Prospective trial of natalizumab personalised extended interval dosing by therapeutic drug monitoring in relapsing-remitting multiple sclerosis (NEXT-MS).

BACKGROUND: Extended interval dosing (EID) of natalizumab is a promising strategy to optimise treatment in multiple sclerosis (MS). Personalised EID by therapeutic drug monitoring can enable further extension of treatment intervals. METHODS: The NEXT-MS trial is an investigator-initiated prospective phase IV non-randomised study. Adults with a diagnosis of relapsing-remitting MS who received ≥6 natalizumab infusions were included in three groups: personalised EID with a target drug trough concentration of 10 µg/mL (EID10), an exploratory group of personalised EID with a target of 5 µg/mL (EID5) and standard interval dosing (SID) of 4 weeks. The primary outcome is radiological disease activity (new/newly enlarged T2 lesions) comparing the EID10 group to a historical cohort of SID (HSID). RESULTS: Results of the first phase of the NEXT-MS trial are reported here (n=376) as the study will continue with an amended protocol. In the EID10 group (n=251), incidence rate of radiological activity was 10.0 per 1000 person-years, which was non-inferior to the HSID cohort (24.7 per 1000 person-years (n=87), incidence rate difference 14.7, 90% CI -4.5 to 34.0). Incidence rate of radiological activity was 10.0 per 1000 person-years in the EID5 group (n=65), and 47.0 per 1000 person-years in the SID group (n=60). Serum neurofilament light levels did not increase over time within the EID groups. There were no cases of progressive multifocal leukoencephalopathy. CONCLUSIONS: MS disease activity is adequately controlled with personalised natalizumab EID. Interval extension to a drug trough concentration of 5 µg/mL is likely a safe target to extend natalizumab treatment intervals >6 weeks. TRIAL REGISTRATION NUMBER: NCT04225312.

The fat embolism syndrome as a cause of paraplegia.

The fat embolism syndrome is a well-known complication in trauma patients. We describe a rare case of traumatic fat embolism that leads to paraplegia. A 19-year-old male motorcycle accident victim was presented to our hospital. After stabilization and trauma survey, he was diagnosed with bilateral femur fractures, a spleen laceration and a tear in the inferior vena cava, for which damage control surgery was performed. Post-operatively, the patient became paraplegic and developed a fluctuating consciousness, respiratory distress and petechiae. Fat embolism syndrome was considered as the most plausible cause of the paraplegia. The fat embolism syndrome is seen in approximately 1% of trauma patients, mostly those with bilateral fractures of the femur. Prevention of the syndrome depends on early stabilization of fractures. However, even with optimal care, this syndrome can still occur and may have dramatic consequences, as we demonstrate in this case.

Natalizumab-associated progressive multifocal leukoencephalopathy is not preceded by elevated drug concentrations.

BACKGROUND: In recent years, a small but increasing number of neurologists choose to extend dose intervals of natalizumab with the aim of reducing the risk of progressive multifocal leukoencephalopathy (PML). This idea is based on the hypothesis that high drug concentrations increase the risk of PML. OBJECTIVE: We investigated the relation between longitudinal natalizumab concentrations in patients who developed PML and patients who did not develop PML. METHODS: In a prospective observational cohort study of 219 patients with relapsing-remitting multiple sclerosis treated with natalizumab, serum samples were taken every 12 weeks prior to natalizumab infusion. In this cohort, 5 patients developed PML and were matched with 10 patients from the cohort who did not develop PML. Natalizumab concentrations were measured in available samples, and the longitudinal results were compared between the two patient groups. RESULTS: Mean natalizumab concentrations in the five patients developing PML was 18.9 µg/mL (standard deviation (SD): ±13.4) versus 23.8 µg/mL (SD: ±11.5) of the control patients. Furthermore, we did not observe a clear rise in concentration levels in patients subsequently developing PML. CONCLUSION: Our results provide preliminary evidence that contradicts the hypothesis that exposure to elevated concentrations of natalizumab is a relevant risk factor of developing PML.

MRI criteria differentiating asymptomatic PML from new MS lesions during natalizumab pharmacovigilance.

