Introduction
Despite significant advances in long-term immunomodulatory therapies for multiple sclerosis (MS), treatment for acute MS relapses has remained largely unaltered for the past 20 years [
1]. International guidelines recommend the administration of high-dose intravenous methylprednisolone (MPS), up to 1000 mg daily for three to five consecutive days, for the alleviation of symptoms [
2,
3]. However, approximately 25% of patients respond insufficiently to the first cycle of MPS [
4] and current guidelines recommend a second, double-dose cycle of up to 2000 mg daily for three to five consecutive days [
5]. Therapeutic apheresis for the clearance of soluble plasma components has entered clinical routine as an alternative, including therapeutic plasma exchange as well as immunoadsorption (IA) [
6,
7]. Although not yet evaluated in randomized clinical trials, IA has been proven to be effective in two prospective [
8,
9] and several retrospective studies [
10‐
13], reporting response rates between 50 and 86% among patients suffering from isolated optic neuritis, clinically isolated syndrome, or relapsing MS (RMS) who had previously responded insufficiently to MPS[
8,
10‐
13]. Consequently, several guidelines recommend IA as an adjunctive [
14] treatment to increasing the chance of recovery from steroid-refractory relapses [
2].
Apheresis treatment is, however, invasive—often requiring the insertion of a central venous catheter—and more expensive compared to MPS. In addition, there is no comparison in the literature between the efficacy of any kind of apheresis treatment and a second cycle of double-dose MPS.
Additionally, substantial differences exist between both treatment regimens in terms of mechanism of action. MPS treatment is thought to target T cells almost exclusively by induction of apoptosis and exert only a minor effect on soluble factors [
15]. IA, however is supposed to largely modulate soluble but not cellular factors [
6]. However, little is known how immunoadsorption alleviates relapse symptoms in detail and thus, predictors for treatment response are unknown yet desired given the abovementioned risks for treatment.
Therefore, we conducted a prospective clinical study comparing the clinical outcomes of a second course of MPS versus six courses of tryptophane column-based IA in patients refractory to the initial cycle of MPS for the treatment of an acute MS relapse. Furthermore, we performed extensive analyses of cellular and soluble factors in peripheral blood to further elucidate the impact of either treatment on the immune system.
Methods
Patients
INCIDENT-MS (ImmuNoadsorption versus high-dose intravenous CorticosteroIDs in RElapsing Multiple Sclerosis—AssessmenT of MechaniSm of Action) was designed as a larger prospective observational study to assess the safety and efficacy of immunoadsorption versus methyl prednisolone for refractory MS relapses and to evaluate the mechanism of action for each treatment. Recruitment included all patients admitted to the Department of Neurology, University Hospital Muenster, Germany.
Patients that underwent a first course of intravenous methylprednisolone (MPS) (1000 mg per day for five consecutive days; “initiation treatment”) yet experienced persistent deficits were identified and were offered to receive either another course of MPS (2000 mg per day for five consecutive days) or tryptophan immunoadsorption (six courses every other day; “escalation treatment”). Treatment regimen was determined by shared-decision making following thorough information of patients by consultants not connected to the study. Patients were enrolled from August 2018—August 2020. In- and exclusion criteria are listed in Table
1.
