Downloaded from http://jnnp.bmj.com/ on August 18, 2015 - Published by group.bmj.com

Neuro-inflammation

RESEARCH PAPER

Limbic encephalitis due to GABAB and AMPA receptor antibodies: a case series M Dogan Onugoren,1 D Deuretzbacher,2 C A Haensch,3 H J Hagedorn,4 S Halve,5 S Isenmann,6 C Kramme,1 H Lohner,7 N Melzer,8 R Monotti,9 S Presslauer,10 W R Schäbitz,11 S Steffanoni,9 K Stoeck,12 M Strittmatter,13 F Stögbauer,14 E Trinka,15 T J von Oertzen,16 H Wiendl,8 F G Woermann,1 C G Bien1 For numbered affiliations see end of article. Correspondence to Dr Müjgan Dogan Onugoren, Krankenhaus Mara, Epilepsy Center Bethel, Maraweg 17, Bielefeld D-33617, Germany; [email protected] Received 24 June 2014 Revised 18 September 2014 Accepted 18 September 2014 Published Online First 9 October 2014

ABSTRACT Background Two novel antibodies (abs) directed to γ-aminobutyric acid B receptor (GABABR) and α-amino3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) in patients with limbic encephalitis (LE) were first described by the Philadelphia/Barcelona groups and confirmed by the Mayo group. We present a novel series for further clinical and paraclinical refinement. Methods Serum and cerebrospinal fluid samples from a diagnostic laboratory were selected if found to be positive for GABABR or AMPAR abs within a broad antineuronal ab panel. Data were retrospectively compiled. Results In 10 patients, we detected abs to GABABR. Median age was 70 years. Five of them were diagnosed with small cell lung cancer (SCLC). Intrathecal GABABR ab synthesis was found in all six patients with sufficient data available (median ab-index: 76.8). On MRI, we found bilateral mediotemporal and in two cases cortical abnormalities. EEG revealed encephalopathy, partly with epileptiform discharges. Five patients received immunotherapy, two patients tumour treatment and three both therapies. Three patients died, in five patients cognitive functions declined, one patient improved slightly and one patient fully recovered. AMPAR abs were detected in three patients with mnestic disturbances. Median age was 60.7 years. The only female patient was diagnosed with ovarian cancer. None of the patients had intrathecal ab synthesis. MRI findings showed bilateral mediotemporal abnormalities. EEG was normal in all patients. Two of the three immunologically treated patients improved, one patient stabilised on a low level. Discussion GABABR and AMPAR abs are well associated with LE. GABABR abs lead to severe clinical, neuroradiological and EEG abnormalities with poorer outcome.

INTRODUCTION

To cite: Dogan Onugoren M, Deuretzbacher D, Haensch CA, et al. J Neurol Neurosurg Psychiatry 2015;86:965–972.

Autoantibodies to extracellular epitopes of neurons are increasingly recognised in clinical neurology. There appear to be effective treatment options, in particular immunosuppression and (if applicable) tumour treatment. Recognition of these autoimmune diseases therefore should be beneficial for patients.1 Recently, autoantibodies to γ-aminobutyric acid B receptor (GABABR) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) were detected. GABABR

antibodies (abs) have been characterised by their discoverers as the causative agents of limbic encephalitis (LE).2 3 Single cases have been reported with cerebellar ataxia,3 4 opsoclonus-myoclonus syndrome or status epilepticus.3 AMPAR abs directed against a subtype of glutamate receptors have been described as defining another form of immunemediated LE.5 Both receptors are involved in synaptic plasticity underlying different processes such as learning, behaviour and memory.6 7 In this study, we comparatively present clinical, cerebrospinal fluid (CSF), EEG and MRI data of patients with GABABR and AMPAR ab-associated LE. This was done to confirm, extend and refine the data from the original series presented by the Philadelphia/Barcelona groups2 5 and confirmed by the Mayo group.8 LE was defined by at least two of the following: seizures of temporal semiology, disturbance of episodic memory, affective disturbances with prominent mood lability, or disinhibition.9 With onconeural abs, the unambiguous identification of different laboratories was once suggested as pre-requisite for ‘well-characterised antibodies’.10

METHODS Sera, CSFs and CSF–serum pairs from 4819 patients from centres across Germany, Austria and Switzerland were tested for antineuronal abs from November 2011 to February 2014. A sample of 300 consecutive patients had the following distribution of suspected diagnoses: encephalitis/encephalopathy 42%, epilepsy 22%, cognitive or psychiatric disorder 9%, peripheral neurological disorder 4%, other or no specified information 23%. Data of the patients with abs to GABABR and AMPAR were contributed for clinical discussion by the referring physicians. For the purposes of this study, data were retrospectively completed. All available brain MRI were reviewed by one author (FW). Specific ab indices (titre of specific ab (CSF)/titre of specific ab (serum))/(total IgG concentration (CSF)/total IgG concentration (serum)) were calculated. Values >2 were taken as evidence for intrathecal synthesis of the specific ab.11 For calculating the specific ab synthesis we took the earliest serum/CSF pair available with all necessary information at hand. Albumin CSF/serum ratio (QAlb) was calculated to determine the integrity of the blood–brain barrier (BBB)7 with QAlb=(4+age/15)×10−3 as the upper limit of the reference range.12

