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The prevalence of neural antibodies in temporal lobe epilepsy and the clinical characteristics of seropositive patients

Open ArchivePublished:September 17, 2018DOI:https://doi.org/10.1016/j.seizure.2018.09.009

      Highlights

      • 5% prevalence of neural surface and GAD antibodies in temporal lobe epilepsy patients.
      • All positive patients had unknown epilepsy aetiology.
      • Positivity associated with higher age at epilepsy onset and autoimmune comorbidity.
      • Immunoresponse observed in half of positive patients.

      Abstract

      Purpose

      Epileptic seizures are a common manifestation of autoimmune encephalitis, but the role of neural antibodies in long-term epilepsy remains unclear. The aim of this study was to assess the prevalence of neural-surface antibodies (NSAbs) and antibodies against glutamic acid decarboxylase (GAD) in patients with chronic temporal lobe epilepsy (TLE).

      Method

      Patients with an electro-clinical diagnosis of TLE and a disease duration longer than one year were included. NSAbs (LGI1, CASPR2, AMPAR1/2, NMDAR, GABABR) and antibodies against GAD were detected. Only patients with significant antibody levels in serum, and/or positivity in CSF (according to antibody subtype), were enrolled in the seropositive group. Cohorts of seropositive and seronegative patients were compared regarding clinical and imaging data.

      Results

      Significant serum levels of antibodies were detected in eight out of 163 (5%) TLE patients (CASPR2 n = 2, GAD n = 3, LGI1 n = 2, and GABABR n = 1). In four of them, antibodies were detected in the CSF as well (CASPR2 in one, GAD in three). Five seropositive patients had uni- or bilateral temporal lobe lesions on MRI and three patients were non-lesional. All seropositive patients had TLE of unknown cause. Seropositive patients had higher age at epilepsy onset and autoimmune comorbidity, but did not differ in other clinical, EEG or neuroimaging characteristics. Response to immunotherapy (seizure reduction >50%) was observed in three of the six patients treated.

      Conclusions

      Besides older age at epilepsy onset and autoimmune comorbidity, seropositive patients cannot be distinguished from seronegative patients on the basis of clinical, EEG or neuroimaging data.

      Abbreviations:

      AMPAR (antibodies against alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor), CASPR2 (antibodies against contactin-associated protein-like 2), CSF (cerebrospinal fluid), GABABR (antibodies against gamma-aminobutyric acid type B receptor), GAD (antibodies against glutamic acid decarboxylase), GLY-R (antibodies against glycine receptor), LGI1 (antibodies against leucine-rich, glioma inactivated 1 protein), NMDAR (antibodies against N-methyl-D-aspartate receptor), NSAbs (neural-surface antibodies), TLE (temporal lobe epilepsy), VGKC complex (antibodies against voltage-gated potassium channel)

