# Long-term clinical and biological prognostic factors of anti-NMDA receptor encephalitis in children

Maxime Mazowiecki, Lorraine Flet-Berliac, Julia Roux, Anne Lépine, Pascale Chretien, Salima Hacein-Bey-Abina, Laetitia Giorgi, Frederic Villega, Emmanuel Cheuret, Marie Benaiteau, et al.

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## To cite this version:

Maxime Mazowiecki, Lorraine Flet-Berliac, Julia Roux, Anne Lépine, Pascale Chretien, et al. Long-term clinical and biological prognostic factors of anti-NMDA receptor encephalitis in children. Neurology Neuroimmunology & Neuroinflammation, 2025, 12 (2), pp.e200346. 10.1212/nxi.0000000000200346. [https://doi.org/10.1212/nxi.0000000000200346](https://doi.org/10.1212/nxi.0000000000200346). [hal-05028880](https:// HAL Id: hal-05028880)

Submitted on 10 Apr 2025

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# Long-Term Clinical and Biological Prognostic Factors of Anti-NMDA Receptor Encephalitis in Children

Maxime Mazowiecki,<fnref n="1" />,* Lorraine Flet-Berliac,<fnref n="1" />,* Julia Roux,<fnref n="1" />,* Anne Lépine,² Pascale Chretien,³,⁴

Salima Hacein-Bey-Abina,³,⁴ Laetitia Giorgi,<fnref n="1" />,¹⁸ Frederic Villega,⁵,⁶ Emmanuel Cheuret,⁷ Marie Benaiteau,⁸

Veronique Rogemond,⁸ Geraldine Picard,⁸ Sarah Baer,⁹,¹⁰ Pierre Cleuziou,¹¹ Elodie Lametery,¹²

Isabelle Desguerre,¹³ Mélodie Aubart,¹³ Mathilde Chevignard,¹⁴,¹⁵,¹⁶ Roger Le Grand,¹⁷ Philippe Horellou,¹⁷,¹⁸

Carole Leroy,¹⁷,¹⁸ Bastien Joubert,⁸ Jerome Honnorat,⁸ and Kumaran Deiva<fnref n="1" />,¹⁷,¹⁸,¹⁹

Neurol Neuroimmunol Neuroinflamm 2025;12:e200346. doi:10.1212/NXJ.0000000000200346

**Correspondence**

Dr. Mazowiecki

maxime.mazowiecki@aphp.fr

## Abstract

### Background and Objectives

Anti-NMDAR encephalitis (NMDARE) is a severe neurologic condition, and recently, the NMDAR Encephalitis One-Year Functional Status (NEOS) score has emerged as a 1-year prognostic tool. This study aimed to evaluate NEOS score and biomarker (neurofilament light chains [NfL], total-Tau protein, glial fibrillary acidic protein, and serum cytokines) correlation with modified Rankin Scale (mRS), cognitive impairment, and clinical recovery in pediatric NMDARE over 2 years.

### Methods

In this French multicenter observational study, 104 pediatric patients with NMDARE were followed for a minimum of 2 years. Clinical data and serum/plasma samples were collected. Biomarker levels, measured using electroluminescence mesoscale discovery (MSD) S-PLEX, were compared between patients and controls and assessed for correlations with disease activity, mRS, cognitive/language impairment, and recovery status at 2 years.

### Results

At a median follow-up of 39.5 months, 68 percent of patients had unfavorable recovery and 54% had significant cognitive impairment. Both outcomes were strongly associated with younger age at diagnosis (OR 6.10 [1.91–27.3] *p* < 0.01 and 5.69 [1.46–27.7] *p* = 0.02, respectively). A higher NEOS score was significantly correlated with increased cognitive impairment (OR 2.53 [1.52–4.21], *p* < 0.001), higher mRS scores (OR 2.12 [1.34–3.57], *p* < 0.01), and unfavorable recovery at 2 years (OR 2.00 [1.30–3.06], *p* = 0.015). Elevated NfL levels were significantly associated with unfavorable recovery (OR 3.62 [1.29–10.9] *p* = 0.012) and severe cognitive impairment (OR 3.77 [1.38–10.9] *p* = 0.012) at 2 years. The combined area under the curve (AUC) for NfL and NEOS was significantly higher than the AUCs of NEOS and NfL alone (*p* = 0.01).

### Discussion

The NEOS score strongly predicts long-term outcomes in NMDARE, with its predictive value extending beyond the first-year mR prediction. NfL levels at disease onset seem to improve accuracy in predicting poor outcomes, providing valuable information for treatment decisions and future clinical trials.

<footnote>*These authors contributed equally to this work.</footnote>

<footnote n="1">¹Pediatric Neurology Departement, Assistance Publique-Hôpitaux de Paris, Paris-Saclay University Hospitals, Bicêtre Hospital, and Paris-Saclay University, Le Kremlin-Bicêtre; ²Pediatric Neurology Department, Assistance Publique des Hôpitaux de Marseille, Hôpital Universitaire, Marseille; ³Clinical Immunology Laboratory, Assistance Publique des Hôpitaux de Paris, Hôpitaux Universitaires Paris-Saclay, Bicêtre Hospital, and Paris-Saclay University, Le Kremlin-Bicêtre; ⁴UTCBS, UMR8258 CNRS-U1267 INSERM, Faculté de Pharmacie de Paris, Université de Paris; ⁵Pediatric Neurology Department, CICp-1401, University Children Hospital, Bordeaux; ⁶Interdisciplinary Institute for Neurosciences, CNRS UMR 5297; ⁷Pediatric Neurology Department, Purpan University Hospital, Toulouse; ⁸Reference Center on autoimmune encephalitis, Hospices Civils de Lyon, Institut MELIS, Inserm U1314/CNRS UMR 5284, Université Claude Bernard Lyon 1; ⁹Department of Neuropediatrics, ERN EpiCare, Hôpitaux Universitaires de Strasbourg; ¹⁰Institute for Genetics and Molecular and Cellular Biology (IGBMC), University of Strasbourg, CNRS UMR7104, INSERM U1258, Illkirch, France; ¹¹Department of Pediatric Neurology, Lille University Hospital; ¹²Pediatric Department, Grenoble Alpes University Hospital, Hôpital Albert Michallon; ¹³Pediatric Neurology Department Necker-Enfants Malades Hospital, University of Paris, AP-HP; ¹⁴Rehabilitation Department for Children with Acquired Neurological Injury, Saint-Maurice Hospitals (M.C.); Saint Maurice Hospitals; ¹⁵Sorbonne Université, CNRS, INSERM, Laboratoire d'Imagerie Biomédicale (LIB); ¹⁶Sorbonne Université, GRC 24 Handicap Moteur Cognitif et Réadaptation (HaMCRE), Paris; ¹⁷Université Paris-Saclay, CEA, INSERM Center for Immunology of Viral, Auto-Immune, Hematological and Bacterial diseases (IMVA-HB/IDMIT); ¹⁸National Referral Center for Rare Inflammatory Brain and Spinal Diseases, Le Kremlin-Bicêtre; and ¹⁹Institut Universitaire de France, France.</footnote>

<footnote>The Article Processing charge was funded by the authors.</footnote>

<footnote>This is an open access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal.</footnote>
# Glossary

**AUC** = area under the curve; **CCSEN** = Class for Children With Special Educational Needs; **GFAP** = glial fibrillary acidic protein; **ICU** = intensive care unit; **IQR** = interquartile range; **mRS** = modified Rankin Scale; **MSD** = mesoscale discovery; **NEOS** = anti-NMDAR Encephalitis One-Year Functional Status; **NfL** = neurofilament light chains; **NMDA** = N-methyl-D-aspartate; **NMDARE** = anti-N-methyl-D-aspartate receptor encephalitis; **ONIND** = other noninflammatory neurologic disease; **t-TAU** = total-TAU protein.

