug antibodies and quantification of biological activity are used as methods of efficacy monitoring for interferon beta and natalizumab therapies. For natalizumab and alemtuzumab treatments, drug level quantification could be an essential component of the overall disease management. Thus, utilization and development of strategies to determine treatment response are vital aspects of multiple sclerosis management given the tremendous clinical and economic promise of this tool.

Natalizumab treatment reduces L-selectin (CD62L) in CD4+ T cells.

BACKGROUND: The purpose of this research was to validate the low expression of L-selectin (CD62L) in natalizumab (NTZ)-treated patients. CD62L is involved in rolling and transmigration of leukocyte cells. A correlation between CD62LCD4+ T cells low expression and progressive multifocal leukoencephalopathy (PML) development has been suggested in multiple sclerosis (MS) patients treated with NTZ. METHODS: We performed a flow cytometric analysis on peripheral blood mononuclear cells (PBMC); we collected from 23 healthy donors and 225 MS patients: untreated (n = 19) or treated with NTZ (n = 113), interferon-beta (n = 26), glatiramer acetate (n = 26), fingolimod (n = 23) and rituximab (n = 18). We have also analysed two PML/IRIS (immune reconstitution inflammatory syndrome) patients and four longitudinal samples of a NTZ-treated patients before and during the development of a clinical asymptomatic magnetic resonance imaging (MRI) lesion confirmed as PML by cerebrospinal fluid (CSF) examination. Thirty-five NTZ-treated patients were studied longitudinally with three samples taken 4 months apart. RESULTS: The NTZ-treated patients showed a lower percentage of CD62L (33.68%, n = 113) than first-line treated patients (44.24%, n = 52, p = 0.0004). NTZ effect was already clear during the first year of treatment (34.68 ; p = 0.0184); it persisted in the following years and disappeared after drug withdrawal (44.08%). Three percent of longitudinally analysed patients showed a percentage of CD62LCD4+ T cells under a hypothetical threshold and one patient with asymptomatic PML belongs to a group which expressed low percentage of CD62LCD4+ T cells. CONCLUSIONS: Our research confirms that NTZ has a specific effect on CD62LCD4+ T cells consisting in decreasing of the number of positive cells. The low level of CD62L found in a clinically asymptomatic PML patient strengthens its potential usefulness as a biomarker of high PML risk in NTZ-treated patients. A larger study is required to better confirm the data.

Drug Holiday of Interferon Beta 1b in Multiple Sclerosis: A Pilot, Randomized, Single Blind Study of Non-inferiority.

Introduction: To compare a schedule with cyclic withdrawal (CW) of interferon beta (IFN-b) 1b, respect to the full regimen (FR), in relapsing-remitting MS (RR-MS). Methods: Participants were randomly assigned to CW or FR schedule and monthly monitored with brain MRI scans for 12 months (three of run-in and 9 of treatment). CW schedule included drug withdrawal for 1 month after two of treatment for a total of three quarters over the 9-month treatment period. The assessing neurologist and the expert neuroradiologists were blind. After the blind phase of the study all participants took their indicated disease modifying therapies in a prospectively planned, open-label extension phase (up to 120 months). Results: Of 60 randomized subjects 56 (29 in FR and 27 in CW group) completed the single-blind phase: the two groups were comparable, except for a non-significant difference in the number of contrast-enhanced lesions (CEL) at the end of run-in. The two-sided 90% CI for the ratio between median number of cumulative CEL was 0.29-1.07, allowing to significantly reject the null hypothesis of a ratio ≥1.2 and to meet the primary end-point of non-inferiority (the threshold and the ratio between median were chosen according to the non-normal distribution of the data). The differences (CW vs. FR) were also non-significant for secondary end points: mean cumulative number of T2-weighted new and enlarging lesions (3.48 ± 5.34 vs. 3.86 ± 6.76); mean number and volume (cm3) of black holes (1.24 ± 1.61 vs. 2.71 ± 4.56; 489.11 ± 1488.12 vs. 204.48 ± 396.98); number of patients with at least an active scan (21 vs. 22); mean relapse rate (0.52 ± 0.89 vs. 0.34 ± 0.66), relapse risk ratio adjusted for baseline variables (2.15 [0.64-7.18]), EDSS score (1.0 [1-1.56] vs. 1.5 [1-1.78]), proportion of patients with antibodies anti-IFN (5 [21%] vs. 8 [36%]). Fifty-four patients (27 for each study arm) completed the open-label phase. The annualized RR, EDSS, proportion of patients shifting to progressive disease and hazard ratio of shifting, adjusting for baseline covariates, were comparable between the two study groups. Conclusions: A calendar with CW was non-inferior than FR at the beginning of IFN-b therapy, and may not affect the long-term outcome. Clinical Trial Registration: www.ClinicalTrials.gov, identifier: NCT00270816.