OBJECTIVE: Differentiation between progressive multifocal leukoencephalopathy (PML) and new multiple sclerosis (MS) lesions on brain MRI during natalizumab pharmacovigilance in the absence of clinical signs and symptoms is challenging but is of substantial clinical relevance. We aim to define MRI characteristics that can aid in this differentiation. METHODS: Reference and follow-up brain MRIs of natalizumab-treated patients with MS with asymptomatic PML (n=21), or asymptomatic new MS lesions (n=20) were evaluated with respect to characteristics of newly detected lesions by four blinded raters. We tested the association with PML for each characteristic and constructed a multivariable prediction model which we analysed using a receiver operating characteristic (ROC) curve. RESULTS: Presence of punctate T2 lesions, cortical grey matter involvement, juxtacortical white matter involvement, ill-defined and mixed lesion borders towards both grey and white matter, lesion size of >3 cm, and contrast enhancement were all associated with PML. Focal lesion appearance and periventricular localisation were associated with new MS lesions. In the multivariable model, punctate T2 lesions and cortical grey matter involvement predict for PML, while focal lesion appearance and periventricular localisation predict for new MS lesions (area under the curve: 0.988, 95% CI 0.977 to 1.0, sensitivity: 100%, specificity: 80.6%). INTERPRETATION: The MRI characteristics of asymptomatic natalizumab-associated PML lesions proved to differ from new MS lesions. This led to a prediction model with a high discriminating power. Careful assessment of the presence of punctate T2 lesions, cortical grey matter involvement, focal lesion appearance and periventricular localisation allows for an early diagnosis of PML.

MRI characteristics of early PML-IRIS after natalizumab treatment in patients with MS.

OBJECTIVE: The early detection of MRI findings suggestive of immune reconstitution inflammatory syndrome (IRIS) in natalizumab-associated progressive multifocal leukoencephalopathy (PML) is of crucial clinical relevance in terms of treatment decision-making and clinical outcome. The aim of this study was to investigate the earliest imaging characteristics of PML-IRIS manifestation in natalizumab-treated patients with multiple sclerosis and describe an imaging pattern that might aid in the early and specific diagnosis. METHODS: This was a retrospective study assessing brain MRI of 26 patients with natalizumab-associated PML presenting with lesions suggestive of PML-IRIS during follow-up. MRI findings were evaluated considering the imaging findings such as mass effect, swelling, contrast enhancement, new perivascular T2 lesions and signs suggestive of meningeal inflammation. RESULTS: Contrast enhancement was the most common imaging sign suggestive of PML-IRIS, seen in 92.3% of the patients (with patchy and/or punctuate pattern in 70.8% and 45.8% respectively), followed by new T2 lesions with a perivascular distribution pattern (34.6%). In those patients with contrast enhancement, the enhancement was present in the lesion periphery in 95.8% of the patients. Contrast-enhancing lesions with a perivascular distribution pattern outside of the PML lesion were observed in 33.3% of the patients. The most common overall pattern was contrast enhancement in the border of the PML lesion with either a patchy or punctuate appearance in 88.5% of all patients. CONCLUSIONS: Contrast enhancement is the most common earliest sign of natalizumab-associated PML-IRIS with a frequent imaging pattern of contrast-enhancing lesions with either a patchy or punctuate appearance in the border of the PML lesion.

Diagnostic performance of brain MRI in pharmacovigilance of natalizumab-treated MS patients.

BACKGROUND: In natalizumab-treated multiple sclerosis (MS) patients, magnetic resonance imaging (MRI) is considered as a sensitive tool in detecting both MS disease activity and progressive multifocal leukoencephalopathy (PML). OBJECTIVE: To investigate the performance of neuroradiologists using brain MRI in detecting new MS lesions and asymptomatic PML lesions and in differentiating between MS and PML lesions in natalizumab-treated MS patients. The secondary aim was to investigate interrater variability. METHODS: In this retrospective diagnostic study, four blinded neuroradiologists assessed reference and follow-up brain MRI scans of 48 natalizumab-treated MS patients with new asymptomatic PML lesions (n = 21) or new MS lesions (n = 20) or no new lesions (n = 7). Sensitivity and specificity for detection of new lesions in general (MS and PML lesions), MS and PML lesion differentiation, and PML detection were determined. Interrater agreement was calculated. RESULTS: Overall sensitivity and specificity for the detection of new lesions, regardless of the nature of the lesions, were 77.4% and 89.3%, respectively; for PML-MS lesion differentiation, 74.2% and 84.7%, respectively; and for asymptomatic PML lesion detection, 59.5% and 91.7%, respectively. Interrater agreement for the tested categories was fair to moderate. CONCLUSION: The diagnostic performance of trained neuroradiologists using brain MRI in pharmacovigilance of natalizumab-treated MS patients is moderately good. Interrater agreement among trained readers is fair to moderate.