# Introduction

Anti-NMDA receptor (NMDAR) encephalitis is an immune-mediated disease characterized by a neuropsychiatric syndrome and the presence of CSF antibodies against the GluN1 subunit of the NMDAR.<sup>1-4</sup> Although a rare disease, it is the second most common immune encephalitis in children.<sup>1,2,3</sup> Despite the initial severity, the classic outcome is often deemed “favorable” after aggressive immunotherapy and tumor removal when it applies.<sup>7</sup> However, a recent study concluded that although most NMDARE children seemed to recover from motor disabilities, more than 45% had persistent long-term cognitive impairments.<sup>8</sup>

Recently, early predictors of poor functional status and risk of relapse at year 1 were validated in NMDARE and defined the NMDAR Encephalitis One-Year Functional Status NEOS score assessed during the first 4 weeks which included clinical, biological, MRI, and early treatment response.<sup>9,10</sup> A recent review summarized all promising biomarkers in NMDARE, but these were mostly studied in CSF.<sup>11,12</sup> Currently, NfL have emerged as promising biomarkers of neuroaxonal integrity or damage.<sup>12-15</sup> If recent studies in adults with NMDARE reported promising results, the prognostic value of NfL in children remains unclear, and long-term assessments are lacking, specifically on cognitive evolution, disease activity, or clinical recovery.<sup>16,18</sup>

Other biomarkers, such as total-Tau protein (t-Tau) or serum glial fibrillary acidic protein (GFAP), are known to be involved in neuronal and glial cell damage. Some studies found that levels of both biomarkers could be elevated in the CSF of patients with autoimmune encephalitis.<sup>11,12,13,19</sup> Therefore, these proteins could suggest brain damage in patients with NMDARE and potentially help in the understanding of the disease pathophysiology. Inflammatory cytokines that lead neuroinflammation may be potential biomarkers of the pathophysiology, clinical outcome, or treatment efficacy.<sup>5,11,20,21</sup>

Our objective was to study several clinical and paraclinical features identified during the routine early diagnostic workup, NEOS score at onset, advanced molecular biomarkers (plasma or serum NfL, t-Tau, sGFAP, and cytokines levels at onset and at 6 months (M6) after NMDARE diagnosis), and their association with the long-term clinical outcomes: mRS score, severity of cognitive impairment, and total or partial clinical recovery status in children with NMDARE.

# Methods

## Patients and Controls

All patients aged 18 years or older at NMDARE diagnosis with a minimum of 2-year follow-up were retrospectively identified and recruited in 2 French national referral center databases. The inclusion period was between December 2007 and April 2022. Some of them have been previously described (81/104).<sup>8</sup> In addition to clinical symptoms, all patients had anti-NMDAR IgG in the CSF detected by cell-based immunofluorescence assays.<sup>23</sup> A standardized case report form was used to collect data on family/personal history of infections/ autoimmune diseases, age and clinical symptoms at diagnosis, and information on admission to the intensive care unit (ICU). CSF analysis at disease onset (pleocytosis, proteins, and/or oligoclonal bands; anti-NMDAR antibodies; herpes simplex virus 1/2; human herpesvirus 6; and varicella-zoster virus) and brain MRI and EEG findings.<sup>6</sup> Brain MRI data were reviewed by the referent radiologist at one of the 2 reference centers: we focused for first MRI data on the first 4 weeks as mentioned by Nosadini et al.<sup>9</sup> and collected abnormalities as described: presence of increased T2 or fluid-attenuated inversion recovery parenchymal signal intensity or contrast enhancement on brain MRI.<sup>9</sup> We looked for the presence of a tumor in each patient. Therapeutic management was studied (successive immunotherapy, date of initiation and end of each treatment, time from symptom onset to treatment initiation, and specifically anti-CD20 therapy) in the light of national and international guidelines.<sup>9,28</sup>

Blood samples were available for 64 of 104 included children in the first 2 weeks after symptom onset and before any treatment and for 15 children at M6 of follow-up. Given the small number of samples available at M6, we compared clinical characteristics at the start of the disease between patients with and without M6 sample. We evaluated the NEOS score based on the assessment of patient characteristics during the first 4 weeks of the disease as previously described<sup>9,10,22</sup>: ICU requirement (1 point), CSF leukocyte count >20 cells/mm<sup>3</sup> (1 point), abnormal findings on brain MRI (1 point), no treatment within 30 days of symptom onset (1 point), and no improvement at 4 weeks after the first immunotherapy (1 point).<sup>9,10,22</sup> We also evaluated the modified NEOS score (mNEOS) without the item “no improvement at 4 weeks after the first immunotherapy”.<sup>9,10,22</sup>

For ethical and regulatory reasons, it was not possible to include a control group of healthy children, including the use
of blood and CSF sample analyses. For this reason, we used
a convenience sample of 17 patients (female:male 0.8:1), who
were followed for other noninflammatory neurologic diseases
(ONINDs) to only assess the intensity of biomarker elevations
at onset and their decline at 6 months, but not the prognostic
evaluation. More than 2 third of patients (78%) had a diagnosis
of somatoform disorder or psychiatric diagnosis, and all MRI
findings were normal. For each patient with ONINDs, we
collected the following data: age, comorbidities, reason for
hospitalization, clinical evaluation and final diagnosis, anti-
myelin oligodendrocyte glycoprotein, anti-NMDAR antibody,
brain imaging, and CSF findings (eTable 1).

Determination of Plasma and Serum NfL, sGFAP, t-TAU, and Cytokines Levels

Serum collection: fresh fasting peripheral blood samples were
collected in test tubes, centrifuged at 1,600×g at 4°C for
10 minutes to obtain fresh serum, and then stored at -80°C
until assay.

Plasma collection: blood samples were collected on heparin-
ized tubes from all patients at the diagnostic CSF punction,
and plasma was obtained after Ficoll gradient and then stored
at -80°C until assay.

All samples from patients with NMDARE were available as
serum, while samples from control patients were collected as
plasma. We therefore compared serum and plasma NfL levels
in a subset of 8 patients who had both sample types available.
The results showed that the mean NfL level was 9.3% higher in
plasma compared with serum. However, this difference was not
statistically significant. A strong and significant correlation was
observed between serum and plasma NfL levels (eFigure 1).