Dogan Onugoren M, et al. J Neurol Neurosurg Psychiatry 2015;86:965–972. doi:10.1136/jnnp-2014-308814

965

Downloaded from http://jnnp.bmj.com/ on August 18, 2015 - Published by group.bmj.com

Neuro-inflammation Antibody determination For detection of abs, commercially available postfixed sagittal mouse brain sections and cell-based assays in the form of commercially available biochips (Euroimmun, Lübeck, Germany) were used. The assays consist of human embryonic kidney (HEK 293) cells transfected with plasmids encoding the following antigens (subsequent fixation with the substances given in brackets): N-methyl-D-aspartate receptor (NMDAR; consisting of NR1 subunits only), NMDAR (NR1-NR2), glutamic acid decarboxylase (GAD) GAD65, GAD67 (acetone), leucine-rich glioma inactivated1 protein (LGI1), contactin-associated protein-like2 (CASPR2), AMPAR1, AMPAR2, GABABR, glycine receptor (GlyR) (paraformaldehyde). Their preparation has been described before (cf. ref. 13) according to the method given in ref. 14. The protocol for indirect immunofluorescence follows the recommendations given by Euroimmun (FA 112d-1005-1, IgG) with few modifications: serum samples were diluted to 1:15 before incubation (Euroimmun: 1:10); the buffer was the phosphate buffer solution (PBS; Euroimmun: PBS-Tween); the secondary system consisted of a goat-antihuman IgG (heavy and light chain) ab conjugated with DyLight 594 produced by Jackson ImmunoResearch, West Grove, Pennsylvania, USA, Code No. 109-515-088 at a dilution of 1:100 (Euroimmun: goat-antihuman IgG, conjugated with fluorescein, no further information given); nuclear counterstaining with Hoechst 33 342 at 1:10 000 (Euroimmun: no nuclear staining); embedding with 1,4-diazabicyclo[2.2.2]octan (Euroimmun: glycerol). The stained biochips were examined using a fluorescence microscope (Leica DM 2000; Wetzlar, Germany) with excitation at 592 nm and emission filter at 616 nm for bound ab and 350/462 nm for the nuclear counterstain. The decision if an ab was present in the tested material was made by one of the three investigators (CGB, CK and MDO) using the signal of the surrounding (supposedly negative) fields as respective negative controls. An end point titration was performed by serial dilutions in a multiple of 1:2. The titre is the concentration at which a signal is still just detectable in comparison with adjacently stained non-transfected cells. Each titration is rated by two of the four investigators (CGB, CK, MDO or an experienced technician) independently. In cases of divergent ratings, the mean of the two ratings is recorded.

RESULTS Using indirect immunofluorescence on mouse brain tissue and transfected HEK293 cells, we found 10 patients to harbour GABAB1+2R abs and 3 AMPAR2 abs (figure 1). AMPAR1 abs were not found in any of our patients. (Besides this, 206 patients were identified as being positive for abs to the following other antigens: GAD65 N=67; NMDAR N=55; LGI1 N=44; CASPR2 at titres >1:100 N=14; glycin receptor N=6; Hu N=5; Ma N=1; amphiphysin N=2; Yo N=4; Sox-1 N=4; amphiphysin and Hu N=1; Hu and Sox-1 N=3.) In the eight patients with CSF–serum pairs available, the abs were found in both materials. All GABABR and all AMPAR ab-positive cases had predominantly limbic syndromes: only one patient with GABABR abs (and Sox-1 abs) had a pure Lambert Eaton myasthenic syndrome; this case is not further considered here and has been reported separately.15