      Keywords

      1. Introduction

      Epileptic seizures are a common symptom of neurological syndromes associated with neural surface antibodies (NSAbs) [
      • Brenner T.
      • Sills G.J.
      • Hart Y.
      • Howell S.
      • Waters P.
      • Brodie M.J.
      • et al.
      Prevalence of neurologic autoantibodies in cohorts of patients with new and established epilepsy.
      ,
      • Iorio R.
      • Assenza G.
      • Tombini M.
      • Colicchio G.
      • Della Marca G.
      • Benvenga A.
      • et al.
      The detection of neural autoantibodies in patients with antiepileptic-drug-resistant epilepsy predicts response to immunotherapy.
      ]. Recent studies agree that NSAbs are pathogenic, even though the exact role of these antibodies in ictogenesis and epileptogenesis remains to be elucidated [
      • Lalic T.
      • Pettingill P.
      • Vincent A.
      • Capogna M.
      Human limbic encephalitis serum enhances hippocampal mossy fiber-CA3 pyramidal cell synaptic transmission.
      ].
      Apart from autoimmune encephalitis syndromes accompanied by acute symptomatic seizures, neural antibodies were also detected in patients with chronic epilepsy without encephalopathic features. Chronic epilepsy was also reported in patients with antibodies against glutamic acid decarboxylase (GAD), a subgroup of antibodies against intracellular epitopes playing important role in neuromediator metabolism [
      • Malter M.P.
      • Frisch C.
      • Zeitler H.
      • Surges R.
      • Urbach H.
      • Helmstaedter C.
      • et al.
      Treatment of immune-mediated temporal lobe epilepsy with GAD antibodies.
      ].
      The presence of neural antibodies in epilepsy patients was reported to range from 9 to 34% depending on the study design and selection criteria. Brenner et al. demonstrated that 11% of patients with established epilepsy were seropositive for at least one of the antibodies – VGKC complex, NMDAR, GAD or GLY-R [
      • Brenner T.
      • Sills G.J.
      • Hart Y.
      • Howell S.
      • Waters P.
      • Brodie M.J.
      • et al.
      Prevalence of neurologic autoantibodies in cohorts of patients with new and established epilepsy.
      ]. Chronic temporal lobe epilepsy (TLE) was described as a possible consequence of limbic encephalitis [
      • Bien C.G.
      • Urbach H.
      • Schramm J.
      • Soeder B.M.
      • Becker A.J.
      • Voltz R.
      • et al.
      Limbic encephalitis as a precipitating event in adult-onset temporal lobe epilepsy.
      ]. In a recent study, NSAbs were detected in as many as 22.5% patients with TLE associated with hippocampal sclerosis [
      • Vanli-Yavuz E.N.
      • Erdag E.
      • Tuzun E.
      • Ekizoglu E.
      • Baysal-Kirac L.
      • Ulusoy C.
      • et al.
      Neuronal autoantibodies in mesial temporal lobe epilepsy with hippocampal sclerosis.
      ]. However, antibodies were found even in cases of non-lesional TLE [
      • Brenner T.
      • Sills G.J.
      • Hart Y.
      • Howell S.
      • Waters P.
      • Brodie M.J.
      • et al.
      Prevalence of neurologic autoantibodies in cohorts of patients with new and established epilepsy.
      ] and in encephalitis with extralimbic involvement [
      • Najjar S.
      • Pearlman D.
      • Najjar A.
      • Ghiasian V.
      • Zagzag D.
      • Devinsky O.
      Extralimbic autoimmune encephalitis associated with glutamic acid decarboxylase antibodies: an underdiagnosed entity?.
      ].
      The aim of our study was, firstly, to assess the prevalence of potentially disease specific neural antibodies (NSAbs and GAD) in a prospectively collected cohort of adult TLE patients, irrespective of MRI findings, and, secondly, to identify typical clinical characteristics of this subgroup.

      2. Methods

      2.1 Study population

      Patients were recruited prospectively between January 2013 and December 2017 from the tertiary Epilepsy Centre, Motol (Prague, Czech Republic). We included adult patients with electro-clinically established diagnosis of TLE. Only patients with a disease duration longer than one year were included. Patients with clinical features of acute encephalopathy were excluded in order to avoid selection bias in favour of patients with acute symptomatic seizures as a manifestation of autoimmune encephalitis. Patients with all TLE aetiologies, except primary neuroglial tumours and brain metastasis were included.
      The study was approved by the Ethical Committee of Motol University Hospital, and informed consent was obtained from all participating patients. Patients were treated according to the best medical care standards and enrolment into the study did not impact the approach to diagnosis or treatment.