Longitudinal JCV serology in multiple sclerosis patients preceding natalizumab-associated progressive multifocal leukoencephalopathy.

The presence of anti-John Cunningham Virus (JCV) antibodies is a risk factor for the development of progressive multifocal leukoencephalopathy (PML) in MS patients treated with natalizumab. It has been suggested that an increase in serum anti-JCV antibody index precedes the development of PML. We here describe extensive longitudinal serum anti-JCV antibody indexes of four MS patients who developed PML. Anti-JCV antibodies were measured using the STRATIFY JCV™DxSelect™ test. All four patients had rather stable high anti-JCV antibody indexes in all samples obtained before developing PML. Possibly caused by reaching the saturation level of the assay, no increase in anti-JCV antibody indexes was seen just before the diagnosis of PML. This study confirms that high serum anti-JCV antibody indexes precede natalizumab-associated PML.

MRI pattern in asymptomatic natalizumab-associated PML.

OBJECTIVE: To investigate the MRI manifestation pattern of asymptomatic natalizumab-associated progressive multifocal leukoencephalopathy (PML) in patients with multiple sclerosis (MS). METHODS: 18 patients with MS with natalizumab-associated PML lesions on MRI were included. In 6 patients, the PML lesions were identified on MRI prospectively and in 12 patients PML lesions were identified retrospectively. MRI sequences were analysed for PML lesion distribution, appearance, grey matter/white matter involvement and possible signs of inflammation. Lesion probability maps were created to demonstrate lesion distribution pattern. RESULTS: The frontal lobe was involved in 14 patients (77.8%) and the parietal lobe in 4 patients (22.2%). Most patients presented with focal lesions (13 patients, 72.2%) involving one single lobe (12 patients, 66.7%). The cortical grey matter was affected in 15 patients (83.3%) and 13 patients (72.2%) presented with a combination of cortical grey and white matter involvement. Signs of inflammation were detected in 7 patients (38.8%). Among patients with available diffusion-weighted imaging, 6 patients (40%) did not show high-signal-intensity lesions. A classical imaging pattern including unilateral and unilobar focal lesions in the frontal lobe affecting the cortical grey matter or the cortical grey and adjacent white matter was observed in 8 patients (44.4%). CONCLUSIONS: Asymptomatic natalizumab-associated PML manifestations on MRI show a rather localised disease, frequently located in the frontal lobes, affecting the cortical grey matter and adjacent juxtacortical white matter. Awareness of this lesion pattern facilitates an earlier diagnosis of natalizumab-associated PML in an asymptomatic stage associated with a more favourable prognosis.

Diagnosis of asymptomatic natalizumab-associated PML: are we between a rock and a hard place?

The diagnosis of natalizumab-associated progressive multifocal leukoencephalopathy (PML) in multiple sclerosis patients in an asymptomatic stage is crucial since it is associated with better clinical outcome measures. Current diagnostic criteria on PML diagnosis in asymptomatic patients require the detection of JC virus and corresponding imaging findings. Magnetic resonance imaging (MRI) is the most sensitive diagnostic method for these purposes. However, the diagnosis of asymptomatic natalizumab-associated PML based on MRI findings can be challenging particularly in case of inconclusive or negative results on JC virus detection in the cerebrospinal fluid. In this report, we present a case series demonstrating different diagnostic scenarios of asymptomatic PML diagnosis based on MRI findings in combination with inconclusive or negative results on JC virus detection in the cerebrospinal fluid. We discuss the challenges of applying current PML diagnostic criteria in asymptomatic natalizumab-associated PML patients and stress the need for specific diagnostic criteria and guidelines regarding managing these diagnostic dilemmas in order to facilitate an early and correct diagnosis of PML presumably leading to a better clinical outcome.

The impact of pre-analytical variables on the stability of neurofilament proteins in CSF, determined by a novel validated SinglePlex Luminex assay and ELISA.