GABABR abs The median age was 70 years (range 51–75), three of the patients were women. Eight patients presented with generalised convulsive seizures being the primary reason for admittance to hospital. Among these eight patients two presented with status epilepticus 966

and one with a series of generalised tonic–clonic seizures. Seizures were accompanied by memory deficits, confusion, in part also disorientation and behavioural abnormalities such as aggressiveness and agitation. One patient developed severe immobility and a catatonic-like condition. Apraxia and aphasia were observed in three patients. Five patients showed cerebellar dysfunction. Three of them presented with gait and stance ataxia, one with limb ataxia and bradydiadochokinesis, two with intentional tremor and two with pathological nystagmus. Two patients (4 and 6) had prodromal symptoms consisting of vertigo and malaise 1–4 days before the onset of neurological symptoms. In five patients, small cell lung cancer (SCLC) was detected, in all of them after manifestation of the neurological symptoms within a median of 18 days (range 0–35). In five patients, tumour searches (CT of the chest and abdomen or whole body fluorodeoxyglucose positron emission tomography (FDG-PET), at minimum two examinations) were initiated without an indicative result. Follow-up period in these patients covered in median 3 months (range 2–12). None of the 10 patients had a history of autoimmune disease. Four patients had additional abs against Sox-1 (6, 8 and 10) and Hu (3; see table 1). These patients did not show significant differences regarding clinical, paraclinical features, response to therapy and outcome compared with the patients without the evidence of intracellular antigens. All patients in our series had CSF studies. Lymphocytic pleocytosis (>4 cells/mL) was found in 6/9 patients with available data (median white cell count was 13/μL; range 2–78) at first spinal tapping. These data were compiled within a median of 4 days after onset of symptoms (range 0–33). Intrathecal IgG synthesis evidenced by elevated IgG indices or unmatched oligoclonal bands was found in 7/10. Extensive microbiology studies were without abnormalities in all cases. GABABR abs were found in serum samples of all patients. In one patient, titration was not possible due to lack of material (5). Median GABABR titre in serum was 1:1000 (range 1:100–1:50 000). Interestingly, patients with a tumour had titre values that were about 50-fold (median value 26 000 (500–50 000)) higher than in patients without a tumour (median value 1000 (100–2000)). GABABR abs were found in CSF samples of six patients with a median titre of 1:250 ranging from 1:32 to 1:1000. As far as data were available, median titre of abs in patients with a tumour was 1:375 (1:250–1:1000) and without a tumour 1:225 (1:32–1:750). In three patients we could not determine GABABR titres in CSF as we did not receive this material. In these three cases diagnostic results were exclusively based on serum tests (table 1). Intrathecal GABABR ab synthesis was found in five of the six patients (1, 3, 6, 8, 9 and 10) with sufficient data available. Median disease duration at determination of the ab-index was 8.5 days (range 4–33). Median-specific index for GABABR abs was 76.8, ranging 57.9–100. Nine of the 10 patients had MRI lesions that were thought to be related to encephalitis. At the earliest available study (median 4 days, range 1–129 days after manifestation), we saw mediotemporal fluid-attenuated inversion recovery (FLAIR)/T2 volume and signal increase (three bilateral, six unilateral, figure 2). Hippocampi (in part, the amygdalae, too) initially exhibited features of acute oedema. We had follow-up examinations of nine patients. There was increasing atrophy and signal increase on T2/FLAIR-weighted images in five of them accounting for evolution of mediotemporal sclerosis. Interestingly, on the outer aspects of the hippocampus on the border to the temporal horn of the lateral

Dogan Onugoren M, et al. J Neurol Neurosurg Psychiatry 2015;86:965–972. doi:10.1136/jnnp-2014-308814

Downloaded from http://jnnp.bmj.com/ on August 18, 2015 - Published by group.bmj.com

Neuro-inflammation

Figure 1 Immunofluorescence of a mouse brain (A, C and E) and cell-based assays (B, D and F) incubated with cerebrospinal fluid (CSF) or serum containing γ-aminobutyric acid B receptor (GABABR) antibodies (abs) (A and B1–3), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor 2 (AMPAR2) ab-positive CSF (C and D1–3) and with serum containing anti-Hu abs of a control individual (E and F1–2). (A) The mouse brain incubated with CSF containing GABABR abs ( patient 8) displays immunostaining of hippocampus, cortical, subcortical regions and stratum moleculare of the cerebellum. (B) Human embryonic kidney (HEK) cells (Euroimmun, Lübeck, Germany) transfected with cDNA for GABAB1+2R are labelled with serum of patient 2 and a commercial ab directed to an extracellular portion of the GABAB1R (monoclonal mouse antihuman antibody clone 2D7, Abnova, Taipei, Taiwan). Merging of both signals (yellow) indicates co-localisation of the abs on the surface of HEK cells (B2) (red: fluorescence signal by patient’s ab (B1); green: fluorescence signal by commercial GABAB1R ab (B3); blue: nuclear staining with Hoechst 33342). (C) Labelling of AMPAR2 in the hippocampus and cortex by using CSF of patient 12 containing abs to AMPAR2. (D) HEK 293 cells transfected with the AMPAR2 subunit are labelled by CSF of patient 12 and a commercial ab directed to the AMPAR2 subunit (monoclonal mouse antihuman antibody, LifeSpan Biosciences, Seattle, Washington, USA) yields green fluorescence signal (D3). Merge of both signals on the surface of HEK cells results in a yellow signal (red: fluorescence signal by patient’s antibody (D1); green: fluorescence signal by commercial AMPAR2 ab (D3); blue: nuclear staining with Hoechst 33 342). (E) Mouse brain immunolabelled with serum of a control individual staining positive for anti-Hu abs. (F) HEK 293 cells transfected with GABABR abs (F1) and HEK 293 cells transfected with AMPAR2 abs (F2) do not show reactivity with control individual’s serum. Bars in A, C and E: 1 mm; in B1–3, D1–3 and F1–2: 25 mm.

