Paraneoplastic anti-N-methyl-D-aspartate receptor encephalitis associated with ovarian teratoma

Abstract

Objective: To report the autoantigens of a new category of treatment-responsive paraneoplastic encephalitis.

Methods: Analysis of clinical features, neuropathological findings, tumors, and serum/cerebrospinal fluid antibodies using rat tissue, neuronal cultures, and HEK293 cells expressing subunits of the N-methyl-D-aspartate receptor (NMDAR).

Results: Twelve women (14-44 years) developed prominent psychiatric symptoms, amnesia, seizures, frequent dyskinesias, autonomic dysfunction, and decreased level of consciousness often requiring ventilatory support. All had serum/cerebrospinal fluid antibodies that predominantly immunolabeled the neuropil of hippocampus/forebrain, in particular the cell surface of hippocampal neurons, and reacted with NR2B (and to a lesser extent NR2A) subunits of the NMDAR. NR2B binds glutamate and forms heteromers (NR1/NR2B or NR1/NR2A/NR2B) that are preferentially expressed in the adult hippocampus/forebrain. Expression of functional heteromers (not single subunits) was required for antibody binding. Eleven patients had teratoma of the ovary (six mature) and one a mature teratoma in the mediastinum; five of five tumors examined contained nervous tissue that strongly expressed NR2 subunits and reacted with patients' antibodies. Tumor resection and immunotherapy resulted in improvement or full recovery of eight of nine patients (paralleled by decreased antibody titers); two of three patients without tumor resection died of neurological deterioration. Autopsies showed extensive microgliosis, rare T-cell infiltrates, and neuronal degeneration predominantly involving, but not restricted to, the hippocampus.

Interpretation: Antibodies to NR2B- and NR2A-containing heteromers of the NMDAR associate with a severe but treatment-responsive encephalitis. Our findings provide a diagnostic test and suggest a model of autoimmune NMDAR-related encephalitis with broad implications for other immune-mediated disorders of memory, behavior, and cognition.

Fig 1

Brain magnetic resonance imaging (MRI) findings in three patients. (A, B) MRI of Patient 1 at symptom presentation (A) and after partial clinical improvement and cerebrospinal fluid normalization with immunotherapy (B); note that the clinical and MRI improvement started to occur before tumor resection. (C, D) MRI of Patient 2 at symptom presentation (C) and 4 months later (D); this patient developed rapidly progressive neurological deterioration that did not respond to immunotherapy. The autopsy demonstrated that the ovarian cyst was a mature teratoma of the ovary. (E, F) MRI of Patient 3 at symptom presentation; note the mild fluid-attenuated inversion recovery hyperintensity in medial temporal lobes and right frontal cortex. After immunotherapy and tumor resection, the MRI was normal (not shown).

Fig 2

Interval increase of tumor size during encephalitis. (A) Computed tomography of Patient 1 shows a 5cm cystic ovarian lesion (arrow) that doubled in size over 2 months (B). The lesion was not initially removed because of the poor clinical condition of the patient and benign appearance of the ovarian mass. After partial clinical improvement with immunotherapy, the mass was removed (immature teratoma).

Fig 3

Reactivity of patients’ antibodies with hippocampus and forebrain, and colocalization with the NR2B subunit of the N-methyl-D-aspartate receptor (NMDAR). (A) Sagittal section of rat brain immunolabeled with a patient’s cerebrospinal fluid. Note the robust reactivity with the hippocampus and milder reactivity with forebrain. The cerebellum is largely spared. (B) Higher magnification of the molecular layer of the hippocampus (arrow in A); this pattern of reactivity is identical to that previously reported in patients with paraneoplastic encephalitis and ovarian teratoma. (C–E) Double immunolabeling of cultures of rat hippocampal neurons using a patient’s antibodies (C, green) and an antibody against NR2B of the NMDAR (E, red); note the significant colocalization of reactivities (D, yellow). These findings suggested that patients’ antibodies were directed against the NMDAR. Subsequent studies demonstrated that the patient also had antibodies against NR2A (not shown), which explains, in part, the partial colocalization of reactivities. Original magnification ×2.5 (A) and ×400 (B), both counterstained with hematoxylin; ×800 (oil lens), immunofluorescence (C–E).

Fig 4

Patients’ antibodies react with heteromers of NR2B and NR2A subunits of the N-methyl-D-aspartate receptor (NMDAR). HEK293 cells expressing heteromers (NR1/NR2B or NR1/NR2A) or transfected with single subunits NR1 or NR2B of the NMDAR were incubated with patients’ serum or cerebrospinal fluid (CSF). (top row) Panels demonstrate that the CSF of Patient 7 reacts with cells expressing heteromers (functional receptors) of NR1/NR2B and NR1/NR2A, but not with cells transfected with single subunits (NR1; NR2B). Cells transfected with single NR2A or plasmid without insert were not reactive with patient’s antibodies (not shown). (bottom row) Panels demonstrate the reactivity of the CSF of Patients 1 and 3 with NR1/NR2B. In the third panel (#7 + NR2B), the cells were coincubated with CSF of Patient 7 and an antibody specific for NR2B, showing colocalization of reactivities. The fourth panel (C (−)) corresponds to the CSF of an individual without paraneoplastic encephalitis (negative control). Original magnification ×800, immunofluorescence; nuclei of cells demonstrated with 4′,6-diamidino-2-phenylindole (DAPI), except “#7 + NR2B,” in which no DAPI was used.

Fig 5

Patients’ antibodies react with NR2 subunits of the N-methyl-D-aspartate receptors (NMDARs) expressed by nervous tissue present in the tumor. (A, B) Panels correspond to the ovarian teratoma of Patients 3 and 5 immunolabeled with MAP2 (brown staining), a marker specific for neurons and dendritic processes. Note the intense reactivity with neuronal-like cells and a network of cell processes that are better developed in A. (B, inset) Some immature neuronal cells at higher magnification. (C–E) Panels correspond to the tumor of Patient 3 immunolabeled with the patient’s antibodies (C, green) and a specific antibody for NR2B (E, red). Note that there is colocalization of reactivities (D, yellow), indicating that the patient’s antibodies react with NR2B expressed in the tumor (similar findings were observed with NR2A, not shown). (F–H) Panels correspond to the tumor of Patient 5 immunolabeled with the patient’s antibodies (F, green) and a specific antibody for NR2B (H, red). There is also colocalization of reactivities (G, yellow), indicating that the patient’s antibodies recognize NR2B expressed in the tumor (similar findings were observed with NR2A, not shown). Original magnification ×200, counterstained with hematoxylin (A, B); ×400, immunofluorescence (C–H).