N-methyl-D-aspartate antibody encephalitis: temporal progression of clinical and paraclinical observations in a predominantly non-paraneoplastic disorder of both sexes

Abstract

Antibodies to the N-methyl-d-aspartate subtype of glutamate receptor have been associated with a newly-described encephalopathy that has been mainly identified in young females with ovarian tumours. However, the full clinical spectrum and treatment responses are not yet clear. We established a sensitive cell-based assay for detection of N-methyl-d-aspartate receptor antibodies in serum or cerebrospinal fluid, and a quantitative fluorescent immunoprecipitation assay for serial studies. Although there was marked intrathecal synthesis of N-methyl-d-aspartate receptor antibodies, the absolute levels of N-methyl-d-aspartate receptor antibodies were higher in serum than in cerebrospinal fluid. N-methyl-d-aspartate receptor antibodies were of the immunoglobulin G1 subclass and were able to activate complement on N-methyl d-aspartate receptor-expressing human embryonic kidney cells. From questionnaires returned on 44 N-methyl-d-aspartate receptor antibody-positive patients, we identified a high proportion without a detected tumour (35/44, 80%: follow-up 3.6-121 months, median 16 months). Among the latter were 15 adult females (43%), 10 adult males (29%) and 10 children (29%), with four in the first decade of life. Overall, there was a high proportion (29%) of non-Caucasians. Good clinical outcomes, as defined by reductions in modified Rankin scores, correlated with decreased N-methyl-d-aspartate receptor antibody levels and were associated with early (<40 days) administration of immunotherapies in non-paraneoplastic patients (P < 0.0001) and earlier tumour removal in paraneoplastic patients (P = 0.02). Ten patients (23%) who were first diagnosed during relapses had no evidence of tumours but had received minimal or no immunotherapy during earlier episodes. Temporal analysis of the onset of the neurological features suggested progression through two main stages. The time of onset of the early features, characterized by neuropsychiatric symptoms and seizures preceded by a median of 10-20 days, the onset of movement disorders, reduction in consciousness and dysautonomia. This temporal dichotomy was also seen in the timing of cerebrospinal fluid, electroencephalographic and in the rather infrequent cerebral imaging changes. Overall, our data support a model in which the early features are associated with cerebrospinal fluid lymphocytosis, and the later features with appearance of oligoclonal bands. The immunological events and neuronal mechanisms underlying these observations need to be explored further, but one possibility is that the early stage represents diffusion of serum antibodies into the cortical grey matter, whereas the later stage results from secondary expansion of the immunological repertoire within the intrathecal compartment acting on subcortical neurons. Four patients, who only had temporal lobe epilepsy without oligoclonal bands, may represent restriction to the first stage.

Figure 1

Detection and characterization of NMDAR antibodies. (A) NMDAR-antibody-positive CSF shows a hippocampal ‘neuropil’ binding pattern on rat brain sections. (B) NMDAR-antibody-positive serum IgG (green) showing surface binding to primary cultures of live hippocampal neurons identified by the neuronal marker microtubule associated protein 2 (MAP2, in red, within merged lower image; 600× magnification). (C) Cell-based assay for NR1/NR2B (NMDAR) antibodies, using transfected human embryonic kidney (HEK293) cells identified by EGFP (enhanced green fluorescent protein) cDNA co-transfection (green), shows surface binding of patient sera (anti-IgG, red). This sample was scored as ‘4’ by visual observation (600× magnification). (D) Cell-based assay scores from 14 paired CSF-serum samples; note one CSF was negative. (E) Values from end-point titrations of the 14 sera and positive paired CSFs in the cell-based assay; note the different vertical scales and that serum levels of NMDAR antibodies were higher than CSF levels. Two of the data pairs overlapped and their values have been slightly adjusted so that the points are visible. Asterisk indicates one data pair could not be plotted as the CSF was negative (D). (F) NMDAR antibodies were found to be predominantly of the IgG1 subclass (n = 11). (G) NMDAR-antibody-positive sera (NMDAR-Ab) but not control sera (HC) were able to deposit complement C3b and C9neo, the membrane attack complex, on human embryonic kidney cells expressing NMDAR (1000× magnification).

Figure 2

Quantitative analysis of NMDAR antibodies and subgroups of NMDAR-antibody-positive patients. (A) The fluorescent immunoprecipitation assay. NR1-specific commercial antibodies (anti-NR1) precipitated large amounts of EGFP-NR1 which was not bound by NR2B-specific (anti-NR2B) or PSD95 (anti-PSD95) antibodies (data from three experiments). Eighty-four percent of 44 NMDAR-antibody-positive patients (as determined by the cell-based assay) precipitated EGFP-NR1 at levels greater than the mean plus three standard deviations (mean + 3SD) of results from 20 healthy controls (HCs). (B) There was a strong correlation between the NMDAR-antibody levels determined by the fluorescent immunoprecipitation assay and by the cell-based assay (r = 0.86, P < 0.0001; Spearman rank correlation). (C) The ages at disease onset of the 44 NMDAR-antibody-positive patients shown as male and female paraneoplastic (PN), non-paraneoplastic (NPN), and children (none of whom had tumours detected). (D) The results of the fluorescent immunoprecipitation assay showed the NMDAR-antibody levels were higher in paraneoplastic when compared to all non-paraneoplastic cases (P = 0.0017, Mann-Whitney U-test). fU = relative fluorescence units precipitated by 25 µl of serum.

