Biological monitoring of IFN-ß therapy in Multiple Sclerosis.

Multiple Sclerosis (MS) is a heterogeneous disease and a variable percentage of patients are non-responders to common treatment. Early diagnosis of non-responders allows change to a more useful therapy for the patient and better allocates a large amount of financial resources. Quantification of Neutralizing antibodies (Nabs) and of biological activity of IFN-ß are recognized approaches to identify immuno-pharmacological non-responders. A consistent number of studies have demonstrated that quantification of Myxovirus-induced protein A (MxA) is a valid biomarker to detect immune-pharmacological non responders after one year of treatment. Persistent high titre of Nabs and absence of biological activity predict abolition of IFN-ß effects in disease activity measured through MRI, number of relapses and disability. Guidelines and flow-charts including both Nabs and MxA quantification are presented.

Antiacquaporin 4 antibodies detection by different techniques in neuromyelitis optica patients.

BACKGROUND: Antibodies against aquaporin-4 (AQP4), a water channel particularly expressed on perivascular astrocytic podocytes, are proposed as a marker for the diagnosis of neuromyelitis optica (NMO). However, a consensus on seroprevalence and optimal detection method has not yet been reached. OBJECTIVES: To investigate the performance of different assays to detect anti-AQP4 antibodies. METHODS: We set up five different assays. Two of them were capable to detect perivascular IgG reactivity on brain tissue by immunofluorescence (NMO-IgG). Other three assays have been set to detect anti-AQP4 antibodies: immunofluorescence and flow cytometry on AQP4-expressing cells, and a radioimmunoprecipitation assay. We assessed sensitivity and specificity of these assays by interrogating sera of 33 NMO patients, 13 patients at high risk to develop NMO (hrNMO), 6 patients affected by acute partial transverse myelitis (APTM), 20 patients with multiple sclerosis (MS), and 67 age- and sex-matched healthy controls. RESULTS: We found that the presence of serum NMO-IgG and anti-AQP4 reactivity is almost exclusively restricted to patients with NMO and hrNMO. Seroprevalence and sensitivity ranged from 30 to 47%, depending on the assay. Specificity ranged from 95 to 100%. Comparing results obtained in the five assays, we noticed lack of concordance in some samples. CONCLUSIONS: Detection of NMO-IgG or anti-AQP4 antibodies may represent a valuable tool to assist neurologists in the differential diagnosis between patients with NMO, hrNMO, APTM, or MS. The current lack of a gold standard to detect anti-AQP4 antibodies implies the necessity to standardize the detection of these antibodies.

Expression and regulation of IFNalpha/beta receptor in IFNbeta-treated patients with multiple sclerosis.

BACKGROUND: The cytokine interferon beta (IFNbeta) is successfully used in the treatment of multiple sclerosis (MS), although there is a high degree of variability in the response. A common mechanism involved in the modulation of responsiveness to cytokines is represented by regulation of their receptor expression through autocrine ligand-mediated loops. The present study is aimed at investigating the regulation of IFNalpha/beta receptor (IFNAR) during IFNbeta therapy in patients with MS and at correlating it with the biologic responsiveness to the cytokine. METHODS: Quantitative PCR measurements of IFNAR-1 and the three IFNAR-2 isoforms were performed in 141 patients after short-term and long-term treatment. Patients were also regularly screened for anti-IFNbeta neutralizing antibodies (NAbs). IFN-inducible myxovirus resistance protein A messenger RNA was used as an indicator of bioactivity. RESULTS: Pretreatment levels of IFNAR-2 in patients were lower overall than in controls (p = 0.038), and high levels correlated with greater bioactivity. Upon prolonged treatment, NAb-negative patients displayed a state of decreased transmembrane IFNAR-2 expression (p < or = 0.025), whereas levels of soluble IFNAR-2 were slightly increased (p < 0.0001). The presence of NAbs reversed these effects (p < or = 0.0056). In NAb-positive patients, pretreatment expression levels of both transmembrane IFNAR-2 isoforms were significantly lower than in NAb-negative patients (p < or = 0.0089). CONCLUSIONS: Findings show that interferon-alpha/beta receptor (IFNAR)-2 isoforms are important regulators of the responsiveness to endogenous and systemically administered interferon beta (IFNbeta). They show a dual action, agonistic and antagonistic, that influences both the magnitude and the nature of the biologic response to IFNbeta. Levels of IFNAR-2 are regulated with the aim of keeping the body in a state of equilibrium, even when nonphysiologic stimuli are present.