      2.2 Clinical, imaging and electrophysiological data

      Clinical variables were collected in all patients, including: age at epilepsy onset, duration of epilepsy, history of initial precipitating injury (meningitis/encephalitis, febrile seizures, traumatic brain injury, perinatal complications), seizure frequency at the time of serum sample collection, seizure semiology, history of status epilepticus and psychiatric, autoimmune and oncological comorbidity. Cancer diagnosed up to five years prior or after the onset of epilepsy was considered a relevant oncological morbidity. Severe depression, psychosis, obsessive-compulsive disorder, mental retardation, psychogenic non-epileptic seizures and panic attacks were recorded as relevant psychiatric comorbidities.
      MRI studies were performed in all patients (with a dedicated epilepsy protocol), as well as one or several EEG investigations and neuropsychological examinations. Some patients with drug-resistant TLE underwent comprehensive preoperative evaluation resulting in epilepsy surgery. Based on the presence of potential epileptogenic lesions, the findings of the MRI studies were categorized as either lesional (unilateral or bilateral pathology), or non-lesional. Based on the presence of an ictal pattern and/or interictal abnormality, the findings of the EEG studies were categorized as either abnormal (unilateral or bilateral pathology) or normal. Cerebrospinal fluid (CSF) analysis was performed only in patients with NSAb or GAD positivity in serum and clinically suspected autoimmune aetiology.

      2.3 Antibody testing

      Serum and CSF samples for antibody testing were collected prior to any immunomodulatory treatment.
      For the detection of NSAbs, a commercial cell-based assay for indirect immunofluorescence was used, with HEK293 cells transfected with cDNAs encoding NMDAR, AMPAR1/2, GABABR, CASPR2 and LGI1 (Euroimmun, Autoimmune Encephalitis Mosaic 1). All CSF samples were used undiluted, serum samples were diluted 1:10. Serum samples positive for NSAbs on the cell-based assays at a 1:10 dilution were subsequently titrated (1:20 and double the prior dilution thereafter) in order to reach endpoint positivity. Results of the indirect immunofluorescence were independently evaluated by two investigators (ME, JH). The following NSAb serum titres were assessed as significantly positive (potentially disease-specific): 1:10 (or higher) in LGI1; >1:128 in CASPR2 [
      • Bien C.G.
      • Mirzadjanova Z.
      • Baumgartner C.
      • Onugoren M.D.
      • Grunwald T.
      • Holtkamp M.
      • et al.
      Anti-contactin-associated protein-2 encephalitis: relevance of antibody titres, presentation and outcome.
      ]; >1:40 AMPAR and GABABR (expert opinion). Lower serum antibody levels were considered to be of unclear significance and these patients were not included in further analysis.
      GAD antibodies were detected by immunoblot (Ravo, PNS 11 Line Assay). Immunoblot GAD seropositivity was confirmed by indirect immunofluorescence on rat brain tissue (Euroimmun, rat hippocampus and cerebellum). In confirmed GAD positive cases, serum levels (concentraions) were measured by radioimmunoassay (CentAK anti-GAD65 M, Medipan). The cut-off of high-positives was set at 100 kU/l for the purposes of this study. GAD concentrations in CSF were omitted as the RIA method is only validated for serum.
      Well-characterized autoantibodies (Hu, Yo, Ri, CV2/CRMP5, amphiphysin, Ma1/2, SOX1, Tr- DNER and Zic4) were also tested, as they are included in the Ravo PNS 11 Line Assay kit. Positivity of onconeural antibodies was confirmed by indirect immunofluorescence on rat brain tissue. Samples with positive immunoblot results that were confirmed by immunofluorescence on rat brain, were considered positive for the respective onconeural antibody (but not included in further clinical analysis). If only the immunoblot was positive, the patient was considered negative for onconeural antibodies.

      2.4 Statistical analyses

      Only patients with significantly positive serum antibodies were enrolled in the statistical analyses. The patients with antibody levels of unclear significance were not included. To compare the clinical characteristics of patients in the seropositive and seronegative group, the Mann-Whitney U test (continuous non-parametric variables) or the Fisher’s exact test (categorical variables) were used as appropriate. The original significance level, p = 0.05, was corrected with Bonferroni correction for multiple testing.

