J Neurol (2010) 257:509–517 DOI 10.

1007/s00415-009-5431-9

REVIEW

Antibodies and neuronal autoimmune disorders of the CNS
Francesc Graus • Albert Saiz • Josep Dalmau

Received: 14 December 2009 / Accepted: 14 December 2009 / Published online: 25 December 2009 Ó Springer-Verlag 2009

Abstract We review the neuronal antibodies described in CNS disorders in order to clarify their diagnostic value, emphasize potentials pitfalls and limitations in the diagnosis of paraneoplastic neurological syndromes (PNS), and examine the current evidence for a possible pathogenic role. We propose to classify the neuronal antibodies associated with syndromes resulting from CNS neuronal dysfunction into two groups according to the location of the antigen: inside the neuron or in the cell membrane. Group I includes antibodies which target intracellular antigens and probably are not pathogenic. They are further subdivided into three groups. Group Ia comprises well-characterized onconeural antibodies (Hu (ANNA1), Yo (PCA1), Ri (ANNA2), CV2 (CRMP5), amphiphysin, Ma2) that are useful for the diagnosis of PNS. Group Ib antibodies (SOX and ZIC) are cancer-specific but there is no evidence that the immune response is in any way pathogenically related to the PNS. Antibodies in group Ic (glutamic acid decarboxylase (GAD), adenylate kinase 5 and Homer 3) identify non-PNS: stiff-person syndrome (SPS), cerebellar ataxia, and limbic encephalitis (LE). Group II antibodies recognize neuronal surface antigens. Antibodies in group IIa associate with characteristic CNS syndromes but their detection does not indicate that the disorder is paraneoplastic. Antibodies to potassium channels, AMPA and GABAB receptors are associated with LE, NMDA receptor antibodies
F. Graus (&) Á A. Saiz ´ Servei de Neurologia, Hospital Clinic, Institut d’ Investigacio ` dica August Pi i Sunyer (IDIBAPS), Villarroel 170, Biome 08036 Barcelona, Spain e-mail: fgraus@clinic.ub.es J. Dalmau Department of Neurology, University of Pennsylvania, Philadelphia, PA, USA

identify a well-defined encephalitis, and antibodies against glycine receptors associate with SPS with encephalitis. A pathogenic role of the antibodies is suggested by the response of symptoms to immunotherapy and the correlation between antibody titers and neurological outcome. Lastly, Group IIb includes antibodies that are found in patients with paraneoplastic cerebellar ataxia associated with lung cancer (P/Q type calcium channels antibodies) or Hodgkin disease (metabotropic glutamate receptor type 1 antibodies). Keywords Antibodies Á Autoimmunity Á Paraneoplastic neurological syndromes Á Encephalitis Á Cerebellar degeneration Á Epilepsy

Introduction The idea that neurons can be the target of an autoimmune attack mediated by antibodies still is not well-acknowledged in the medical community. Initial evidence that this may occur was reported more that 20 years ago when Hu and Yo antibodies were described in some paraneoplastic neurological syndromes (PNS) and glutamic acid decarboxylase (GAD) antibodies in stiff-person syndrome (SPS) [1–3]. However, failure to model these disorders in animals prevented clarification of the immunological mechanisms involved in the neuronal damage, and the fact that the antigens were intracellular questioned the pathogenic role of the antibodies. Nevertheless, these antibodies and others described in the next decades were found useful in the diagnosis of PNS [4]. In recent years, the description of antibodies against neuronal surface antigens in patients with paraneoplastic and idiopathic encephalitis has bolstered the concept that antibodies against CNS antigens can

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be pathogenic, indicating that the autoimmune hypothesis of neuronal damage should not be limited to the PNS [5]. On a practical level, the constant description of novel neuronal antibodies and the expanding spectrum of associated symptoms make it difficult for neurologists to decide which antibody or set of antibodies is important for the diagnosis of a given syndrome and which antibodies are markers of PNS or non-cancer related disorders. The aim of this study is to review the neuronal antibodies described in CNS disorders in order to clarify their diagnostic value, emphasize potential pitfalls and limitations in the diagnosis of PNS, and examine the current evidence for a possible pathogenic role. In addition, we propose a classification of neuronal antibodies that may help neurologists in the diagnosis of these disorders. The study is focused only on neuronal antibodies with wellcharacterized target antigens.

as paraneoplastic or non-paraneoplastic markers of CNS syndromes.

