MxA mRNA quantification and disability progression in interferon beta-treated multiple sclerosis patients

Abstract

Even though anti-interferon beta (IFNβ) antibodies are the main determinants of IFNβ bioactivity loss and Myxovirus-resistance protein A (MxA) is the most established marker of IFNβ biological activity in IFNβ-treated multiple sclerosis patients, their usefulness in the routine clinical practice is still debated. Therefore, 118 multiple sclerosis patients naïve for treatment were enrolled for a 3-year longitudinal observational study mimicking the conditions of a real-world setting. In order to evaluate the kinetics of bioactivity loss in blood samples obtained every 6 months after therapy initiation, MxA and interferon receptor isoform/subunit mRNA were quantified by real-time PCR, anti-IFNβ binding antibodies were detected by radioimmunoprecipitation, and neutralizing antibodies by cytopathic effect inhibition assay. Clinical measures of disease activity and disability progression were also obtained at all time points. We found that, at the individual-patient level, the response to IFNβ therapy was extremely heterogeneous, including patients with stable or transitory, early or late loss of IFNβ bioactivity, and patients with samples lacking MxA mRNA induction in spite of absence of antibodies. No interferon receptor isoform alterations that could explain these findings were found. At the group level, none of these biological features correlated with the measures of clinical disease activity or progression. However, when MxA mRNA was evaluated not at the single time point as a dichotomic marker (induced vs. non-induced), but as the mean of its values measured over the 6-to-24 month period, the increasing average MxA predicted a decreasing risk of short-term disability progression, independently from the presence of relapses. Therefore, a more bioactive treatment, even if unable to suppress relapses, reduces their severity by an amount that is proportional to MxA levels. Together with its feasibility in the routine laboratory setting, these data warrant the quantification of MxA mRNA as a primary tool for a routine monitoring of IFNβ therapy.

Figure 1. Kinetics of change of MxA, BAbs, NAbs during the study period.

MxA values (A) in patients treated with IFNβ-1a i.m. (clear circles, dashed grey line), IFNβ-1a s.c. (clear squares, solid grey line), IFNβ-1b s.c. (clear diamonds, solid black line). Average kinetics of BAb production in all samples (B) and of NAb (C) production in selected samples in patients treated with IFNβ-1a i.m. (dashed grey line), IFNβ-1a s.c. (solid grey line), IFNβ-1b s.c. (solid black line). Lines connect predicted means. Arrows indicate the cut-offs. For BAbs, 95% confidence intervals are shown; for NAbs, standard deviations are shown because confidence intervals were not calculated (no statistical inference was performed due to selection bias). MxA: myxovirus-resistance protein A; NR: normalization ratio; BAbs: binding antibodies; NAbs: neutralizing antibodies; IFNβ: interferon beta.

Figure 2. Role of IFNAR2.2 and IFNAR2.3 isoforms on IFNβ bioactivity.

Average decrease of IFNAR2.2 mRNA (A) in patients treated with IFNβ-1a i.m. (clear circles, dashed grey line), IFNβ-1a s.c. (clear squares, solid grey line), IFNβ-1b s.c. (clear diamonds, solid black line). Lines connect predicted means; 95% confidence intervals are shown. Average change in MxA mRNA induction over time in dependence of different levels of expression of IFNAR2.3 mRNA (B), as determined by multivariable mixed-model regression. IFNAR: interferon receptor; IFNβ: interferon beta;MxA: myxovirus-resistance protein A; NR: normalization ratio.

Figure 3. Average MxA as a potential marker of disability progression.

Comparison of the log₂MxA values (A) and of the area under the curve for non-transformed MxA mRNA levels calculated over the first two years of treatment (B) between patients with or without at least 1-point EDSS increase in the same period. Predicted probability of 1-point EDSS increase after 2 years of treatment in patients with at least one relapse (filled circles) vs. relapse-free patients (clear circles) in the same period (C). In (A) 95% confidence intervals are shown, while the shown p-value refers to the main effect of the ANOVA factor “1-point EDSS increase” (the interaction with the “time-point” factor was non-significant). In (B) the median, interquartile range, and range are shown as box-and-whisker plot. AUC: area under the curve; MxA: myxovirus-resistance protein A; NR: normalization ratio; EDSS: Expanded Disability Status Scale.