Table 1
In- and exclusion criteria of the INCIDENT-MS study
Inclusion criteria |
• Signed informed consent form |
• Established diagnosis of relapsing MS according to the 2017 revised McDonald-criteria |
• Incomplete remission of symptoms after administration of 1000 mg intravenous (methyl-) prednisolone as measured by the EDSS value: •EDSS value baseline + 1, if pre-treatment EDSS value is ≤ 3.5; EDSS value baseline + 0.5, if pre-treatment EDSS value is > 3.5 |
• Absence of clinically apparent fever or concomitant infection. Asymptomatic urinary tract infection is not considered as significant infection unless it leads to an at least two-fold increase of C-reactive protein levels above ULN (upper level of normal) |
Exclusion criteria |
• Patients with a documented EDSS > 6.5 prior to recent relapse. Patients that are suspicious to having entered a secondary-progressive course of the disease at the time point of screening |
• Patients that previously received either escalation treatment for refractory MS relapses |
• Female patients known to be pregnant or unwilling to perform a pregnancy test |
• Patients that receive immunosuppressive treatment for diseases other than RRMS or that receive long-term corticosteroid treatment |
• Patients that received less than 3 g or more than 5 g (methyl-)prednisolone prior to initial admission or that received (methyl-)prednisolone for more than 8 days |
• Patients with verified infection by human-immunodeficiency-virus or hepatitis-c-virus |
• Patients with medical, psychiatric, cognitive, or other conditions that, in the investigator’s opinion, compromise the patient's ability to understand the patient information, to give informed consent, or to complete the study |
• Patients with significant psychiatric comorbidities with the necessity of specific treatment during administration of intravenous steroids at the investigators discretion |
• Patients on regular medication with inhibitors of angiotensin-converting-enzyme (ACE) inhibitors |
• Patients with major impairment of the blood coagulation system with increased risk during establishment of central venous catheters as follows: •therapy with anticoagulants for any purpose other than prevention of deep vein thrombosis •elevation of INR above 1.5, elevation of PTT above 50 s •thrombocytopenia below 50.000/μL •intake of dual antiplatelet therapy |
Initially, the study was designed to incorporate a larger cohort of 204 patients in order to allow confirmation of various secondary endpoints as well as immunologic analyses but health insurances temporarily halted reimbursement of IA treatment for RMS patients. Thus, we here present results from the core study population which were sufficient to evaluate the primary outcome at common significance parameters (α level: 0.05, power: 80%).
Within the core population, a minimal sample size of 15 patients per group was calculation upon results from a previous retrospective analysis of patients undergoing treatment for refractory MS relapses in our department [
7]. However, the decision was made to include all suitable patients during the core study period to avoid selection bias, even if 15 patients per group were exceeded.
Treatment
MPS (2000 mg per day for five consecutive days) was administered according to clinical guidelines and was accompanied by prophylaxis against gastric ulcers, venous thrombosis and osteoporosis.
IA was performed using jugular central venous catheters. Plasma separation was performed using the Octo Nova extracorporeal circuit technology (SV 4.30.6, Front 4.30.6) and the polyethylene plasma separator OP-05W (Asahi Kasei Kuraray, Tokyo, Japan). Plasma filtrate passes through a tryptophan column (Immunosorba TR-350, Diamed, Germany). For all treatments unfractionated heparin was used for anticoagulation. Six sessions with a treated plasma volume of 2.5 L were performed within 6–8 days.
Outcome measurements
Patients were examined by two trained neurologists at baseline (prior to initiation of escalation treatment), including the assessment of expanded disability status scale (EDSS) score, multiple sclerosis functional composite (MSFC) testing, and the SF36 questionnaire to assess health-related quality-of-life (QoL). Relapses were determined to be either “visual”, “motor” or “sensory”, depending on the clinical presentation. Within the first two days, patients underwent electrophysiology testing, including evoked potentials (full-field visual-evoked potentials and somatosensory-evoked potentials from tibial nerves). These tests were repeated at discharge as well as at three-month follow-up.
Physicians involved in testing were given no information regarding treatment and CVC were removed prior testing in IA patients. All tests were conducted in a standardized environment at our scientific outpatient clinic for further reduction of bias. Patients were screened for adverse events daily. Patients with incomplete remission following escalation treatment were offered to receive the respective treatment as rescue therapy. Patients undergoing this procedure were again examined as described above at the beginning and end of rescue therapy.