Mesoscale discovery (MSD) technique: all levels of blood NfL, sGFAP, t-Tau, and cytokines were measured in duplicate by using the electroluminescence technique MSD S-PLEX (ultrasensitive), blinded to clinical data in 1 single center (Clinical Immunology Laboratory, Assistance Publique -Hôpitaux de Paris, University Hospitals Paris-Saclay, Bicêtre Hospital). The human proinflammatory II S-plex kit and S-plex neurology kit were used for cytokine assessments, and we strictly followed the manufacturer's protocols. The lower limit of quantification for TNFα was 0.05 pg/mL; IL-2, 0.08 pg/mL; IL-6, 0.054 pg/mL; IL-4, 0.027 pg/mL; IFNγ, 0.13 pg/mL; IL-10, 0.170 pg/mL; IL-1β, 0.170 pg/mL; IL-17A, 0.130 pg/mL; and IL-12p70, 0.49 pg/mL, with intra-assay coefficients of variation ranging from 3% to 6%. Sensitivities for neurology panel were for sGFAP 1.34 pg/mL; t-Tau, 0.180 pg/mL; and NfL, 3.4 pg/mL, with intra-assay coefficients of variation ranging from 8% to 14%.

Outcome Assessment

Overall Disability Level

The mRS score was designed to assess the global level of
disability and dependence as previously described.⁸,¹⁰ Patient

scores were calculated by a local referral center investigator at
disease onset, 12, 24 months, and at last follow-up. A good
outcome was defined as a score strictly below 2 at last
follow-up.

Neuropsychological Assessment and Speech and Language Assessment

We collected all available neuropsychological and language
assessments: age-adapted Wechsler scale evaluations of in-
tellectual ability (WPPSI for children from 3 to 5 years and
11 months old, WISC for children from 6 to 16 years and
11 months old, and WAIS for young adult from 17 to 18 years
old) and all other tests performed by psychologists, speech
and language therapists, and psychomotor therapists to in-
dividually assess specific cognitive functions. Attention was
assessed using the Conners Rating Scales and the TEA-Ch
psychometric evaluations (for children aged 6–13 years).
Evaluations were performed at 6, 12, and 24 months after
NMDARE onset. For this study, we only focused on the
available data at 24 months. Given the age heterogeneity and
the performed assessments by each center, based on patient’s
clinical needs, we established a cognitive severity score at least
2 years after NMDARE diagnosis, according to 6 criteria:
language, memory, attention, executive function, praxis, and
psychomotor impairment, each of which was rated “impaired”
or “within the normal range” based on test results, on the
conclusions of the therapists and on the adaptations or
rehabilitations implemented. Cognitive disorders were con-
sidered significant with impairment of ≥2 criteria and minor
or nonexistent with impairment of 0 or 1 criterion.

Academic Performance

We recorded patients' academic performance at 2-year follow-
up, which is an indirect indicator of cognitive or learning
difficulties. By noting any school challenges identified by the
teaching staff, academic performance was deemed to be af-
fected if patients required any school accommodations, ex-
perienced difficulties in completing their grade, repeated
grades, needed a specialized assistant, or attended a class for
children with special educational needs (CCSEN).

Rehabilitation

We recorded the need for rehabilitative care (physiotherapy,
psychomotricity, occupational therapy, psychological follow-
up, and speech and language therapy) during the disease and
after at least 2 years of follow-up.

Treatment

We collected all immunotherapy (IT) received with the date
of initiation and duration. We considered as described by
Nosadini et al. the line and treatment response.⁹ “First-line”
therapy was high-dose corticosteroids, IV immunoglobulin
and/or therapeutic plasma exchanges, “second-line” was rit-
uximab (anti-CD20), cyclophosphamide and/or therapeutic
apheresis, and escalation therapy or “third-line” was tocilizu-
mab, bortezomid, and/or intrathecal injection of methotrex-
ate. We evaluated treatment response as previously described
at 6, 12, and 24 months from diagnosis, stratifying patients into 3 responder groups based on the duration of IT from disease onset: 3–6 months for the best responders, 6–12 months for the average responders, and 12–24 months for the poorest responders.⁹

## Recovery Assessment

To assess the global outcome of the disease at 2-year follow-up, we defined a composite recovery outcome based on the absence of treatment needed for more than 6 months at last follow-up, the mRs assessment following definition of favorable outcome in previous studies, and the cognitive severity score.²,⁴,⁷,⁹,¹⁰,¹³

Recovery with no sequelae was defined by an mRS of 0, absence of immunotherapy for more than 6 months, and a cognitive severity score of 0. Minor sequelae was defined by an mRS of 1 and/or a cognitive severity score of 1, and no treatment for more than 6 months. Moderate sequelae were characterized by an mRS of 2 and/or a cognitive severity score of 2, and no treatment for more than 6 months. Any other outcomes were categorized as poor recovery. A favorable recovery outcome was defined for patients who met the criterion of no or minor sequelae at 2-year follow-up.

## Statistical Methods

Categorical and quantitative data are described with median (interquartile range [IQR]), mean (SD), and frequency (%) and were analyzed using Microsoft Excel and R-Studio, with parametric and nonparametric tests: Welch test, Student t test, Mann-Whitney test, or Pearson correlation for quantitative variables and $\chi^2$ test or Fisher exact test for categorical variables. If there were more than 2 groups, the Kruskal-Wallis test or ANOVA was performed. Multivariate logistic regression analysis was used to identify categorical predictors or linear regression analysis for quantitative predictors. Statistical significance was set at a $p < 0.05$. Odd ratios (ORs) are reported with 95% CIs. We selected potential adjustment variables from the set of collected variables so that less than 20% of patients had missing data or each variable had less than 5% missing values. To assess the feasibility of using the prognostic factor, we used the receiver operating characteristic (ROC) curve, assessing the discriminatory power with the area under the curve (AUC) and 95% CIs. An AUC of 1.0 indicated perfect prediction and 0.5 no better than random prediction, AUC comparisons were performed by the Delong test.

## Standard Protocol Approvals, Registrations, and Patient Consents

The study was conducted in accordance with French law relevant to clinical noninterventional research. According to French law on bioethics (July 29, 1994; August 6, 2004; and July 7, 2011, Public Health Code), patients' written informed consent for patient participation was collected. Data confidentiality was ensured in accordance with the recommendations of the French commission for data protection

(Commission Nationale Informatique et Liberté, CNIL decision DR-2014-558).

## Data Availability

Anonymized data not published within this article will be made available by request from any qualified investigator.

# Results

## Demographic Characteristics of Patients With NMDARE

### Clinical Features

We included 104 patients and among them 7 were post-HSVE NMDARE (Table 1). As per our inclusion criteria, anti-NMDAR antibodies were detected in the CSF of all patients. All underwent a brain MRI at diagnosis, and findings were abnormal for 39 (37%). The paraneoplastic workup revealed an associated tumor in 12 (11%) (mature ovarian teratomas $n = 9$, cystic ovarian teratoma $n = 2$, and pineal dysgerminoma $n = 1$). These patients underwent tumor resection with other treatments. In the 64 patients with a biological sample available, only 2 were post-HSVE NMDARE. Comparison between NMDARE patients' clinical features at disease onset with or without biological sample at M6 (15 vs 89/104) did not show any statistical difference (eTable 2).