Predictive markers for response to interferon therapy in patients with multiple sclerosis.

BACKGROUND: Prolonged therapy with interferon beta (IFN beta) often leads to the development of anti-IFN beta binding antibodies (BAbs). A subset of the BAbs is of a neutralizing nature (neutralizing antibodies, NAbs) and is associated with reduced clinical efficacy of therapy. Myxovirus-resistance-protein A (MxA) has proven to be a reliable biomarker of IFN beta bioactivity. We analyzed the prognostic value of MxA mRNA, NAbs, and BAbs on the risk of having a new relapse in IFN beta-treated patients. METHODS: A 3-year study was conducted in 137 IFN beta-treated patients. Blood samples for BAbs, NAbs, and MxA mRNA measurements were taken after 12 +/- 3 months of therapy. Analysis of relapse-free survival (RFS) was performed for all measures by using known thresholds, generating "positive" and "negative" groups. Also, time between sampling and following relapse and risk of new relapses were calculated. RESULTS: The MxA-negative group showed poorer RFS rates than the MxA-positive group [p < 0.0001, hazard ratio (HR) = 2.87]. Likewise, the NAb-positive group showed poorer RFS rates than the NAb-negative group (p =0.0013; HR = 2.49). On the contrary, BAb measurement did not show a clear clinical significance. CONCLUSIONS: Findings indicate that measurements of both myxovirus-resistance-protein A (MxA) and neutralizing antibodies (NAbs) predict the risk of new relapses; however, the slightly stronger prognostic significance of MxA mRNA and the easier method for it measurement make MxA mRNA the preferred biomarker for monitoring interferon beta (IFN beta)-treated patients. This information can be used to better tailor treatment to the individual patient with MS.

Variable responses to rituximab treatment in neuromyelitis optica (Devic's disease).

We have described two cases of Devic's disease patients treated with rituximab with different outcomes. The results indicate that there may be early unresponsiveness in very aggressive cases. Well designed clinical trials are needed to assess treatment effects in such a rare disease.

Development and validation of a real time PCR-based bioassay for quantification of neutralizing antibodies against human interferon-beta.

There are two commonly employed types of bioassays for the detection of neutralizing antibodies (NAbs) against interferon-beta (IFNbeta): the cytopatic effect assay (CPE), and the MxA (myxovirus resistance protein A) protein assay (MPA). This article describes a bioassay based on the real time PCR measurement of mRNA that results from the induction, in cultured human cells, of the MxA gene by IFNbeta. Serum samples from 104 patients with multiple sclerosis (MS) treated with IFNbeta were tested for NAbs using our real time PCR bioassay. NAbs also were measured in the same specimens by the MPA assay and CPE assay. The calibration range of the real time PCR bioassay is 0.125-30 LU/mL. The range of the intra- and inter-assay variations (coefficients of variation in log(10)) were 4.05% (range 0.88%-7.90%) and 4.42% (range 0.31%-9.15%), respectively. Samples of the three commercial preparations of IFNbeta-1a and -1b were measured showing dose-response curves parallel to that of the NIH reference IFNbeta (mean SD at the midpoint of the dose-response curve=5%). In addition, the assay was robust with respect to number of cells plated (i.e., increasing cell densities from 12x10(3)/well to 384x10(3)/well resulted in 3.03% variability in MxA expression normalized with glyceraldehyde-3 phosphate dehydrogenase). NAbs titers measured were closely comparable to those obtained by the MPA [r(spearman)=0.899; 89% of observed agreements; K=0.779] and the CPE [r(spearman)=0.7899); 86%; K=0.729] assays. Despite the obvious disadvantage of cost, when carried out according to quality assurance guidelines for molecular diagnostics the new MxA gene-expression assay (MGA) has significant advantages over the other methods for testing NAbs: it has excellent reliability and reproducibility, and utilizes equipment and methodologies already accessible in many clinical laboratories.