For determination of the treatment response, we performed analysis of EDSS function score changes according to the system proposed by Conway and colleagues [
17]. Briefly, treatment response was stratified according to relative function system score changes (“full/best” vs. “average” vs. “worse/none”) and proportions of patients within each response group were compared. We defined the proportion of patients with “full/best” + ”average” recovery vs. the proportion of patients with “worse/none” response per treatment group assessed upon discharge from escalation treatment as primary endpoint. Secondary endpoints comprised the treatment response at follow-up, EDSS scores at discharge and follow-up, MSFC scores, SF-36 scores, and evoked potentials. MSFC and SF-36 results were interpreted using the reference manuals. Evoked potential outcomes were categorized using a six-step ordinal system as described by Jung et al. [
16]. Tertiary analyses comprised evaluation of cellular and soluble factors from peripheral blood (see below).
Multiparameter flow cytometry
Peripheral blood mononuclear cell (PBMC) samples analyzed by flow cytometry were generated by density gradient centrifugation using Lymphoprep (Stemcell technologies) and subsequent cryo-preservation in serum-free medium (CTL-Cryo ABC Media Kit, Immunospot) in the vapor phase of a liquid nitrogen tank.
For flow cytometry, PBMC were thawed by placing in a 37 °C water bath for 8 min. The cell suspension was transferred to a 50 ml conical tube and 9 ml pre-warmed RPMI-medium (RPMI (Sigma Aldrich), 10% FCS Gold Plus (BioSell), 1% Glutamax (Gibco), 1% Na-Pyruvate (invitrogen)) was added prior to centrifugation at 300
g for 10 min. Supernatant was discarded and the cell pellet was resuspended in RPMI-medium. PBMC were counted and viability was assessed using a Countess II automated cell counter (Invitrogen). Subsequently, PBMC were subjected to immune phenotyping by flow cytometry. Therefore, PBMC were directly stained with fluorochrome-conjugated antibodies (for complete list see Additional file
1: Table S1) In addition, intra-cellular/-nuclear epitopes were investigated by incubation of PBMC with Perm/Fix buffer (BD Biosciences) for 20 min at room temperature and subsequent staining for 30 min at 4 °C in Perm buffer (BD Biosciences). Samples were acquired on a Cytoflex 13-color flow cytometer (Beckman Coulter) under daily quality control by CytoFlex Daily QC Fluorospheres (Beckman Coulter). Resulting data was analyzed using Kaluza 2.1 (Beckman Coulter) by manual gating on PBMC subsets. Absolute cell counts were calculated from differential blood counts which were acquired during clinical routine upon sampling of study blood.
Serum analysis
Serum samples were collected following standard procedure. After 30–45 min (min) at room temperature, separation of serum was achieved by differential centrifugation at 2000
g for 10 min at room temperature. Samples were aliquoted in polypropylene tubes and stored at − 80 °C until further analysis. sNfL was measured by single molecule array with a SiMoA HD-1 (Quanterix) using the NF-Light Advantage Kit (Quanterix) according to the manufacturer’s instructions. Samples were measured in duplicate. Blinded sNfL measurements were performed, without information about clinical data. For cytokine analysis, serum samples were sent to Olink (Uppsala, Sweden) using the “Olink target 48-cytokine” assay containing 45 selected cytokines (see Additional file
1: Table S2).
Statistical analysis
Epidemiological data at baseline were analysed using descriptive statistics and comparisons among groups were made using the Mann–Whitney U test or the Kruskal–Wallis test for continuous variables and Fisher’s exact test for categorical variables. To assess recovery, function system scores at baseline and discharge were categorized as “best”, “average”, or “worse”, according to previous work published by Conway and colleagues [
17]. For adjustment, logistic regression models were established using “best/average vs. worse recovery” (discharge) or “best vs. average/worse recovery” (follow-up) as dependent variables and “sex (male/female)”, “baseline-EDSS”, “affected function system (visual/motor/sensory)”, and “first demyelinating event (yes/no)” as covariates in an enter method (with p-values derived from a likelihood-ratio test). Differences of the MSFC at discharge or follow-up compared to baseline were analysed using linear regression models using these covariates.