The Soluble Form of Toll-Like Receptor 2 Is Elevated in Serum of Multiple Sclerosis Patients: A Novel Potential Disease Biomarker.

Multiple sclerosis (MS) is an immune-mediated inflammatory demyelinating disease of the central nervous system. It was previously shown that toll-like receptor (TLR)-2 signaling plays a key role in the murine experimental autoimmune encephalomyelitis (EAE) model of MS, and that TLR2-stimulation of regulatory T cells (Tregs) promotes their conversion to T helper 17 (Th17) cells. Here, we sought potential sources of TLR2 stimulation and evidence of TLR2 activity in MS patient clinical samples. Soluble TLR2 (sTLR2) was found to be significantly elevated in sera of MS patients (n = 21), in both relapse and remission, compared to healthy controls (HC) (n = 24). This was not associated with the acute phase reaction (APR) as measured by serum C-reactive protein (CRP) level, which was similarly increased in MS patients compared to controls. An independent validation cohort from a different ethnic background showed a similar upward trend in mean sTLR2 values in relapsing-remitting MS (RRMS) patients, and significant differences in sTLR2 values between patients and HC were preserved when the data from the two cohorts were pooled together (n = 41 RRMS and 44 HC, P = 0.0006). TLR2-stimulants, measured using a human embryonic kidney (HEK)-293 cells transfectant reporter assay, were significantly higher in urine of MS patients than HC. A screen of several common urinary tract infections (UTI)-related organisms showed strong induction of TLR2-signaling in the same assay. Taken together, these results indicate that two different markers of TLR2-activity-urinary TLR2-stimulants and serum sTLR2 levels-are significantly elevated in MS patients compared to HC.

Anti-interferon-beta neutralising activity is not entirely mediated by antibodies.

Many multiple sclerosis (MS) patients treated with interferon-beta (IFNbeta) develop anti-IFNbeta antibodies (BAbs), which can interfere with both in vitro and in vivo bioactivity of the injected cytokine. Objective of this study was to correlate these measures. Among the 256 enrolled patients, 11 (4.3%) showed a significant inhibition of the IFNbeta activity in vitro, but no measurable BAbs. As a whole, in vivo bioactivity was inhibited in 9/11 (82%) of these patients. A minority of IFNbeta treated patients have a non-antibody mediated neutralising activity, which competitively inhibits the bioactivity both in vitro and in vivo.

Detection of potassium channel KIR4.1 antibodies in Multiple Sclerosis patients.

The presence of KIR4.1 antibodies has been proposed to be a characteristic of Multiple Sclerosis (MS). This could have a significant impact on disease management. However, the validation of the initial findings has failed till date. Conflicting results have been attributed to difficulties in isolating the lower-glycosylated (LG) KIR4.1 expressed in oligodendrocytes, the putative target antigen of autoantibodies. The aim of this study is to verify the presence of KIR4.1 antibodies in MS patients, by independently replicating the originally-described procedure. Assay procedure consisted of KIR4.1 expression in HEK293 cells, 3-step elution to isolate LG-KIR4.1 in elution fraction 3, and ELISA. Sera of 48 MS patients and 46 HCs were studied in 21 working sessions. In a preliminary analysis, we observed different KIR4.1 antibody levels between MS patients and Healthy Controls (HCs). However, a high variability across working sessions was observed and the sensitivity of the assay was very low. Thus, stringent criteria were established in order to identify working sessions in which the pure LG-KIR4.1 was isolated. As per these criteria, we detected LG-KIR4.1 antibodies in 28% of MS patients and 5% of HCs. Unlike previous findings, this study is in agreement with the original report. We propose further efforts be made towards the development of a uniform method to establish the detection of KIR4.1 antibodies in MS patients.

Role of Anti-Osteopontin Antibodies in Multiple Sclerosis and Experimental Autoimmune Encephalomyelitis.