Figure 3

Imaging, cerebrospinal fluid and EEG results. (A) The extent of CSF lymphocytosis (>5 cells/mm³, red dots) observed at different time points across all patients. Thirt-two percent of patients had persistently negative CSF lymphocytosis (<5 cells/mm³, blue dots). (B) MRI was commonly normal (white) and abnormalities were mainly restricted to white matter tracts (n = 6, red) and hippocampi (n = 4, blue) both at initial (median Day 1) and subsequent (median Day 25) imaging. (C) Serial electroencephalograms (EEGs) of a 17-year-old non-paraneoplastic female NMDAR-antibody-positive patient, not within our cohort of 44 cases. Bipolar transverse EEG recordings are shown in the scheme below. (a) EEG was normal after the patient had experienced a generalized convulsion. From Day 21–24, she suffered from frequent complex partial seizures; (b) EEG showed frequent, in part long-lasting frontal epileptiform spike-wave activity; (c–d) subsequently, there was continuous diffuse high-amplitude slowing without epileptiform potentials. From Day 39 on, no further seizures were observed. On Day 42, anti-epileptic therapy consisted of phenytoin (blood level: 25.4 µg/ml), phenobarbital (29.7 µg/ml) and lorazepam (2 mg daily dose). Glucocorticosteroids were instituted on Day 38. From Day 56, the patient recovered; and (e) normal EEG appearances recurred in sections in between abnormally high waves.

Figure 4

(A–F) Effects of immunotherapy [plasma exchange (downward arrow), glucocorticosteroids (solid lines) and intravenous immunoglobulins (upward arrow)] and oophorectomy (asterisk) on NMDAR-antibody titres [fluorescent units precipitated (fU), black] and on clinical outcomes (modified Rankin scores, red). Death is indicated by ‘dagger’. Age, sex and neoplasm status (paraneoplastic = PN, non-paraneoplastic = NPN) are shown within each figure. (A) and (B) show reductions in NMDAR antibodies correlating with clinical outcome after successful immunotherapy, although antibodies persist in (B). (C) and (D) show two patients who died with very high NMDAR-antibody levels persisted throughout the illness despite immunotherapies (C and D) and late oophorectomy (C). (E) and (F) show patients with clinical relapses after no immunotherapy (E) or only 3 days of intravenous glucocortocosteroid therapy (F) during their first episode. Both patients showed clinical and serological improvement after immunotherapy administered during their second episode. In (F), the dotted line represents unknown titres between episodes. More detailed vignettes of these cases are available in the Supplementary data. MRS = modified Rankin score.

Figure 5

Clinical outcomes and therapeutics. (A) The change in modified Rankin score (MRS) at 120 days after onset of symptoms divided into subgroups. (B) The improvement in modified Rankin scores at 120 days after onset of symptoms in non-paraneoplastic patients according to administration of no immunotherapy (No IT), any immunotherapy after 40 days (IT > 40 days), or before or at 40 days (IT ≤ 40 days). The other immunotherapies were intravenous lg (n = 4), plasma exchange (n = 2), intravenous lg and plasma exchange (n = 3) or cyclophosphamide (n = 1). (Kruskal–Wallis, P < 0.0001; ***Dunn’s multiple comparison test P < 0.0001 for no IT or IT > 40 days compared to IT ≤ 40 days. For those administered IT at ≤40 days, there was a trend towards better outcomes when steroids plus other immunotherapies were given (Steroids alone v. Steroids + other IT; Mann Whitney, P = 0.02). (C) The correlation between percentage change in NMDAR-antibody levels determined by fluorescent immunoprecipitation assay and the corresponding change in modified Rankin scores over the time the two samples were taken (r = 0.54, P = 0.005; n = 25). Symbols as in (A).

Figure 6

Temporal progression of clinical features and investigations. (A) The times of first appearance of the main clinical features for each patient, up to 120 days after onset of neurological symptoms. (B) The times of all CSF pleocytosis or oligoclonal bands, and all abnormal EEG or MRI findings. Median and interquartile ranges are shown. P-values in B represent Mann–Whitney U-tests for each blue-red pairing. OCB = CSF-specific oligoclonal bands. Percentages (bracketed) show the frequency of each feature or investigation within the total NMDAR-antibody patient cohort.