Experimental data were analysed using the Mann–Whitney U test or Kruskal–Wallis test including Dunn’s post-test. For comparison of longitudinal data sets, Friedman’s test was used. Volcano plots were generated by plotting log2 values of the relative difference between the medians (continuous) or means (categorical parameters) against the p-values, calculated using the Mann–Whitney U test. Outside from pre-defined clinical endpoints (proportion of patients with a response to treatment), data were considered exploratory. Bonferroni-correction was applied to immunologic analysis where appropriate and is shown in the respective figures. A p-value below 0.05 was considered significant.
Ethical approval and study registration
Ethical approval was given by local authorities (Medical Council Westphalia-Lippe; 2018-261-f-S) and the study was listed in the National Institute of Health’s registry [clinicaltrials.gov; NCT04450030].
Consent for publication
Not applicable.
Data availability statement
Anonymized data will be shared with any qualified investigator upon reasonable request.
Discussion
IA is often regarded as alternative to escalated MPS or therapeutic plasma exchange in patients with acute RMS, yet high-level evidence regarding its effectiveness and safety profile is lacking [
6]. Our data demonstrate favourable outcomes in patients having received tryptophan-IA compared to escalated MPS and these results persisted at three-month follow-up, including clinical function scores, health-related QoL assessments and serum NfL levels.
The safety profile was in accordance with previous reports, with complications of CVC usage (dislocation, necessity of a femoral CVC) requiring the most clinical attention. Our study also highlighted safety concerns regarding escalated MPS, including not only hyperglycaemia, hypokalaemia and hypertension but also the development of severe psychosis and infection.
Furthermore, we documented profound changes in peripheral immune cell composition, most notably a decrease of B cells and reduction of T cell activation markers following apheresis treatment contrasting isolated changes in the T cell compartment following double-dose MPS. We also documented depletion of various soluble factors including immunoglobulins, coagulation factors and cytokines following immunoadsorption.
Apheresis treatment was first established for inflammatory demyelinating disorders of the central nervous system following a randomized, sham-controlled trial by Weinshenker and colleagues [
19]. Several studies have been reported since, but were mostly retrospective and/or uncontrolled. IA was first studied for treatment of diseases thought to be primarily driven by autoantibodies, such as myasthenia gravis or autoimmune encephalitis [
20,
21]. However, several studies reported alleviation of MS relapses following IA treatment [
8,
13]. Notably, a blinded trial even demonstrated the superiority of IA over plasma exchange in MS [
9]. Unfortunately, a larger randomized multicentre clinical trial comparing IA and escalated MPS was registered in 2018 yet results are pending (EudraCT: 2017-000635-13).
Several questions remain unanswered regarding immunoadsorption. First of all, it remains unclear by which mechanism IA alleviates relapse symptoms. Clearance of immunoglobulins has been deemed as most likely mechanism of apheresis treatment and indeed was supported by previous findings. The use of protein A columns was shown to be equally effective to the use of tryptophan columns and those columns are deemed highly specific for immunoglobulin G [
6,
8]. Previous studies also identified the presence of immune complexes in the context of type II MS lesions essential for success of apheresis treatment in smaller studies further pointing towards antibody clearance as mechanism of action [
22]. The other way around, experimental studies showed that passive transfer of antibodies separated from RMS patient’s plasma by protein A immunoadsorption was capable to aggravate rodent experimental autoimmune encephalitis [
23].
However, apart from the study from Keegan and colleagues [
22], apheresis treatment was successful in the vast majority of patients it was applied to and a substantial proportion of “non-responders” was absent although it should have been present considering the supposed association to a specific histotype. Furthermore, previous studies indicated that depletion of autoantibodies alone did not influence production of new antibodies indicating absence of relevant feedback-loops [
24].