Osteopontin (OPN) is highly expressed in demyelinating lesions in multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). OPN is cleaved by thrombin into N- (OPN-N) and C-terminal (OPN-C) fragments with different ligands and functions. In EAE, administering recombinant OPN induces relapses, whereas treatment with anti-OPN antibodies ameliorates the disease. Anti-OPN autoantibodies (autoAbs) are spontaneously produced during EAE but have never been detected in MS. The aim of the study was to evaluate anti-OPN autoAbs in the serum of MS patients, correlate them with disease course, and recapitulate the human findings in EAE. We performed ELISA in the serum of 122 patients collected cross-sectionally, and 50 patients with relapsing-remitting (RR) disease collected at diagnosis and followed longitudinally for 10 years. In the cross-sectional patients, the autoAb levels were higher in the RR patients than in the primary- and secondary-progressive MS and healthy control groups, and they were highest in the initial stages of the disease. In the longitudinal group, the levels at diagnosis directly correlated with the number of relapses during the following 10 years. Moreover, in patients with active disease, who underwent disease-modifying treatments, autoAbs were higher than in untreated patients and were associated with low MS severity score. The autoAb displayed neutralizing activity and mainly recognized OPN-C rather than OPN-N. To confirm the clinical effect of these autoAbs in vivo, EAE was induced using myelin oligodendrocyte glycoprotein MOG35-55 in C57BL/6 mice pre-vaccinated with ovalbumin (OVA)-linked OPN or OVA alone. We then evaluated the titer of antibodies to OPN, the clinical scores and in vitro cytokine secretion by spleen lymphocytes. Vaccination significantly induced antibodies against OPN during EAE, decreased disease severity, and the protective effect was correlated with decreased T cell secretion of interleukin 17 and interferon-γ ex vivo. The best effect was obtained with OPN-C, which induced significantly faster and more complete remission than other OPN vaccines. In conclusion, these data suggest that production of anti-OPN autoAbs may favor remission in both MS and EAE. Novel strategies boosting their levels, such as vaccination or passive immunization, may be proposed as a future strategy in personalized MS therapy.

Vitamin D Binding Protein Isoforms and Apolipoprotein E in Cerebrospinal Fluid as Prognostic Biomarkers of Multiple Sclerosis.

BACKGROUND: Multiple sclerosis (MS) is a multifactorial autoimmune disease of the central nervous system with a heterogeneous and unpredictable course. To date there are no prognostic biomarkers even if they would be extremely useful for early patient intervention with personalized therapies. In this context, the analysis of inter-individual differences in cerebrospinal fluid (CSF) proteome may lead to the discovery of biological markers that are able to distinguish the various clinical forms at diagnosis. METHODS: To this aim, a two dimensional electrophoresis (2-DE) study was carried out on individual CSF samples from 24 untreated women who underwent lumbar puncture (LP) for suspected MS. The patients were clinically monitored for 5 years and then classified according to the degree of disease aggressiveness and the disease-modifying therapies prescribed during follow up. RESULTS: The hierarchical cluster analysis of 2-DE dataset revealed three protein spots which were identified by means of mass spectrometry as Apolipoprotein E (ApoE) and two isoforms of vitamin D binding protein (DBP). These three protein spots enabled us to subdivide the patients into subgroups correlated with clinical classification (MS aggressive forms identification: 80%). In particular, we observed an opposite trend of values for the two protein spots corresponding to different DBP isoforms suggesting a role of a post-translational modification rather than the total protein content in patient categorization. CONCLUSIONS: These findings proved to be very interesting and innovative and may be developed as new candidate prognostic biomarkers of MS aggressiveness, if confirmed.

Comparison of three PCR assays for the evaluation of interferon-beta biological activity in patients with multiple sclerosis.

BACKGROUND: The gene expression of the myxovirus-resistant protein A (MxA) gene is a sensitive measure of the biological response of therapeutically applied interferon-beta (IFNbeta) and of its reduced bioavailability due to inhibiting factors such as IFNbeta-induced neutralizing antibodies (NAbs). METHODS: We compared three methods for MxA mRNA quantification in 826 peripheral blood mononuclear cell (PBMC) samples obtained from patients with multiple sclerosis (MS). MxA mRNA measurements were performed using quantitative-competitive (qc)-PCR, real time-PCR, and the new semi-quantitative (sq)-PCR assay (MxA IBRIDOGEN). RESULTS: According to the treatment status (untreated samples versus NAb-negative treated samples), real time-PCR gave the highest specificity (93%). Slightly lower specificities were obtained with qc-PCR and sq-PCR (both 91%). qc-PCR showed the highest sensitivity (97%) compared with both real time-PCR (94%) and sq-PCR (95%). A positive correlation was found between qc-PCR and real time-PCR measurements (rspearman=0.776; p<0.0001), which also showed 90% agreement based on a statistically calculated threshold. Likewise, sq-PCR evaluations showed 84% and 79% agreement with qc-PCR and real time-PCR measurements, respectively. In addition, we showed a concordance of 89% between three sq-PCR kits. CONCLUSIONS: All three methods displayed high specificity for MxA gene expression analysis, allowing the detection of patients in whom IFNbeta did not have any biological action. qc-PCR and real time-PCR are both useful during clinical trials demanding quantitative data of biological activity, whereas sq-PCR could prove useful for routine screening purposes because it is easy to perform and can be done in not specialized laboratories.