      3. Results

      Potentially disease specific neural antibodies in serum were detected in 5% (eight 8/163) of TLE patients investigated (seropositive group); CASPR2 antibodies > 1:128 were found in two cases, GAD antibodies in three, LGI1 antibodies in two, and GABABR antibodies in one patient. Low serum titres of NSAbs (unclear clinical significance) were found in three cases (CASPR2 < 1:128 in two, AMPAR < 1:40 in one). One patient was positive for onconeural antibodies by immunoblot, confirmed by indirect immunofluorescence using tissue-based analyses. The seronegative group consists of 151 patients.
      The combined clinical, imaging and EEG characteristics of the seropositive (n = 8) and seronegative (n = 151) groups are outlined in Table 1. Four patients with antibody levels of unclear significance and the patient positive for onconeural antibodies were not included in the group statistical analysis.
      Table 1Comparison of the clinical data of seropositive and seronegative patients.
      Seropositive patients

      (n = 8)
      Seronegative patients

      (n = 151)
      Statistical significance
      using the Bonferroni correction for multiple comparisons, p < 0.0033 was set as the statistically significant difference (bold).
      Sex (M/F)2M/6F72 M/80Fp = 0.2274
      Age at epilepsy onset (median, range)54 (19–64)25 (1k64)p = 0.0028
      Epilepsy duration - years (median, range)4.5 (1–39)14 (1–65)p = 0.1803
      History of initial precipitating injury
      perinatal hypoxia, febrile seizures, meningitis/encephalitis, traumatic brain injury.
      337p = 0.9663
      Seizure frequency (per month)0.52p = 0.3505
      Aura075p = 0.0600
      FBTCS history7111p = 0.6810
      Status epilepticus history211p = 0.0731
      Pharmacoresistance691p = 0.5299
      EEG bi-temporal327p = 0.7026
      MRI non-lesional335p = 0.7193
      MRI bi-temporal lesion218p = 0.6270
      Autoimmune comorbidity416p = 0.0010
      Psychiatric comorbidity243p = 0.7292
      Oncological comorbidity
      5 years prior/after epilepsy onset.
      04p = 0.6411
      FBTCS - focal seizure evolving to bilateral tonic-clonic.
      a using the Bonferroni correction for multiple comparisons, p < 0.0033 was set as the statistically significant difference (bold).
      b perinatal hypoxia, febrile seizures, meningitis/encephalitis, traumatic brain injury.
      c 5 years prior/after epilepsy onset.

      3.1 Clinical characteristics of seropositive patients

      The clinical, MRI, EEG and laboratory characteristics of seropositive patients, i.e. those with high NSAbs or GAD levels, are shown in Table 2. Meningoencephalitis or encephalitis as an initial precipitating injury was reported in the history of two patients and both of them fulfilled the limbic encephalitis criteria.
      Table 2Clinical characteristics of seropositive patients.
      No

      (sex/age)
      Antibodies

      (serum titres/

      CSF)
      Epilepsy onset

      (y)
      Initial precipitating injuryOnset to antibodies detection

      (y)
      Seizure frequency

      (per month)
      Seizure semiologyMRIEEGOCB CSF/serumComorbidity (immune, oncology)Psychiatric comorbidityImmunotherapy

      (type and dose / response)
      1 (M/68)CASPR2 > 1:40960

      /CASPR2
      64no40FIAS (behav arrest) FBTCSISI lateral T bilatbilat0/0nonoMP 4.5 g + taper* / responder
      2 (M/58)CASPR2 1:320

      /negative
      19possible LE

      acute psychosis, encephalopathy, seizures (19y)
      395FIAS, FBTCSHS bilat + ISI T lateral bilatleft0/0nosevere mood disorderMP 4.5 g + taper* / no response
      3 (F/29)GAD 120 kU/l/28no15FIAS (emotional) FBTCSISI mesial T leftleft0/0autoimmune thyroiditisnoIvIg 175 g /

      no response
      4 (F/73)GAD 187 kU/l68no510FIAS (emotional) FBTCSISI mesial + lateral T leftleft20/3DM 1anxiety disorderMP 4.5 g + taper*/

      no response
      5 (F/28)GAD 360 kU/l26possible LE- acute memory deficit, seizures (26y)21FIAS (cognitive)non-lesionalleft7/0DM 1noMP 3 g /

      responder
      6 (F/71)GABABR 1:80

      /negative
      55no160FIAS (sensory) FBTCS, NCSEISI mesial + lateral T leftleft4/4nonono
      7 (F/66)LGI1 1:320