### Treatments

Eighty-two (78%) received “second-line” or “third-line” treatment. Overall, 32% ($n = 34$) received rituximab early, defined as a first infusion before 30 days after disease onset. We found no significant differences between the time of initiation of first-line immunotherapy (corticosteroids [$p = 0.5$], IVIg [$p = 0.13$], or plasmapheresis [$p = 0.44$]) and second-line immunotherapy (rituximab [$p = 0.12$]), according to the year of management (before and after 2021). At month 6, about half of the patients (46%, $n = 48$) had immunotherapy, 33% at 1 year ($n = 34$) and 8% at 2 years ($n = 8$).

## NEOS

We evaluated the NEOS score at onset in all the 104 patients. The median score was 2.00 (IQR 1.00–3.00). For the mNEOS score, the median score was 2.00 (1.00–3.00).

## Modified Rankin Scale

A total of 53 of 104 patients (51%) had an initial mRS of 5. At 2 years, 12 of 97 (12%) had disabilities with an mRS score >2. The mean mRS at last follow-up was 1 (SD 1.34). In addition, 1 patient died 38 months after diagnosis for an unknown reason.

## Recovery

In our cohort, 21% ($n = 22/102$) of patients exhibited recovery with no sequelae at 2 years, and 11% ($n = 11/102$) recovery with minor sequelae only. Overall, 32% ($n = 33/102$) met the criteria of a favorable recovery outcome (no or minor sequelae).
<table><caption>**Table 1** Demographic Characteristics of Patients With NMDARE</caption><thead><tr><th></th><th>Patients with NMDARE (n = 104)</th><th>NMDARE patients with favorable recovery (n = 33, 32%)</th><th>NMDARE patients with unfavorable recovery (n = 69, 68%)</th></tr></thead><tbody><tr><td>Sex ratio (female:male)</td><td>2.4:1</td><td>3.1:1</td><td>2.45:1</td></tr><tr><td>Age at diagnosis, (y), median (IQR)</td><td>8.70 (4.50-14.2)</td><td>9.58 (6.6-14.0)</td><td>6.3 (3.3-14.1)</td></tr><tr><td>Follow-up duration, (m), median (IQR)</td><td>39.5 (26.0-55.8)</td><td>43.3 (24.8-54.5)</td><td>41.0 (27.8-55.7)</td></tr><tr><td>Clinical symptoms, n (%)</td><td></td><td></td><td></td></tr><tr><td>Behavioral impairment</td><td>90 (91)</td><td>28 (84)</td><td>64 (93)</td></tr><tr><td>Seizures</td><td>84 (85)</td><td>30 (90)</td><td>55 (79)</td></tr><tr><td>Abnormal movements</td><td>82 (83)</td><td>27 (82)</td><td>58 (84)</td></tr><tr><td>Consciousness impairment</td><td>67 (68)</td><td>21 (64)</td><td>47 (68)</td></tr><tr><td>Language impairment</td><td>65 (66)</td><td>20 (61)</td><td>48 (69)</td></tr><tr><td>Dysautonomia</td><td>40 (40)</td><td>10 (30)</td><td>29 (42)</td></tr><tr><td>Memory impairment</td><td>38 (38)</td><td>14 (42)</td><td>26 (37)</td></tr><tr><td>Central hypoventilation</td><td>13 (13)</td><td>2 (6)</td><td>11 (16)</td></tr><tr><td>Number of symptoms, median (IQR)</td><td>5.00 (5.00-6.00)</td><td>5.00 (4.0-6.0)</td><td>6.00 (5.0-6.0)</td></tr><tr><td>mRS score at onset, median (IQR)</td><td>5.00 (4.00-5.00)</td><td>4.00 (3.0-5.0)</td><td>5.0 (4.0-5.0)</td></tr><tr><td>Abnormal cerebral MRI onset, n (%)</td><td>39 (37)</td><td>7 (21)</td><td>31 (45)</td></tr><tr><td>Diffuse T2-weighted/fluid-attenuated inversion recovery</td><td>35 (89)</td><td></td><td></td></tr><tr><td>Supratentorial lesions</td><td>23 (65)</td><td></td><td></td></tr><tr><td>Infratentorial lesions</td><td>6 (17)</td><td></td><td></td></tr><tr><td>Thalamic lesions</td><td>5 (14)</td><td></td><td></td></tr><tr><td>Cortical atrophy</td><td>3 (8)</td><td></td><td></td></tr><tr><td>Cortical necrosis</td><td>1 (2)</td><td></td><td></td></tr><tr><td>CSF leukocyte count >20, n (%)</td><td>56 (53)</td><td>20 (60)</td><td>35 (51)</td></tr><tr><td>Abnormal EEG results at onset, n (%)</td><td>81 (83)</td><td>26 (79)</td><td>57 (83)</td></tr><tr><td>Time to diagnosis (d), median (IQR)</td><td>24.0 (14.0-43.0)</td><td>26.0 (13.7-43.2)</td><td>23.0 (14.5-45.0)</td></tr><tr><td>Time symptom onset—first IT (d), median (IQR)</td><td>18.0 (7.00-33.8)</td><td></td><td></td></tr><tr><td>ICU, n (%)</td><td>55 (53)</td><td>14 (42)</td><td>40 (58)</td></tr><tr><td>No tumor, n (%)</td><td>92 (89)</td><td>26 (79)</td><td>61 (88)</td></tr><tr><td>“First-line” immunotherapy,<sup>a</sup> n (%)</td><td>91 (87)</td><td>29 (88)</td><td>64 (92)</td></tr><tr><td>“Second-line” immunotherapy,<sup>b</sup> n (%)</td><td>82 (79)</td><td>25 (76)</td><td>59 (86)</td></tr><tr><td>NEOS score, median (IQR)</td><td>2.00 (1.00-3.00)</td><td>2.0 (1.0-3.0)</td><td>3.0 (2.0-3.5)</td></tr><tr><td>Relapse, n (%)</td><td>12 (12)</td><td>4 (12)</td><td>8 (11)</td></tr><tr><td>mRS score at M24 of follow-up, median (IQR)</td><td>1.00 (0-1.00)</td><td>0.0 (0.0-0.0)</td><td>1.0 (0.0-2.0)</td></tr><tr><td>Treatment at 1 y of follow-up, n (%)</td><td>34 (33)</td><td>6 (18)</td><td>26 (38)</td></tr><tr><td>Treatment at 2 y of follow-up, n (%)</td><td>8 (8)</td><td>0 (0)</td><td>8 (11)</td></tr><tr><td>Altered academic performance, n/85 (%)</td><td>59 (69)</td><td></td><td></td></tr><tr><td>Rehabilitation, n/97 (%)</td><td>82 (84)</td><td>22 (66)</td><td>60 (86)</td></tr><tr><td>Speech and language therapy</td><td>68 (70)</td><td>13 (39)</td><td>55 (79)</td></tr></tbody></table>