One-year evaluation of factors affecting the biological activity of interferon beta in multiple sclerosis patients.

MxA is an antiviral protein induced by type I interferons (IFN) and some viruses; MxA gene expression is an appropriate marker for measuring biologic activity of exogenous IFNß, as its induction indicates IFNAR receptor stimulation. A recent study has shown that measurement of MxA mRNA, after 1 year of treatment, predicts clinical responsiveness to IFNß therapy. Loss of IFNß bioactivity is mostly due to anti-IFNß antibodies (both neutralizing and binding), non-compliance and receptor saturation. The aim of this study was to evaluate all possible causes of loss of IFNß bioactivity after 1 year in treated patients. One hundred sixty-seven multiple sclerosis (MS) patients were included. One year after beginning IFNß therapy, each patient underwent a blood test; MxA gene expression was measured by real time PCR, antiviral CPE assay to detect neutralizing antibodies (NAbs), and capture-ELISA (cELISA) to measure binding antibodies (BAbs). For MxA an upper normal threshold of 87 (RE) was considered, 20 TRU/mL was the threshold for NAbs, and 1 U for BAbs positivity. Thirty-seven out of 167 patients (22%) were MxA-negative; of these, 22 were both BAbs and NAbs+, whereas 12 were BAbs+ but Nabs-, and three were both BAbs and NAbs-. The following conclusions were drawn from the study: (1) MxA mRNA should be measured after 1 year of IFNß therapy; (2) after 1 year of IFNß treatment, absence of IFNß bioactivity was detected in 22% of the patients; (3) different biological phenomena and reduced compliance explain this absence; (4) identification of the reason for absence of IFN bioactivity improves patients' management.

Loss of braking signals during inflammation: a factor affecting the development and disease course of multiple sclerosis.

BACKGROUND: In a recent genome-wide transcriptional analysis, we identified a gene signature for multiple sclerosis (MS), which reverted back to normal during pregnancy. Reversion was particularly evident for 7 genes: SOCS2, TNFAIP3, NR4A2, CXCR4, POLR2J, FAM49B, and STAG3L1, most of which encode negative regulators of inflammation. OBJECTIVES: To corroborate dysregulation of genes, to evaluate the prognostic value of genes, and to study modulation of genes during different treatments. DESIGN: Comparison study. SETTING: Italian referral center for MS. PATIENTS: Quantitative polymerase chain reaction measurements were performed for 274 patients with MS and 60 healthy controls. Of the 274 patients with MS, 113 were treatment-naive patients in the initial stages of their disorder who were followed up in real-world clinical settings and categorized on the basis of disease course. The remaining 161 patients with MS received disease-modifying therapies (55 patients were treated with interferon beta, 52 with glatiramer acetate, and 54 with natalizumab) for a mean (SD) of 12 (2) months. MAIN OUTCOME MEASURES: Gene expression levels, relapse rate, and change in Expanded Disability Status Scale. RESULTS: We found a dysregulated gene pathway (P ≤ .006), with a downregulation of genes encoding negative regulators. The SOCS2, NR4A2, and TNFAIP3 genes were inversely correlated with both relapse rate (P ≤ .002) and change in Expanded Disability Status Scale (P ≤ .005). SOCS2 was modulated by both interferon beta and glatiramer acetate, TNFAIP3 was modulated by glatiramer acetate, and NR4A2 was not altered at all. No changes were induced by natalizumab. CONCLUSIONS: We demonstrate that there is a new molecular pathogenic mechanism that underlies the initiation and progression of MS. Defects in negative-feedback loops of inflammation lead to an overactivation of the immune system so as to predispose the brain to inflammation-sensitive MS.