      /negative
      64no20FIAS (behav arrest) FBTCSnon-lesionalbilat0/0IgG and IgA deficiencynoMP 3 g + taper * / responder
      8 (F/71)LGI1 1:10

      /negative
      52no190FIAS (cognitive) FBTCS, NCSEnon-lesionalbilat0/0nonono
      behav – behaviour, bilat – bilateral, CBZ – carbamazepine, CLB – clobazam, CSF – cerebrospinal fluid, DM 1 – diabetes mellitus type 1, FBTCS – focal evolving to bilateral tonic-clonic seizure, FIAS – focal impaired awareness seizure, HS – hippocampal sclerosis, ISI – increased signal intensity, IvIg - intravenous immunoglobulins, LE – limbic encephalitis, LEV – levetiracetam, LTG – lamotrigine, ME – meningoencephalitis, MP – methylprednisolone, NCSE – non-convulsive status epilepticus, ND – not done, OCB – oligoclonal bands, PER – perampanel, PGB – pregabaline, T – temporal, SCLC – small cell lung cancer, VPA – valproic acid, ZNS – zonisamide, * taper – prednisone 1 mg/kg, 6–12 weeks, seizure frequency is given at the time of antibody testing.
      MRI revealed possible epileptogenic lesions in the temporal lobe of five seropositive patients. MRI findings in all lesional seropositive cases could be interpreted as sequelae of autoimmune encephalitis. CSF was abnormal in three out of eight investigated patients due to the presence of isolated oligoclonal bands. Pleocytosis was not observed in any of our patients. CSF antibodies were detected in four out of eight tested patients (GAD n = 3, CASPR2 n = 1).

      3.2 Immunotherapy in seropositive patients

      Immunotherapy was considered in six out of eight seropositive patients (five patients had drug-refractory TLE, while patient No. 7 had pharmaco-dependent epilepsy and memory deficit). One patient refused immunotherapy (No. 6), and one patient was seizure free at the time of antibody detection (No. 8). The type of treatment regimen was selected based on the disease severity – intravenous methylprednisolone, in some cases followed by prednisone oral taper (1 mg/kg discontinued gradually over the subsequent five weeks), and/or intravenous immunoglobulins. The type and dosage of immunotherapy are shown in Table 2. All patients were treated with antiepileptic drugs during immunotherapy administration. Neither the antiepileptic drugs or their dosing were changed during the three months after immunotherapy initiation. Median duration from epilepsy onset to initiation of immunotherapy was five years (range 1–21.5 years). Three patients responded favourably to immunotherapy. Two of the six immunotherapy treated patients became seizure-free (No. 1, CASPR2; No. 7, LGI1), including non-disabling seizures. One patient had seizure reduction >50% (No.5, GAD). Patient No. 1 (CASPR2) is seizure free five years after immunotherapy, but still receives antiepileptic drugs in reduced dosages. Patient No. 7 (LGI1) is seizure free five years after immunotherapy and two years after antiepileptic drug withdrawal. The others did not respond.

      3.3 Patients with antibody levels of unclear significance and the patient with positivity for isolated onconeural antibodies

      The clinical, MRI, EEG and laboratory characteristics of these patients are shown in Table 3.
      Table 3Clinical characteristics of patients with antibody levels of unclear significance and of the patient positive for isolated onconeural antibodies.
      No

      (sex/age)
      Antibodies

      (serum titres/

      CSF)
      Epilepsy onset (y)Initial precipitating injuryOnset to antibodies detection (y)Seizure frequency

      (per month)
      Seizure semiologyMRIEEGOCB CSF/serumComorbidity (immune, oncology)Psychiatric comorbidityImmunotherapy

      (type and dose / response)
      Patients with antibody levels of unclear significance
      9 (M/54)CASPR2 1:80/