Continued
<table><caption>**Table 1** Demographic Characteristics of Patients With NMDARE (continued)</caption><thead><tr><th></th><th>Patients with NMDARE (n = 104)</th><th>NMDARE patients with favorable recovery (n = 33, 32%)</th><th>NMDARE patients with unfavorable recovery (n = 69, 68%)</th></tr></thead><tbody><tr><td>Physiotherapy</td><td>37 (38)</td><td>7 (21)</td><td>30 (43)</td></tr><tr><td>Psychological care</td><td>46 (47)</td><td>8 (24)</td><td>38 (55)</td></tr><tr><td>Significant cognitive severity score at M24, n/97 (%)</td><td>55 (56)</td><td></td><td></td></tr><tr><td>Favorable recovery at M24, n/97 (%)</td><td>33 (34)</td><td></td><td></td></tr><tr><td>Recovery with no sequelae<sup>c</sup></td><td>22 (22)</td><td></td><td></td></tr><tr><td>Recovery with minor sequelae<sup>d</sup></td><td>11 (11)</td><td></td><td></td></tr><tr><td>Unfavorable recovery at M24, n/97(%)</td><td>69 (71)</td><td></td><td></td></tr><tr><td>Recovery with moderate sequelae<sup>e</sup></td><td>13 (17)</td><td></td><td></td></tr><tr><td>Poor recovery<sup>f</sup></td><td>56 (57)</td><td></td><td></td></tr></tbody></table>

Abbreviations: ICU = intensive care unit; IQR = interquartile range; mRS = modified Rankin Scale; M = month; NMDARE = anti-N-methyl-D-aspartate receptor encephalitis.

## Cognitive Evaluation

At 2-year follow-up, 55 of 97 patients (56%) had significant cognitive impairment. Academic performance was altered in 69% (n = 59/85), and 50% (n = 30/59) attended regular school with or without human help (30% and 20%, respectively).

## NfL Assays

The median serum/plasma NfL level for patients with NMDARE was 156 pg/mL (IQR 76.6–337) at disease onset and 106 pg/mL (55–180) at M6. For both time points, it was significantly higher than in patients with ONIND (27.2 pg/mL, 18.9–35.7, *p* < 0.001). There was no significant correlation between NfL levels and age (*r* = 0.06, *p* = 0.69) (Figure 1).²⁶

## sGFAP and t-TAU Assays

The median serum GFAP levels were significantly higher for patients with NMDARE than for controls at disease onset but not at M6. GFAP levels at onset were positively and significantly correlated with the NfL level (*r* = 0.636, *p* = 0.011). t-TAU levels were 3 times lower in patients with NMDARE than in patients with ONIND at disease onset and at M6 (*p* < 0.001) (Figure 1).

## Cytokine Assays

All cytokines were significantly increased in all patients with NMDARE at onset as compared with patients with ONIND (Figure 2, eTable 3). High levels of proinflammatory cytokines were found, such as IL-6 (10,536 fg/mL [IQR 3587–58969]), IL-17A (1,255 fg/mL [682–2,893]), and TNF-α (1,484 fg/mL [682–2,893]). At M6, levels were

significantly lower as compared with disease onset for TNF-α, IL-6, IL-1β, and IL-10, with no significant difference with patients with ONIND (eTable 3).

## Long-Term Outcome Predictors

### Clinical Characteristics

Younger age at onset (before 5 years) was significantly associated with the cognitive impairment score at M24 (OR 5.69 [1.46–27.7] *p* = 0.02) and with an unfavorable recovery outcome (OR 6.10 [1.91–27.3] *p* < 0.01). Two other major predictors that are included in the NEOS score seemed to be associated with long-term outcomes: abnormal cerebral MRI findings and the absence of clinical improvement at 4 weeks after treatment initiation (Table 2).

### Treatments

Starting immunotherapy after 30 days led to more severe cognitive scores and prolonged treatment at 1 year. For patients still requiring treatment at 1 year, the time between symptom onset and day 1 of rituximab was longer than in those who no longer required treatment (48 vs 31 days, *p* = 0.015) (Table 2).

### NEOS Score

We found a significant positive association between the increase of NEOS score and the unfavorable recovery outcome, cognitive severity score at 2 years, poor academic performance, and the need to continue immunotherapy at M6 and M12. No significant association was found between NEOS score and relapse (Table 3).

<footnote>a First-line IT was considered for IV corticosteroids (n = 82, 78%), IVIG (n = 83, 79%), and therapeutic plasma exchange (n = 29, 28%).</footnote>

<footnote>b Second-line IT was considered for rituximab (n = 82, 79%), immunoadsorption (n = 14, 13%), oral azathioprine (n = 14, 13%), IV cyclophosphamide (n = 10, 10%), oral mycophenolate mofetil (n = 9, 9%). Third-line IT was considered for IV tocilizumab (n = 7, 7%), IV bortezomib (n = 4, 4%), and intrathecal methotrexate with steroids (n = 19, 18%).</footnote>

<footnote>c Recovery with no sequelae, indicated by an mRS of 0, absence of the need for immunotherapy for more than 6 mo, and a cognitive severity score of 0, was considered optimal.</footnote>

<footnote>d Minor sequelae were defined by an mRS of 1 and/or a cognitive severity score of 1, and no treatment beyond 6 mo.</footnote>

<footnote>e Moderate sequelae were characterized by an mRS of 2 and/or a cognitive severity score of 2, and no treatment exceeding 6 mo.</footnote>

<footnote>f Any other outcomes were categorized as poor recovery.</footnote>
<figure><img src="image_3.png" /><figcaption>**Figure 1** Box Plot Comparison of Median Neurofilament Light Chain, sGFAP, and t-TAU Levels Between Patients With ONIND and Those With NMDARE at Onset and at 6 Months</figcaption></figure>

NfL = neurofilament light chain; NMDARE = anti-N-methyl-D-aspartate receptor encephalitis; ONIND = other noninflammatory neurologic disease. Horizontal line is median, box edges are interquartile range, and whiskers are range. Results adjusted by age, sex, and body mass index (*p* < 0.05, **p** < 0.01, ***p* < 0.001).