ventricle, T1-weighted not-enhanced images convey the impression of laminar necrosis that is usually seen as an MRI correlate of necrosis of neurons and astrocytes in vulnerable brain regions.16 17 This feature was observed in 3/10 patients (4, 6 and 10), 2 weeks to 4 months into the disease (shown for an AMPAR ab case, figure 2C2). Involvement of extramediotemporal areas was found in 2/10 patients, one in other parts of the limbic system, that is, the insula and the fronto-orbital cortex (8, figure 2A), one had diffuse hyperintensity involving the whole temporal lobe (2; not shown). EEG revealed generalised or focal slowing in six patients, in two cases combined with foci of epileptic activity in temporal lobe or more extended in one hemisphere. Epileptic activity without slowing occurred in two patients (1 and 6; table 2).

AMPAR abs In three serum or CSF samples we found abs to AMPAR. All reacted with the subunit 2. Looking at the age, there were no

significant differences among the patients. The median age was 61 years (range 60–62), one of the patients was a woman. This patient was the only one with a tumour. The most prominent symptoms were short-term memory deficits. In all three patients, a two-stage progression of disease could be observed. Two patients (12 and 13) showed initially mild, partially transient and later on prolonged, more prominent symptoms. The only female patient (13) presented Hashimoto thyroiditis and showed ovarian cancer in the diagnostic work-up. After resection of the tumour, memory worsened and a psychosyndrome combined with anxiety and thoughts of suicide manifested itself. Immunotherapy and chemotherapy were applied. After 14 months memory deficits and the psychosyndrome regressed. The patient was able to live independently. The other two patients (11, 12) did not show a concomitant autoimmune disorder, a tumour in the diagnostic work-up or additional autoimmune-abs. Patient 12 presented a multiphase

Dogan Onugoren M, et al. J Neurol Neurosurg Psychiatry 2015;86:965–972. doi:10.1136/jnnp-2014-308814

967

Demographic data, presenting symptoms and CSF/serum data of patients with GABABR and AMPAR abs Ab proving study

Number

Latency (days)

Earliest CSF

Dogan Onugoren M, et al. J Neurol Neurosurg Psychiatry 2015;86:965–972. doi:10.1136/jnnp-2014-308814

Serum titre

CSF titre

Ab index

Latency (days)

Cells/ mL

100 100 500

32 ND 250

57.9 ND 100

8 26 2

78 10 47

+ + +

+ + +

− − Hu (S+CSF)

50 000

ND

ND

11

23

+

+



Positive* 50 000

1000 375

ND 2.6

0 4

13 4

+ −

ND −

Limbic symptoms

Extralimbic symptoms

Abs

1 2 3

M; 51 M; 65 M; 72

8 0 9

M; 75

GABABR

31

5 6

F; 60 F; 74

Apraxia, psychotic symptoms Cognitive decline Cognitive decline, gait and stance difficulties Catatonia, aphasia, nystagm, oculomotor disorder Psychosyndrome Gait difficulties, psychomotor agitation

GABABR GABABR GABABR

4

Memory deficits Seizures, memory deficits Seizures, memory deficits, confusion, Seizures, confusion, psychomotor agitation Seizures (SE) Memory deficits, confusion

GABABR GABABR

8 4

7 8 9

M; 71 M; 62 M; 69

Change of personality Apraxia, aphasia, intention tremor

GABABR GABABR GABABR

266 8 33

2000 2000 1000

ND 750 250

ND 84.6 86.2

7 0 33

ND 39 11

+ + +

+ + −

10

M; 70

Seizures Seizures, confusion Seizures (SE), memory deficits, confusion Seizures, memory deficits,

Word-finding difficulties

GABABR

4

1000

200

69.0

4

2

+



11

M; 61

Memory deficits (multi-phase)

9

32

ND

ND

9

Limbic encephalitis due to GABAB and AMPA receptor antibodies: a case series.

Two novel antibodies (abs) directed to γ-aminobutyric acid B receptor (GABA(B)R) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AM...
667KB Sizes 0 Downloads 6 Views