      Negative
      20no343FIAS, FBTCSISI mesial + lateral T rightbilat0/0nonoMP 3.5g /

      no response
      10 (F/68)CASPR2 1:80/

      ND
      56ME

      (3 y)
      123FIAS (emotional) FBTCSHS rightbilatNDretinoblastoma (1y)nono
      11 (F/70)AMPAR1 1:10/

      ND
      58no128FIAS (autonomic) FBTCSISI mesial T rightleft0/0SCLC (4y after epilepsy onset)nono (chemotherapy)
      Patient positive for isolated onconeural antibodies
      12(M/71)Ma2/

      ND

      no tumour found
      67possible LE – memory deficit, seizures61FIAS (autonomic)

      FBTCS
      atrophy mesial T bilatbilat9/1autoimmune thyroiditisnoMP5 g + taper* /

      responder
      Abbreviations – see Table 2.
      Patient No. 9 had low-titre CASPR2 antibodies (1:80). He had focal cortical dysplasia in the temporal lobe on histopathology. This patient originally had refractory epilepsy, repeatedly tested positive for CASPR2 antibodies, but became seronegative after being seizure-free for a year post epilepsy surgery (anteromedial temporal lobe resection). Immunotherapy prior to epilepsy surgery was ineffective for this patient and histopathology did not reveal any inflammatory changes in the brain tissue.
      Patient No. 11 had a low titre of serum AMPAR antibodies (dilution 1:10) and she presented with TLE. Oncological screening subsequently identified a small-cell lung carcinoma – the tumour was diagnosed four years after the onset of epilepsy.
      Patient No. 12 had isolated Ma2 positivity and repeated oncological screening did not reveal any tumour. A significant response to immunotherapy was observed in this patient.