<figure><img src="image_4.png" /><figcaption>**Figure 2** Cytokines Levels at Disease Onset and 6 Months in Patients With NMDARE and ONIND</figcaption></figure>

IFN = interferon; IL = interleukin; NMDARE = anti-N-methyl-D-aspartate receptor encephalitis; ONIND = other noninflammatory neurologic disease; TNF = tumor necrosis factor (*p* < 0.05, **p* < 0.01, ***p* < 0.001).
<table><caption>**Table 2** Clinical Outcome Predictors at Disease Onset for Patients With NMDARE</caption><thead><tr><th></th><th>Age at diagnosis<br>below 5 years old</th><th>Time onset to<br>first-line<br>treatment, >30 d</th><th>ICU requirement,<br>n (%)</th><th>No clinical<br>improvement 4 wk<br>after treatment</th><th>Abnormal cerebral MRI<br>findings at disease<br>onset</th></tr></thead><tbody><tr><td>Cognitive severity<br>at M24</td><td>5.69 (1.46-27.7)</td><td>0.02</td><td>4.63 (1.12-19.2)</td><td>0.018</td><td>2.43 (0.933-6.33)</td><td>0.069</td><td>6.15 (2.56-15.7)</td><td>b</td><td>4.09 (1.65-11.0)</td><td>a</td></tr><tr><td>Treatment at M6</td><td>1.05 (0.97-1.13)</td><td>0.23</td><td>2.32 (0.989-5.63)</td><td>0.056</td><td>2.07 (0.94-4.60)</td><td>0.071</td><td>4.48 (1.95-10.9)</td><td>b</td><td>2.30 (1.03-5.25)</td><td>0.022</td></tr><tr><td>Treatment at 1 y</td><td>1.22 (0.45-3.11)</td><td>0.68</td><td>2.63 (1.08-6.48)</td><td>0.02</td><td>1.03 (0.45-2.36)</td><td>0.94</td><td>4.21 (1.69-11.7)</td><td>a</td><td>2.76 (1.18-6.54)</td><td>0.019</td></tr><tr><td>mRS score ≥2 at M24</td><td>2.24 (0.63-7.72)</td><td>0.2</td><td>2.32 (0.725-8.99)</td><td>0.18</td><td>1.49 (0.53-4.32)</td><td>0.45</td><td>2.18 (0.678-8.50)</td><td>0.22</td><td>5.00 (1.75-15.5)</td><td>a</td></tr><tr><td>Unfavorable recovery<br>at 2 y</td><td>6.10 (1.91-27.3)</td><td>a</td><td>1.67 (1.03-9.94)</td><td>0.37</td><td>1.94 (0.840-4.57)</td><td>0.12</td><td>4.29 (1.81-10.6)</td><td>a</td><td>3.75 (1.37-12.1)</td><td>0.021</td></tr></tbody></table>

Abbreviations: ICU: intensive care unit; mRS = modified Rankin Scale; NMDARE = anti-N-methyl-D-aspartate receptor encephalitis. Data are odds ratios (ORs) and 95% CIs. ORs are adjusted by age and sex.

## NfL Levels

The NfL level was significantly associated with poor outcome, with each 100 pg/mL increase associated with 3.09 and 3.38 increased odds of a cognitive severity score >2 at 24 months (p = 0.032) and absence of favorable recovery at 2 years (p = 0.025), respectively. As NfL levels may be associated with age and BMI, we have also calculated the NfL level Z-score within the NMDARE group which showed similar results.²⁹ Increased levels of NfL at M6 also showed significant association with the need for immunotherapy continuation (at M6 and M12, p < 0.001 and p = 0.026) and recovery with moderate sequelae (p = 0.031). A significant association between higher NfL levels and presence of cognitive impairment was observed (174 pg/mL, IQR 89.4-486 vs 92.4 pg/mL, IQR 54.1-234, p = 0.016). Similarly, patients who recovered with sequelae at 24 months had significantly higher levels of NfL than those without sequelae (171 pg/mL, IQR 78.9-402 vs 88.9 pg/mL, IQR 57-153, p = 0.023) (Table 4).

## sGFAP and t-TAU Assays

The sGFAP level at onset was significantly increased in the NMDARE group for whom treatment was continued at M12 (p < 0.01), which was similar although not significant at M6 and 2 years. We found no association between t-TAU level at disease onset or at M6 and the different outcome subgroups.

## Cytokine Assays

At disease onset, levels were not correlated with outcomes, and although all patients had increased levels of cytokines, tendencies were recorded (eTable 4). Patients with more aggressive clinical presentation at onset or poor long-term outcomes had notably higher IL-6 and IL-17a levels (approximately 6 and 2 times higher, respectively). By contrast, higher IL-10 levels at onset often indicated milder initial clinical presentations and better long-term outcomes.

## Associations Between Serum NfL and NEOS With Clinical Outcomes

Serum NfL level and NEOS score were significant predictors of a substantial subsequent poor recovery status prognosis,

cognitive impairment, or recovery without sequelae, and the combination of both criteria was a better predictor (Figure 3). AUCs were similar with NfL and NEOS score (NfL AUC: 0.720 and NEOS AUC: 0.682, p = 0.36) and higher with combination of both (AUC = 0.836, p = 0.01). Specifically, for predicting poor 2-year recovery, the AUCs for NfL (AUC = 0.720) and NEOS (AUC = 0.682) were not significantly different from each other (Delong test p-value = 0.3). The combined AUC for NfL and NEOS was 0.836, which was significantly higher than the AUCs of the individual predictors (Delong test p-value = 0.01).

## Discussion

In our extensive pediatric cohort of NMDARE, we have shown that the NEOS score, initially designed to predict poorer neurologic outcomes in mRS at 1 year, remains valid for long-term outcomes at 2 years. Notably, we found that the NEOS score also correlates with worse cognitive outcomes at the 2-year time point, and incorporating NfL into the NEOS score may enhance its predictive ability for unfavorable outcomes. Although not significant, we observed that increased plasma proinflammatory cytokines seem to be associated with poor outcome, while IL-10 is associated with favorable outcome.

We evaluated the NEOS score, originally developed to predict mRS at 1 year, to assess its validity in long-term predictions, including mRS at 2 years, as well as other critical outcomes such as cognition. In this extensive pediatric cohort, we confirmed that the NEOS score remains a robust predictor of outcomes at 2 years in children with NMDARE. It effectively predicted the 2-year cognitive severity score, the need for immunotherapy at 1 and 2 years, and the recovery status with no and minor sequelae at 2 years. These findings align with previous studies, which primarily focused on assessing the mRS score at 1 year. However, it is important to note that patients with NMDARE often present