We found that IA not only influenced immunoglobulin levels but also exerted profound effects on blood lymphocytes. Specifically, a profound reduction of nearly all examined B cell subsets immediately following IA treatment was observed. Surface expression of the rapid-responding activation marker CD69 [
25] on T cells also declined rapidly, whereas the slower-reacting HLA-DR expression remained unaffected indicating that the recent treatment was responsible here.
As the reduction of B cell subsets in the periphery correlated to clinical function outcomes, we assume that B cell-modulation is a central mechanism of IA. Notably, we found that administration of two courses of MPS prior to IA abrogated not only the observed correlation between B cell depletion and clinical outcomes but also hampered clinical recovery reflected by MSFC and EP scores, as well as being associated with a smaller reduction in NfL levels, at follow-up. The cause for this observation is unclear; however, it is known that high-dose MPS may modulate the blood–brain-barrier [
26] and impair protein re-distribution in the blood, which might impair IA efficacy, as this therapy is thought to clear serum protein and lower the protein concentration including antibody levels [
27]. Furthermore, patients in the MPS + IA group were latest to receive IA since relapse onset and thus, one could also assume that the “window of opportunity” during which modulation of the immune system can result in alleviation of neurologic deficits, had closed.
Previously, a threshold of six weeks from relapse onset was discussed as suitable for initial corticosteroid treatment [
4,
28,
29]. yet no data regarding escalation treatment exist. Median time from relapse onset to apheresis treatment in our MPS + IA patient was still below this (median: 39 days), still, this subgroup of patients was refractory to two courses of treatment already und thus, persistent structural damage is already likely.
Interestingly, IA treatment shifted the cytokine repertoire compared to MPS treatment and reduced the cytokines necessary for B cell maturation as well as B cell-derived cytokines that are supposed to maintain neuroinflammation. For example, IA was associated with reduction of IL7 and IL15, which are both known to promote B cell-mediated recruitment of CD4 + and CD8 + T cells [
30,
31]. Furthermore, IL27, which is thought to also contribute to B cell development [
32], was reduced. Conversely, lymphotoxin alpha, which is considered as pro-inflammatory B-cell derived cytokine in MS [
33], was reduced following MPS treatment but increased following IA treatment.
MPS-associated changes in the cytokine network comprised cytokines such as IL4, which is also pivotal to B cell maturation [
34], and IL6, which is known as important effector cytokine secreted by several B cell subsets in MS patients [
35]. However, these findings did not result in a substantial reduction of B cells.
Some of our findings regarding cytokines involved in B cell maturation and activation have been described in RMS already. Anti-CD20 therapy also induces changes in IL-7 and IL-15 levels [
36] further implying that B-cell-mediated T cell activation is an important mechanism of B-cell-dependent inflammatory demyelination. In line with this, clinical data from anti-CD20 antibody trials showed that B cell depletion reduced the burden of contrast-enhancing MRI lesions early after treatment [
37], supporting that B cell modulation can indeed resolve acute inflammation.
However, we observed that the specific effects of IA on B cell subsets disappeared within three months. Since re-emergence of peripheral B cell subsets is associated with disease reactivation in patients receiving B cell-depleting anti-CD20 treatment [
38], protective effects of IA beyond month 3 appear unlikely. Conversely, effects of IA on the immune system including impaired response to vaccines as observed following B cell-depletion [
39], are reversible.
Although not conventionally randomized, we aimed for reduction of potential bias by various mechanisms. First of all, treatment decision was made independently from study conduction using a standardized decision-making process led by neutral consultants. Second, we evaluated only patients with their first refractory MS relapse and moreover, more than half of patients experienced their first clinical demyelinating event ever further reducing a potential treatment-bias. In line with this, the majority of patients were treatment-naïve. Among patients already receiving disease-modifying treatment, substances were equally distributed. None of the patients received cell-depleting therapy. Although sample size of previously-treated patients remains too low for distinct subgroup analysis, those patients showed courses and laboratory findings similar to their naïve counterparts. Further evaluation of baseline epidemiological parameters showed no relevant differences among groups. We of course cannot rule out that indeed, a certain degree of restitution is a delayed effect of initial MPS treatment; however, this would not explain differences between IA and MPS escalation treatment.