      4. Discussion

      Significantly high serum levels of neural antibodies (NSAbs or anti-GAD) were detected in 5% of prospectively recruited adult patients with TLE in this cohort from a tertiary epilepsy centre. The proportion of seropositive patients in this study is lower compared to other studies [
      • Brenner T.
      • Sills G.J.
      • Hart Y.
      • Howell S.
      • Waters P.
      • Brodie M.J.
      • et al.
      Prevalence of neurologic autoantibodies in cohorts of patients with new and established epilepsy.
      ,
      • Iorio R.
      • Assenza G.
      • Tombini M.
      • Colicchio G.
      • Della Marca G.
      • Benvenga A.
      • et al.
      The detection of neural autoantibodies in patients with antiepileptic-drug-resistant epilepsy predicts response to immunotherapy.
      ,
      • Vanli-Yavuz E.N.
      • Erdag E.
      • Tuzun E.
      • Ekizoglu E.
      • Baysal-Kirac L.
      • Ulusoy C.
      • et al.
      Neuronal autoantibodies in mesial temporal lobe epilepsy with hippocampal sclerosis.
      ]. Studies focusing on "autoimmune epilepsy" tend to over-represent patients with encephalopathic features, often patients with acute autoimmune encephalitis, where epileptic seizures may be regarded as acute symptomatic and the proportion of seropositive patients tends to be higher [
      • Quek A.M.L.
      • Britton J.W.
      • McKeon A.
      • So E.
      • Lennon V.A.
      • Shin C.
      • et al.
      Autoimmune epilepsy: clinical characteristics and response to immunotherapy.
      ,
      • Toledano M.
      • Britton J.W.
      • McKeon A.
      • Shin C.
      • Lennon V.A.
      • Quek A.M.L.
      • et al.
      Utility of an immunotherapy trial in evaluating patients with presumed autoimmune epilepsy.
      ]. Other studies investigated unselected populations of patients with epilepsy [
      • Brenner T.
      • Sills G.J.
      • Hart Y.
      • Howell S.
      • Waters P.
      • Brodie M.J.
      • et al.
      Prevalence of neurologic autoantibodies in cohorts of patients with new and established epilepsy.
      ,
      • Iorio R.
      • Assenza G.
      • Tombini M.
      • Colicchio G.
      • Della Marca G.
      • Benvenga A.
      • et al.
      The detection of neural autoantibodies in patients with antiepileptic-drug-resistant epilepsy predicts response to immunotherapy.
      ]. Our study focused on patients with chronic temporal lobe epilepsy.
      The most common types of antibodies detected in our patients were CASPR2 and anti-GAD, followed by anti-LGI1 and individual cases of anti-GABABR and anti-AMPAR. Only patients with serum titres previously described as clinically relevant (LGI1: 1:10 or higher, CASPR2: >1:128 [
      • Bien C.G.
      • Mirzadjanova Z.
      • Baumgartner C.
      • Onugoren M.D.
      • Grunwald T.
      • Holtkamp M.
      • et al.
      Anti-contactin-associated protein-2 encephalitis: relevance of antibody titres, presentation and outcome.
      ]; AMPAR, GABABR: >1:40), were included in further analyses in order to avoid bias due to antibody positivity of unclear significance. However, these cut-off titres were described in patients with autoimmune encephalitis and we cannot exclude the possibility that even low titres of certain antibodies may play a role in patients with chronic epilepsy. Patient No. 11 with low titre AMPAR antibodies and small cell lung cancer apparently had a paraneoplastic syndrome with TLE. This case illustrates that even low levels of antibodies may sometimes be of clinical significance.
      The role of neural antibodies in patients with long-term epilepsy is controversial. TLE, especially in cases associated with hippocampal sclerosis, may be the result of an earlier limbic encephalitis. [
      • Bien C.G.
      • Urbach H.
      • Schramm J.
      • Soeder B.M.
      • Becker A.J.
      • Voltz R.
      • et al.
      Limbic encephalitis as a precipitating event in adult-onset temporal lobe epilepsy.
      ]. In this situation, antibody positivity may reflect an active chronic inflammatory process or it can be residual. In our study, we identified two patients with possible limbic encephalitis in their history and potentially disease-specific neural antibodies: one female (No. 5, GAD) with non-lesional MRI and CSF oligoclonal bands, who responded favourably to immunotherapy with > 50% seizure reduction, and one male (No. 2, CASPR2) with acute onset of epilepsy together with major psychiatric symptoms 20 years prior to antibody testing. Despite immunological treatment and stereotactic thermocoagulation of his left atrophic hippocampus, this patient continues to suffer from drug-resistant TLE. Duration of the disease may be important in this respect (39 years vs. one year in patient No. 5, GAD). The very long disease duration in patient No. 2 (CASPR2) and the young age at onset is striking. The patient did not have an inflammatory CSF and no peripheral tumour was found despite repeated detailed oncoscreening. We suggest that TLE (together with severe psychiatric comorbidity) in this patient could be a consequence (end-stage) of untreated limbic encephalitis at an earlier age with persisting positivity for neural antibodies in the serum.
      Hypothetically, the production of neural antibodies (e.g. CASPR2) could, in some cases, be secondary to active epilepsy rather than being the primary cause [
      • Wright S.
      • Geerts A.T.
      • Maria Jol-van Cornelia
      • Jacobson L.
      • Lang B.
      • Waters P.
      • et al.
      Neuronal antibodies in pediatric epilepsy: clinical features and long-term outcomes of a historical cohort not treated with immunotherapy.
      ]. This might explain the low titre CASPR2 seropositivity in patient No. 9 with temporal lobe focal cortical dysplasia without inflammatory signs on histopathology. This patient originally had refractory epilepsy, repeatedly tested positive for CASPR2 antibodies, but became seronegative after being seizure–free for one year after epilepsy surgery (anteromedial temporal lobe resection). Immunotherapy prior to epilepsy surgery was ineffective for this patient and histopathology did not reveal any inflammatory changes in the brain tissue.
      There was no significant difference between seropositive and seronegative groups regarding seizure frequency. This observation is in agreement with a recently published study comparing the detection of neural antibodies in drug-responsive and resistant focal epilepsies of unknown cause [
      • Gozubatik-Celik G.
      • Ozkara C.
      • Ulusoy C.
      • Gunduz A.
      • Delil S.
      • Yeni N.
      • et al.
      Anti-neuronal autoantibodies in both drug responsive and resistant focal seizures with unknown cause.
      ]. Similarly we did not detect significant levels of NSAbs or GAD antibodies in patients with any other defined aetiology of their epilepsy (malformation of cortical development n = 29, posttraumatic n = 4, hamartoma n = 2, vascular malformation n = 2, meningioma n = 1).
      Results regarding CSF antibody positivity were available only in some patients from the seropositive or seronegative group, which is one of the limitations of this study.
      Analysis of different characteristics of our cohort confirmed the well-known difficulty in distinguishing between seropositive and seronegative patients on the basis of clinical, EEG or neuroimaging data. However, onset of TLE at an older age seems to be more common among the seropositive patients, as well as autoimmune comorbidity. Furthermore, none of the seropositive patients reported febrile seizures, perinatal complications or trauma in their history. Presence of these features (late-onset TLE without typical initial precipitating injury in early life, autoimmune comorbidity) should raise the suspicion of an autoimmune aetiology and lead to antibody testing. The relevance of antibody testing is emphasized by the fact that three out of six patients in whom immunotherapy was indicated, responded favourably to treatment.
      The most important goal is to identify patients who will respond to immunotherapy. In previous studies (2, 9), a favourable response to immunotherapy was associated with a relatively short duration of epilepsy (in this series: ≤4 years) and NSAb positivity (this series: LGI1 and CASPR2 antibodies). In agreement with these studies, none of our immunotherapy responders had an epilepsy duration of longer than four years. Seizure freedom or a significant reduction in seizure frequency was observed in patients with CASPR2, GAD and LGI1 antibodies. Both CASPR2 and GAD responders had antibodies detectable in the CSF as well. We did not escalate immunotherapy in most of our seropositive patients due to the absence of clinical guidelines and concerns about treatment side-effects. Thus, we cannot rule out the potential benefit of second line immunotherapy in those who did not respond at all, or only partially. Interestingly, the patient with Ma2 antibodies responded to immunotherapy.
      The main limitation of our study is the relatively low number of patients in the seropositive group, which may influence the statistical results regarding clinical characteristics. Furthermore, the diagnosis of TLE is based on clinical findings in most patients. Video-EEG studies with further characterisation of epilepsy are available only in patients with refractory TLE, in whom epilepsy surgery was discussed as a treatment option.
      The absence of CSF analyses in seronegative patients is another limitation of the study, however, CSF analyses is not a standard part of clinical evaluation in TLE patients, even in presurgical candidates with refractory TLE. The use of commercially available kits for the detection of NSAbs may raise some (although very low) possibility of false positive and false negative results. Antibody testing with methods other than the commercial kits currently available would be necessary to identify possible false negative patients.