<footnote>a p < 0.01.</footnote>

<footnote>b p < 0.001.</footnote>
<table><caption>**Table 3 NEOS Score in Patients With NMDARE by Clinical Outcome Subgroup**</caption><thead><tr><th></th><th>NEOS score median (IQR)</th><th>p Value</th><th>OR (95% CI)</th><th>p Value</th></tr></thead><tbody><tr><td><strong>Cognitive severity at 2 y</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Yes</td><td>3.00 (2.00-3.00)</td><td>&lt;0.01</td><td>2.53 (1.52-4.21)</td><td>&lt;0.01</td></tr><tr><td>No</td><td>2.00 (1.00-3.00)</td><td></td><td></td><td></td></tr><tr><td><strong>Immunotherapy at M6</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Yes</td><td>3.00 (2.00-4.00)</td><td>&lt;0.001</td><td>2.13 (1.46-3.24)</td><td>&lt;0.01</td></tr><tr><td>No</td><td>2.00 (1.00-2.25)</td><td></td><td></td><td></td></tr><tr><td><strong>Immunotherapy at M12</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Yes</td><td>3.00 (2.00-4.00)</td><td>&lt;0.01</td><td>1.87 (1.28-2.84)</td><td>&lt;0.01</td></tr><tr><td>No</td><td>2.00 (1.00-3.00)</td><td></td><td></td><td></td></tr><tr><td><strong>Immunotherapy at M24</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Yes</td><td>3.50 (2.75-4.00)</td><td>0.024</td><td></td><td></td></tr><tr><td>No</td><td>2.00 (1.00-3.00)</td><td></td><td></td><td></td></tr><tr><td><strong>mRS score at M24</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>≥2</td><td>2.00 (1.00-3.00)</td><td>&lt;0.01</td><td>2.12 (1.34-3.57)</td><td>&lt;0.01</td></tr><tr><td>&lt;2</td><td>3.00 (2.50-4.00)</td><td></td><td></td><td></td></tr><tr><td><strong>Unfavorable recovery at 2 y</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Yes</td><td>2.00 (1.00-3.00)</td><td>&lt;0.01</td><td>2.00 (1.30-3.06)</td><td>0.015</td></tr><tr><td>No</td><td>3.00 (2.00-4.00)</td><td></td><td></td><td></td></tr><tr><td><strong>Academic difficulties</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Yes</td><td>2.00 (1.00-3.00)</td><td>&lt;0.01</td><td>2.04 (1.37-3.19)</td><td>&lt;0.01</td></tr><tr><td>No</td><td>3.00 (2.00-3.00)</td><td></td><td></td><td></td></tr><tr><td><strong>Relapse</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Yes</td><td>3.00 (1.00-3.00)</td><td>0.81</td><td></td><td></td></tr><tr><td>No</td><td>2.00 (1.25-3.00)</td><td></td><td></td><td></td></tr></tbody></table>

Abbreviations: NEOS = Anti-NMDAR Encephalitis One-Year Functional Status score; NMDARE = anti-N-methyl-D-aspartate receptor encephalitis, mRS = modified Rankin Scale, treatment defined active immunotherapy, M = month.

All data show are multivariable analysis and are adjusted by age and sex. Odds ratios (ORs) are given with their 95% CIs.

cognitive and/or behavioral impairments that may not be adequately captured by the mRS alone.¹⁶,¹⁷ We have also observed that younger age and specifically below 5 years old are associated with worse cognitive outcome, supporting that NMDAR activity is crucial in early steps of a developing brain.²⁷

In addition, the assessment of blood NfL levels as well it's Z-score to avoid any interference due to age and BMI, at disease onset yielded promising results because it was significantly correlated with cognitive severity status and recovery at the 2-year time point.¹⁶⁻¹⁸ Although previous research has yielded varying results regarding the prognostic value of NfL on mRS scores at 1 year, none have assessed its prognostic

effect on cognitive impairment, duration of immunotherapy, and overall recovery status in such a large pediatric cohort at the 2-year milestone. Despite previous reports, no statistically significant correlation was found between NfL levels at onset and mRS scores at 2 years, likely due to the low sensitivity of the mRS in the pediatric NMDARE population to accurately discern favorable outcomes and clinically significant impairments.¹⁶,¹⁷

This study seems to be the first to assess the combined prognostic value of these 2 biomarkers across various outcome parameters over an extended follow-up period. This finding is valuable because it can guide the initial choice of immunotherapy, potentially allowing for a more aggressive
<table><caption>**Table 4** Serum NfL Levels in Patients With NMDARE and Predictive Value Across Clinical Outcome Subgroups</caption><thead><tr><th></th><th>NfL level (onset) (pg/mL), median (IQR)</th><th>p Value</th><th>Z-score NfL level (onset) mean (SD)</th><th>p Value</th><th>OR (95% IC)</th><th>p Value</th></tr></thead><tbody><tr><td><strong>Cognitive severity at 2 y</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Yes</td><td>174 (89.4-486)</td><td>0.016</td><td>10.2 (26.7)</td><td>0.016</td><td>3.77 (1.38-10.9)</td><td>0.012</td></tr><tr><td>No</td><td>92.4 [54.1-234]</td><td></td><td>2.59 (3.03)</td><td></td><td></td><td></td></tr><tr><td><strong>Recovery with no sequelae at 2 y</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Yes</td><td>88.9 (57-153)</td><td>0.023</td><td>2.17 (2.28)</td><td>0.023</td><td>3.38 (1.33-11.2)</td><td>0.014</td></tr><tr><td>No</td><td>171 (78.9-402)</td><td></td><td>9.05 (24.2)</td><td></td><td></td><td></td></tr><tr><td><strong>mRS score at M24</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>≥2</td><td>207 (133-379)</td><td>0.16</td><td>14.6 (38.8)</td><td>0.25</td><td>3.00 (0.901-11.9)</td><td>0.08</td></tr><tr><td>&lt;2</td><td>116 (75.3-241)</td><td></td><td>4.07 (5.37)</td><td></td><td></td><td></td></tr><tr><td><strong>Favorable recovery status at 2 y</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Yes</td><td>92.4 (54.1-234)</td><td>0.011</td><td>2.42 (2.69)</td><td>0.011</td><td></td><td></td></tr><tr><td>No</td><td>174 (87.1-500)</td><td></td><td>8.91 (23.9)</td><td></td><td>3.62 (1.29-10.9)</td><td>0.012</td></tr><tr><td><strong>Recovery with no sequelae at 2 y</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Yes</td><td>88.9 (57-153)</td><td>0.023</td><td>2.17 (2.28)</td><td>0.023</td><td>3.38 (1.33-11.2)</td><td>0.014</td></tr><tr><td>No</td><td>171 (78.9-402)</td><td></td><td>9.05 (24.2)</td><td></td><td></td><td></td></tr><tr><td><strong>Relapse</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Yes</td><td>74.1 (49.6-209)</td><td>0.09</td><td>2.04 (2.56)</td><td>0.08</td><td></td><td></td></tr><tr><td>No</td><td>156 (78.3-339)</td><td></td><td>7.47 (21.3)</td><td></td><td></td><td></td></tr><tr><td><strong>Immunotherapy at M6</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Yes</td><td>190 (78.9-349)</td><td>0.082</td><td>9.28 (26.2)</td><td>0.082</td><td>3.93 (1.44-11.3)</td><td>&lt;0.01</td></tr><tr><td>No</td><td>93.8 (65.0-234)</td><td></td><td>3.32 (5.26)</td><td></td><td></td><td></td></tr><tr><td><strong>Immunotherapy at M12</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Yes</td><td>262 (150-473)</td><td>0.13</td><td>4.50 (5.22)</td><td>0.13</td><td></td><td></td></tr><tr><td>No</td><td>117 (79.8-290)</td><td></td><td>7.38 (25.3)</td><td></td><td></td><td></td></tr><tr><td><strong>Immunotherapy at M24</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Yes</td><td>226 (173-270)</td><td>0.87</td><td>3.03 (2.49)</td><td>0.96</td><td></td><td></td></tr><tr><td>No</td><td>196 (180-448)</td><td></td><td>6.75 (21.5)</td><td></td><td></td><td></td></tr></tbody></table>

Abbreviations: mRS = modified Rankin Scale, M = month; NfL = neurofilament light chain.