Biological markers of interferon-beta therapy: comparison among interferon-stimulated genes MxA, TRAIL and XAF-1.

Biological activity of interferon-beta (IFNbeta) can be assessed by measuring IFN-stimulated genes (ISGs). Among them, myxovirus resistance protein A (MxA) appears to have the highest specificity, but it has no role in the pathogenesis of multiple sclerosis (MS). To investigate the reliability of MxA as a biomarker, we compared its expression to that of two other ISGs: TNF-related apoptosis-inducing ligand (TRAIL) and X-linked inhibitor of apoptosis factor-1 (XAF-1). Both were shown to be involved in immunoregulatory mechanisms and might play a role in MS. Quantitative-PCR measurements were performed in peripheral blood mononuclear cells from 73 MS patients after short-term and long-term treatment with IFNbeta. A time-dependent response for multiple ISGs was observed in all patients after short-term treatment. In contrast, long-term treatment induced concurrent inhibition of ISGs in 12.3% (9/73) of patients, in whom neutralizing antibodies (NAbs) were detectable. Besides, 22% (16/73) of chronically treated patients showed a non-NAbs-related abrogation of TRAIL expression. In summary, 1) MxA expression was significantly higher than both TRAIL and XAF-1, and 2) MxA was the most sensitive gene to detect decreased bioavailability due to NAbs. These findings identify MxA as an appropriate biomarker for IFNbeta, although there is no evidence for a functional role of it in MS.

Qualitative and quantitative analysis of antibody response against IFNbeta in patients with multiple sclerosis.

To date, inter- and intra-laboratory consistency of binding assays for measuring anti-interferon (IFN)beta antibodies has not been assessed. In this investigation, two independent laboratories tested a library of 80 serum specimens obtained from multiple sclerosis (MS) patients treated with IFNbeta. For binding antibodies (BAbs) evaluations, each laboratory used both a capture-ELISA (cELISA) and an enzyme-immuno-assay (EIA), which is commercially available. Samples were also tested for neutralizing antibodies (NAbs). Data demonstrated good intra-laboratory reliability (r(pearson) > or = 0.86), and a good overall agreement between the results obtained from the two centers, using both the cELISA (69/80 of observed agreements) and the EIA (67/80). Accordingly, kappa coefficients (K) showed good concurrence (K > or = 0.651). There was also substantial agreement between cELISA and EIA measurements, as performed in both centers (Orbassano, 66/80, K = 0.631; Basel, 70/80, K = 0.717). However, by comparing NAbs and BAbs titers obtained with both assays, we found that a high degree of BAb-negative samples were positive in NAb-assay. Thus, our study does not support the usefulness of ELISA-based BAb assays as a screening tool for NAbs. Otherwise, BAb-assays can be used as a confirmation test, indicating that the decrease of the biological effects is due to antibodies. In this context, both ELISA-based assays are equally reliable techniques.

Fate of multiple sclerosis patients positive for neutralising antibodies towards interferon beta shifted to alternative treatments.

Relapsing-remitting multiple sclerosis (MS) has a very fluctuating course and responsiveness to interferon beta (IFN-beta) treatment in each patient is extremely difficult. Agreement exists about the negative role of neutralising antibodies (NAbs) on clinical efficacy and markers of IFN-beta bioavailability have been studied; no guidelines exist yet about what to do when a patient becomes NAbs positive or IFN biological activity is lost. In this study 405 MS patients have been longitudinally studied for NAbs and MxA expression. A spontaneous disappearance of NAbs was observed in a few patients with low antibody titre; according to the clinical course, a therapeutic modification has been made in patients persistently NAbs positive; in these patients NAbs persisted over time despite the interruption of IFN therapy.

Biological responsiveness to first injections of interferon-beta in patients with multiple sclerosis.