      5. Conclusion

      The distinction between antibody positive and antibody negative patients is difficult based on clinical, EEG or neuroimaging characteristics. Testing should be considered especially in patients with unknown aetiology, higher age at epilepsy onset and autoimmune comorbidity. A favourable response to immunotherapy was observed in half of the treated seropositive TLE patients, especially when immunotherapy was used early in the disease course.

      Declarations of interest

      Christian Bien gave scientific advice to UCB (Monheim, Germany) and obtained honoraria for speaking engagements from Eisai (Frankfurt, Germany), UCB (Monheim, Germany), Desitin (Hamburg, Germany), Biogen (Ismaning, Germany) and Euroimmun (Lübeck, Germany). He received research support from the Deutsche Forschungsgemeinschaft (Bonn, Germany), Gerd-Altenhof-Stiftung (Deutsches Stiftungs-Zentrum, Essen, Germany). He is a consultant to the Laboratory Krone, Bad Salzuflen, Germany, regarding neural antibodies and therapeutic drug monitoring for antiepileptic drugs. Petr Marusic received honoraria from UCB, Eisai and Egis as a speaker and from UCB as advisory board member.

      Funding

      This work was supported by the Charles University project GA UK No 189215 , by the Alzheimer nadacni fond and nadacni fond AVAST .

      Conflict of interest

      None.

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