Z-scores are calculated using the mean and SD NfL levels from the entire ONIND sample. All data show are multivariable analysis and are adjusted by age, sex, and body mass index. Odds ratios (ORs) are given according to NfL increase, when NfL (pg/mL) increases by 100 unit(s), the rating is multiplied on average by the given OR. OR are given with their 95% CIs.

protocol for patients at high risk of unfavorable outcomes or facilitating initial screening for future therapeutic studies.

We observed significantly higher sGFAP levels in patients with NMDARE at onset compared with levels at 6 months and ONIND. In addition, there was a positive correlation between sGFAP and NfL levels, suggesting the role of astrocytes in the pathophysiology of NMDARE. Indeed, under certain pathologic conditions, astrocytes may become a primary source of detrimental reactive oxygen species and

reactive nitrogen species, which can activate microglia or directly induce neural damage.¹⁹,²⁴,²⁵

The cytokine analysis revealed a prominent proinflammatory state during the acute phase of the disease, characterized by upregulation of various cytokine axes including T helper 1 cell (Th1) axis (IFN-γ and TNF-α), Th2 axis (IL-2, IL-4), Th17 axis (IL-6, IL-17a), broad-spectrum cytokines (IL-1β, IL12p70), and the T regulatory (Treg) cell axis (IL-10). These findings align with previous CSF studies, indicating
<figure><img src="image_5.png" /><figcaption>**Figure 3** Receiver Operating Characteristic (ROC) Curve According to Serum Neurofilament Light Chain (NfL) Level, Anti-NMDAR Encephalitis One-Year Functional Status (NEOS) Score, and Association of Both</figcaption></figure>

(A) Predicting 2-year unfavorable recovery: $NfL_{AUC} = 0.720$, $NEOS_{AUC} = 0.682$ (Delong test *p* value = 0.36). $NfL + NEOS_{AUC} = 0.836$ (Delong test *p* value = 0.01). (B) Predicting 2-year recovery without sequelae: $NfL_{AUC} = 0.695$, $NEOS_{AUC} = 0.684$ (Delong test *p* value = 0.94). $NfL + NEOS_{AUC} = 0.767$ (Delong test *p* value = 0.097). (C) Predicting 2-year cognitive impairment: $NfL_{AUC} = 0.644$, $NEOS_{AUC} = 0.678$ (Delong test *p* value = 0.93). $NfL + NEOS_{AUC} = 0.77$ (Delong test *p* value = 0.067). NEOS = anti-NMDAR Encephalitis One-Year Functional Status score; $NfL$ = neurofilament light chain; NMDARE = anti-N-methyl-D-aspartate receptor encephalitis; ROC = receiver operating characteristic.

that in NMDARE, plasma/serum levels of cytokines may potentially accurately reflect intracerebral immune activation.¹¹,¹⁹,²¹

Although all patients exhibited marked proinflammatory activation, no significant differences were found between initial clinical severity and subsequent clinical outcomes. However, trends were noted, such as a more inflammatory profile at diagnosis for patients with aggressive presentations and less favorable outcomes, and contrasting trends for IL-10 levels, suggesting a stronger immunomodulatory response in patients with less severe disease. Although similar trends have been described previously, samples at M6 did not yield significant results, possibly due to early sampling in the disease course and the limited number of available samples.²⁰,²¹

Despite several limitations, including its retrospective design and the rarity of NMDARE, this study benefits from being based on 2 national centers for rare autoimmune encephalitis, ensuring comprehensive data collection and allowing for the largest pediatric long-term follow-up cohort of NMDARE to date. In addition, although the small number of samples at 6 months limits interpretation and precludes drawing conclusions, the clinical characteristics of patients with NMDARE at this time point with or without available samples were similar, albeit with caution due to lack of power. Another limitation was the inclusion of patients with ONIND who, while noninflammatory, were not strictly healthy, although they allowed us to assess the intensity of biomarker elevations at onset and their decline at 6 months in patients with NMDARE. However, this group was not intended for prognostic evaluation, which was conducted exclusively among patients with NMDARE. Furthermore, the comprehensive evaluation of clinically significant criteria at 2 years, including a global assessment of recovery for children with NMDARE, adds strength to the study.

We have demonstrated in this large pediatric cohort that the NEOS score is a strong tool to predict several long-term important clinical outcomes in NMDARE. The blood NfL level at onset in children may be an interesting biomarker of 2-year prognosis, and a combination of both could improve the prediction accuracy. We confirm the evidence of marked blood inflammatory activation at disease diagnosis, involving B-cell and T-cell activation. No cytokine was strongly associated with initial severity or follow-up outcomes, but our data support the need for further studies to confirm their role in the assessment of NMDARE.

## Author Contributions

M. Mazowiecki: drafting/revision of the manuscript for content, including medical writing for content; major role in the acquisition of data; study concept or design; analysis or interpretation of data. L. Flet-Berliac: major role in the acquisition of data. J. Roux: major role in the acquisition of data. A. Lépine: major role in the acquisition of data; drafting/revision of the manuscript for content, including medical writing for
content. P. Chretien: analysis or interpretation of data. S. Hacein-Bey-Abina: major role in the acquisition of data. L. Giorgi: analysis or interpretation of data. F. Villega: drafting/ revision of the manuscript for content, including medical writing for content. E. Cheuret: drafting/revision of the manuscript for content, including medical writing for content. M. Benaiteau: major role in the acquisition of data. V. Rogemond: major role in the acquisition of data. G. Picard: major role in the acquisition of data. S. Baer: drafting/revision of the manuscript for content, including medical writing for content. P. Cleuziou: drafting/revision of the manuscript for content, including medical writing for content. E. Lametery: drafting/revision of the manuscript for content, including medical writing for content. I. Desguerre: drafting/revision of the manuscript for content, including medical writing for content. M. Aubart: drafting/revision of the manuscript for content, including medical writing for content. M. Chevignard: drafting/revision of the manuscript for content, including medical writing for content. R. Le Grand: drafting/ revision of the manuscript for content, including medical writing for content. P. Horellou: analysis or interpretation of data. C. Leroy: major role in the acquisition of data. B. Joubert: drafting/revision of the manuscript for content, including medical writing for content. J. Honnorat: drafting/ revision of the manuscript for content, including medical writing for content. K. Deiva: drafting/revision of the manuscript for content, including medical writing for content; study concept or design; analysis or interpretation of data.

## Acknowledgment

The authors thank all patients and families who participated in this study.

## Study Funding

This work was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM) and the Assistance Publique-Hôpitaux de Paris (AP-HP). The authors thank the ENMDR association for funding a part of this study. MB, VR, GP, BJ, and JH are supported by the BETPSY, project as part of the second Investissements d'Avenir programme (ANR- 18-RHUS-0012) supported by a public grant overseen by the Agence Nationale de la Recherche (ANR).

## Disclosure

The authors report no relevant disclosures. Go to Neurology.org/NN for full disclosures.

## Publication History

Received by Neurology: Neuroimmunology & Neuroinflammation June 28, 2024. Accepted in final form November 22, 2024. Submitted and externally peer reviewed. The handling editor was Editor Josep O. Dalmau, MD, PhD, FAAN.

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