BACKGROUND: Neurofilament (Nf) proteins have been shown to be promising biomarkers for monitoring and predicting disease progression for various neurological diseases. The aim of this study was to evaluate the effects of pre-analytical variables on the concentration of neurofilament heavy (NfH) and neurofilament light (NfL) proteins. METHODS: For NfH an in-house newly-developed and validated SinglePlex Luminex assay was used; ELISA was used to analyze NfL. RESULTS: For the NfL ELISA assay, the intra- and inter-assay variation was respectively, 1.5% and 16.7%. Analytical performance of the NfH SinglePlex Luminex assay in terms of sensitivity (6.6pg/mL), recovery in cerebrospinal fluid (CSF) (between 90 and 104%), linearity (from 6.6-1250pg/mL), and inter- and intra-assay variation (<8%) were good. Concentrations of both NfL and NfH appeared not negatively affected by blood contamination, repeated freeze-thaw cycles (up to 4), delayed processing (up to 24hours) and during long-term storage at -20°C, 4°C, and room temperature. A decrease in concentration was observed during storage of both neurofilament proteins up to 21days at 37°C, which was significant by day 5. CONCLUSIONS: The newly developed NfH SinglePlex Luminex assay has a good sensitivity and is robust. Moreover, both NfH and NfL are stable under the most prevalent pre-analytical variations.

Safety, anxiety and natalizumab continuation in JC virus-seropositive MS patients.

The use of natalizumab in multiple sclerosis has been restricted by the risk of progressive multifocal leukoencephalopathy (PML). JC virus carriership, duration of natalizumab treatment and past immunosuppression are known risk factors. This has allowed for calculated risk assessment for individual patients to be implemented. Not much data are available about the effect of JCV carriership on patient willingness to continue natalizumab. Here, we evaluated the impact of JCV seropositivity on safety feelings, anxiety and treatment continuation for patients treated with natalizumab, using a visual analog scale, the Hospital Anxiety and Depression Scale and a decisional conflict scale. Seropositivity led to an elevated anxiety level for PML (p = 0.004). However, so far only 3% of patients have discontinued natalizumab because of JCV positivity in our cohort.

Drug interference in immunogenicity assays depends on valency.

Direct comparison of immunogenicity data is hampered by differential drug interference in different assay formats. In this paper we identify a drug-related factor that influences the extent of drug interference. We systematically investigated the influence of drug valency of different antibody-derived biologicals on the drug interference, using mono- and bivalent formats of adalimumab as a model system. Our results indicate that compared to regular bivalent antibodies, antibody-derived drugs that are monovalent result in less drug interference. Two real-life examples were examined: natalizumab, an IgG4 antibody that becomes effectively monovalent in vivo due to Fab arm exchange, and certolizumab pegol, a pegylated Fab fragment. For both drugs it was demonstrated that drug interference is less pronounced in an antigen-binding test compared to similar assays for other therapeutic antibodies. When comparing immunogenicity data obtained for different biologicals this phenomenon should be taken into account.

Clinical relevance of serum natalizumab concentration and anti-natalizumab antibodies in multiple sclerosis.

BACKGROUND: Antibodies against natalizumab have been found in 4.5-14.1% of natalizumab-treated multiple sclerosis (MS) patients. If antibodies persist, they are associated with an adverse effect on treatment response. However, it has proved to be difficult to standardize anti-drug antibody measurements. OBJECTIVES: The purpose of this study was to evaluate the clinical and radiological impact of serum natalizumab concentrations and their relation with anti-natalizumab antibodies in MS patients. METHODS: In this prospective observational cohort study of 73 consecutive patients treated with natalizumab, we measured serum natalizumab levels and antibody titers before the start of natalizumab treatment, at weeks 12 and 24 and annually after natalizumab initiation. Antibodies against natalizumab were measured by radioimmunoassay and serum natalizumab concentrations using a newly developed enzyme linked immunosorbent assay (ELISA). Magnetic resonance imaging (MRI) scan and clinical evaluation were performed before the start of natalizumab treatment and subsequently every year. RESULTS: Antibodies were detected in 58% of the natalizumab-treated patients. All patients developed their antibodies before week 24. The large majority of these patients reverted to neutralizing antibody (NAb) negative status during follow-up. The presence of antibodies was inversely correlated with serum natalizumab concentration (p<0.001). Only high antibody titers are associated with very low or undetectable serum natalizumab concentration. Both high antibody titers and low serum natalizumab concentrations are associated with relapses and gadolinium-enhancing lesions on MRI. CONCLUSIONS: Our data show that both low natalizumab serum concentration and high antibody titers are associated with a lack of efficacy of natalizumab. Measuring serum natalizumab, using a highly specific assay, might lead to more enhanced precision using natalizumab in individual patients.

Natalizumab remains detectable in patients with multiple sclerosis long after treatment is stopped.