In conclusion, IA proved to be a promising strategy for steroid-refractory RMS and thus should be considered early in treatment algorithms. Since the safety profile appeared advantageous to plasma exchange in previous reports and effectiveness appeared superior in outcomes such as MSFC, its use in routine clinical practice should be considered, especially in specialized centres. Furthermore, our findings indicate that modulation of B cells potentially represents a major mechanism of action of IA treatment.
Declarations
Competing interests
Steffen Pfeuffer: received travel grants from Sanofi Genzyme and Merck Serono, lecturing honoraria from Sanofi Genzyme, Mylan Healthcare, and Biogen, and research support from Diamed, Merck Serono, and the German Multiple Sclerosis Society Northrhine-Westphalia. Leoni Rolfes: received travel grants from Merck Serono and Sanofi-Genzyme. Timo Wirth: has no competing interests. Falk Steffen: has no competing interests. Marc Pawlitzki: received speaker honoraria and travel support from Novartis. He received research support from the DMSG Landesverband NRW and the IMF program of the University Münster. Andreas Schulte-Mecklenbeck: has no competing interests. Catharina C. Gross: received speaker honoraria from DIU Dresden International University GmbH, Bayer Healthcare, and Mylan. She received travel/accommodation/meeting expenses from Bayer Healthcare, Biogen, EUROIMMUN, and Novartis. She receives research support from the European Union, the German Research Foundation, the IZKF Münster, Biogen, Novartis, and Roche. Marcus Brand: has no competing interests. Stefan Bittner: has no competing interests. Tobias Ruck: received travel grants and financial research support from Genzyme and Novartis and received honoraria for lecturing from Roche, Merck, Genzyme, Biogen, and Teva. Luisa Klotz: received compensation for serving on Scientific Advisory Boards for Alexion, Genzyme, Janssen, Merck Serono, Novartis and Roche. She received speaker honoraria and travel support from Bayer, Biogen, Genzyme, Grifols, Merck Serono, Novartis, Roche, Santhera and Teva. She receives research support from the German Research Foundation, the IZKF Münster, IMF Münster, Biogen, Immunic AG, Novartis and Merck Serono. Heinz Wiendl: received compensation for serving on Scientific Advisory Boards/Steering Committees for Bayer Healthcare, Biogen Idec, Sanofi Genzyme, Merck Serono, and Novartis. He received speaker honoraria and travel support from Bayer Vital GmbH, Bayer Schering AG, Biogen, CSL Behring, EMD Serono, Fresenius Medical Care, Genzyme, Merck Serono, Omniamed, Novartis, and Sanofi Aventis. He received compensation as a consultant from Biogen Idec, Merck Serono, Novartis, Roche, and Sanofi-Genzyme. Heinz Wiendl also received research support from Bayer Healthcare, Bayer Vital, Biogen Idec, Merck Serono, Novartis, Sanofi Genzyme, Sanofi US, and Teva. Sven G. Meuth: received honoraria for lecturing and travel expenses for attending meetings from Almirall, Amicus Therapeutics Germany, Bayer Health Care, Biogen, Celgene, Diamed, Genzyme, MedDay Pharmaceuticals, Merck Serono, Novartis, Novo Nordisk, ONO Pharma, Roche, Sanofi-Aventis, Chugai Pharma, QuintilesIMS, and Teva. His research is funded by the German Ministry for Education and Research (BMBF), Deutsche Forschungsgemeinschaft (DFG), Else Kröner Fresenius Foundation, German Academic Exchange Service, Hertie Foundation, Interdisciplinary Center for Clinical Studies (IZKF) Muenster, German Foundation Neurology and by Almirall, Amicus Therapeutics Germany, Biogen, Diamed, Fresenius Medical Care, Genzyme, Merck Serono, Novartis, ONO Pharma, Roche, and Teva.
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