This study is the first to evaluate biological response to first injections of interferon-beta (IFNbeta) in patients with multiple sclerosis. MxA mRNA was measured in 96 patients receiving IFNbeta-1a (Avonex, n=32), IFNbeta-1b (Betaferon, n=19), IFNbeta-1a (Rebif) 22 microg (n=30), or IFNbeta-1a 44 microg (n=15). Patients were analysed before, 3 and 24 h after the first injection, and 12 h after the second administration. Results showed that up-regulation was evident within 3 h of IFNbeta injection, peaked 12 h after injection, and progressively declined 24 h after administration. The cumulative responses were similar after a single administration, regardless of product/dose. Moreover, data indicate that the abolition of the biological activity detected during IFNbeta therapy is due to underlying phenomena (e.g., neutralizing antibodies), because all patients were constitutively responders to IFNbeta at treatment initiation.

Biological activity of interferon betas in patients with multiple sclerosis is affected by treatment regimen and neutralising antibodies.

BACKGROUND: MxA gene expression is one of the most appropriate markers of biological activity of exogenous interferon (IFN) beta. METHODS: We quantified MxA mRNA for five consecutive days in 62 patients treated with IFN beta (16, Avonex; 10, Betaferon; 24, Rebif 22; 12, Rebif 44), by quantitative-competitive polymerase chain reaction. Every three months, IFN beta induced neutralising antibodies (NAbs) were evaluated in sera using a cytopathic effect assay. RESULTS: Two categories of patients were identified: one group (49/62) had a sharp post-injection increase in MxA expression (defined as "IFN beta biological responder"), whereas the other group (13/62) had no MxA induction after IFN beta administrations (defined as "IFN beta biological non-responder"). In 11/13 biological non-responders, the persistent presence of NAbs correlated with abolished biological activity, independently of treatment regimen. The two remaining IFN beta biological non-responders were NAb-. Among the 49 IFN beta biological responders, biological activity was comparable between the four preparations on day 2 and 3 (+12 and +36 hours post-injection), but it was greater in Betaferon and both Rebif preparations on day 1, 4, and 5. In biological responders treated three times a week, only 82% (59/72) of injections were considered effective, compared with 100% (13/13) of Avonex injections. CONCLUSION: Our results suggest that an optimal IFN beta regimen is not yet available: Avonex, given once a week, shows lower cumulative biological activity. On the other hand, both Betaferon and Rebif, given three times a week, show 18% biologically ineffective injections and higher risk of developing NAbs, which abolish biological activity.

Persistent neutralizing antibodies abolish the interferon beta bioavailability in MS patients.

BACKGROUND: MxA is an antiviral protein exclusively induced by type I interferons (IFN) and some viruses, and MxA gene expression is one of the most appropriate markers for measuring the biologic activity of exogenous IFNbeta. METHODS: A new quantitative-competitive PCR method was used to quantify MxA mRNA in peripheral blood mononuclear cells of 99 treatment-naïve and 92 IFNbeta-treated patients with MS (22 Avonex, 17 Betaferon, and 53 Rebif-22). Every 3 months, IFNbeta-induced neutralizing antibodies (NAb) were evaluated in sera using a cytopathic effect assay. Three categories of patients were identified: NAb negative (NAb-), persistent NAb positive (NAb+, >or=2 consecutive positive samples), and isolated NAb+ (one positive sample). RESULTS: Treatment-naïve patients expressed detectable MxA mRNA levels (mean = 36 +/- 32 fg MxA/pg glyceraldehyde-3-phosphate dehydrogenase (GAPDH); range 1 to 160) and an upper normal threshold was established (mean + 3 SD = 132 fg MxA/pg GAPDH). IFNbeta-treated patients exhibited more than 11-fold higher levels (mean = 412 +/- 282 fg MxA/pg GAPDH; range 16 to 1,172). However, 17 patients did not exhibit an increase in MxA mRNA level; 15 of these 17 patients showed a concurrent Nab+ titer. Moreover, 13 were persistent NAb+. Isolated NAb+ patients did not show a decrease in bioavailability of IFNbeta (n = 9; mean = 567 +/- 366 fg MxA/pg GAPDH; range 83 to 1,120). In NAb- patients, bioavailability was comparable among the three different IFNbeta preparations 12 hours after injection. CONCLUSION: During IFNbeta therapy, the presence of NAb reduced or abolished bioavailability in a relevant percentage of patients. These data could be important for the early detection of patients with MS who are not responsive to IFNbeta therapy.