Natalizumab is frequently used as a treatment for multiple sclerosis (MS). The occurrence of progressive multifocal leukoencephalopathy (PML) in natalizumab-treated patients indicates that its prominent beneficial effects need to be balanced against the risks. Also, cessation of the drug seems to be associated with recurrence of disease activity. Both the moment of rebound disease activity and the outcome of PML are related to clearance of the drug. Specific features of this IgG4 antibody (i.e. half-antibody exchange) may result in underestimated drug levels. Here, we demonstrate natalizumab levels in 10 patients with relapsing MS, using a recently developed sensitive assay. Remarkably, natalizumab was detectable up to 200 days after cessation of therapy.

Antibodies against aquaporin-4 in neuromyelitis optica: distinction between recurrent and monophasic patients.

The detection of antibodies against aquaporin-4 (AQP4) has improved the diagnosis of neuromyelitis optica (NMO). We evaluated a recently established cell-based anti-AQP4 assay in 273 patients with inflammatory CNS demyelination. The assay had a specificity of 99% and a sensitivity of 56% to detect all NMO patients and of 74% to detect the recurrent NMO patients, similar to the initial studies reported. AQP4 antibodies were absent in monophasic NMO patients, while samples in recurrent cases remained positive during follow-up. We conclude that the pathogenesis of monophasic NMO may be different from that of relapsing NMO.

Measurement of serum levels of natalizumab, an immunoglobulin G4 therapeutic monoclonal antibody.

Human immunoglobulin G4 (IgG4) is a poor trigger of effector functions and, therefore, is the preferred subclass for therapeutic monoclonal antibodies that merely aim to block their in vivo targets. An example is natalizumab, a recombinant IgG4 antibody directed against α4-integrin and used for treatment of multiple sclerosis. Efficient treatment requires that the pharmacokinetics of therapeutic monoclonal antibodies can be accurately monitored. For natalizumab, this requires special precautions due to recently reported structural peculiarities of human IgG4. Here we describe the development of an assay to determine serum levels of natalizumab. Compared with other IgG subclasses, human IgG4 possesses unique structural properties that influence its interactions in both in vivo and in vitro settings. Thus, IgG4 undergoes Fab arm exchange in vivo, resulting in effectively monovalent antibodies. Furthermore, IgG4 is able to bind to other human and nonhuman IgG via Fc interactions. We demonstrate how these features can interfere with measurement of specific IgG4 and describe how we addressed these issues, resulting in an assay that is not sensitive to Fab arm exchange by natalizumab or to IgG4 Fc interactions.

Natalizumab drug holiday in multiple sclerosis: poorly tolerated.

It has been suggested that natalizumab-associated progressive multifocal leukoencephalopathy may be prevented by structured interruptions of treatment. Evidence supporting such a drug holiday is not yet available. Here we present initial observations in 10 multiple sclerosis patients who were stringently monitored up to 6 months after discontinuation of the infusions. Cumulatively, a combination of clinical relapse and new and/or enhanced lesions on magnetic resonance imaging had occurred in 7 of 10 patients. Although numbers are small, our data suggest that in patients who were switched to natalizumab because of disease activity despite first-line treatment, a natalizumab drug holiday without reinstatement of alternate disease-modifying therapy is poorly tolerated.

T lymphocytes impair P-glycoprotein function during neuroinflammation.

The ATP-binding cassette (ABC) transporter P-glycoprotein (P-gp; ABCB1) is highly expressed at the blood-brain barrier (BBB). P-gp actively secretes and keeps the central nervous system (CNS) safe from body-born metabolites, but also from drugs and food components, emphasising the importance of its optimal function to maintain brain homeostasis. Here we demonstrate that vascular P-gp expression and function are strongly decreased during neuroinflammation. In vivo, the expression and function of brain endothelial P-gp in experimental allergic encephalomyelitis (EAE), an animal model for multiple sclerosis (MS), were significantly impaired. Strikingly, vascular P-gp expression was decreased in both MS and EAE lesions and its disappearance coincided with the presence of perivascular infiltrates consisting of lymphocytes. Our data strongly suggest that activated CD4(+) T cells induce impaired function of brain endothelial P-gp. Notably, lymphocyte interaction through endothelial intracellular adhesion molecule -1 (ICAM-1) resulted in activation of a nuclear factor kappa B (NF-kappaB) signaling pathway, which resulted in endothelial P-gp malfunction. Our study provides first evidence that CD4(+) T cells are able to affect endogenous molecular protection mechanisms of brain endothelium. Loss of vascular P-gp function during neuroinflammation may disturb brain homeostasis and thereby aggravate disease progression via exposure of vulnerable CNS cells to detrimental compounds.