Group I: Antibodies against intracellular antigens Group Ia: PNS-related onconeuronal antibodies The term ‘‘onconeural’’ antibodies was introduced by tumor immunologists to designate antibodies that target antigens present in neuroectodermal tissues and tumors [6]. Concerning PNS, well-characterized onconeural antibodies were defined as those that, in addition to reacting with neural antigens, also present in tumors, served to establish the associated neurological disorder as definite PNS (Table 1). These antibodies are unambiguously demonstrated by standardized tests, associate with limited subsets of tumors, and are present in several CNS syndromes. Some of these syndromes, such as limbic encephalitis, had been known to occur as PNS long before the discovery of onconeural antibodies. However, the discovery of these antibodies facilitates the prompt diagnosis of these disorders and has been crucial for the identification of new PNS. Two examples are chorea associated with CV2 antibodies and the mesodiencephalic encephalitis of patients with Ma2 antibodies [12, 13]. An important issue is that a positive report for any wellcharacterized onconeural antibody has to be assessed

Antineuronal antibodies and CNS autoimmunity We propose to classify the neuronal antibodies associated with syndromes resulting from CNS neuronal dysfunction into two groups according to the location of the antigen: inside the neuron or in the cell membrane. This classification provides an indication of the potential pathogenic role of the antibodies. Additionally, each group of antibodies is further classified according to its diagnostic value

Table 1 Well-characterized onconeuronal antibodies and CNS paraneoplastic neurological syndromes (PNS) Antibody Hu (ANNA1) [7] Predominant tumors Small cell lung cancer (SCLC) CNS syndromes Encephalomyelitis Paraneoplastic cerebellar degeneration (PCD) Limbic encephalitis Brainstem encephalitis CV2 (CRMP5) [8] SCLC, thymoma Encephalomyelitis Chorea PCD Limbic encephalitis Amphiphysin [9] Breast, SCLC Stiff-person syndrome Myelopathy and myoclonus Ri (ANNA2) [10] Yo (PCA1) [11] Ma2 [12]
a

Antibody positive patients without cancera (%) 2

Frequency in cancer without PNSa (%) 16

4

9

5

1

Breast, SCLC Ovary, breast Testicular

Encephalomyelitis Brainstem encephalitis Opsoclonus myoclonus PCD Limbic encephalitis Brainstem encephalitis

3 2 4

4 1 0

For review see reference [4]

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according to the clinical setting. All these antibodies, particularly those associated with SCLC (Hu, CV2, amphiphysin), can be found in patients with cancer without PNS [14]. Therefore, one should still rule out other potential causes of the neurological syndrome that is being evaluated. This concern increases when the presence of a cancer is already known or when the antibody titers are low. Up to 16% of patients with SCLC without PNS have low titers of Hu antibodies, whereas in patients with PNS and Hu antibodies the titers are substantially higher [15]. A typical example is a patient with SCLC treated with cisplatinum who develops a delayed sensory neuropathy [16]. In this case, the detection of low titer Hu or other onconeural antibody probably reflects the presence of the cancer and it cannot be used to support the paraneoplastic origin of the neuropathy, unless other clinical clues exclude a toxic origin. Well-characterized onconeural antibodies are markers of specific subsets of tumors, usually SCLC, breast, ovarian and testicular cancer (Table 1). If another tumor is diagnosed, one cannot establish that the tumor is the cause of the PNS unless the antigen recognized by the onconeural antibody is found expressed in the tumor. If this is not the case, the search for a second neoplasm, directed by the type of onconeural antibody, is warranted. In some PNS, the clinical significance of the antibodies is difficult to establish. One can argue that the antibodies are unrelated to the PNS and simply indicate a tumorinduced immune response [17]. In this scenario, the PNS could be caused by additional, pathogenically more relevant antibodies against cell surface antigens (see below) [18] or other immunological mechanisms. However, there are at least two important findings indicating that wellcharacterized onconeural antibodies are not just biological markers of cancer. First, the frequency of these antibodies is much higher in patients with some PNS than in those with isolated cancer (Table 1). For example, the frequency of Yo antibodies is much higher in patients with PCD and ovarian cancer that in patients with ovarian cancer alone or with other PNS associated with ovarian cancer, such as dermatomyositis. Second, and most important, specific intrathecal synthesis has been demonstrated for all types of well-characterized onconeural antibodies associated with PNS of the CNS [19]. This should not occur unless a specific immunologic activation against neuronal antigens operates in the CNS. Despite these data, the direct role of well-characterized onconeural antibodies in the pathogenesis of PNS is unclear. The intracellular location of the antigen and multiple failed attempts to produce an animal model by passive transfer experiments or active vaccination with the antigen strongly suggest these antibodies are not pathogenic [20, 21]. A possible exception is amphiphysin

antibodies. In one study, rats injected with IgG from a patient with SPS and amphiphysin antibodies developed transient spasms and rigidity. Although one cannot rule that other IgG antibodies caused the symptoms, amphiphysin is a protein involved in the recycling of synaptic vesicles, and perhaps it is transiently exposed on the cell membrane to the binding of antibodies [22]. Well-characterized onconeural antibodies probably represent the humoral component of a complex immune response, likely mediated by cytotoxic T cell mechanisms against the same onconeural antigens. This is supported by several lines of evidence: First, postmortem studies demonstrate prominent perivascular and parenchymatous inflammatory infiltrates [23]. Second, immunophenotyping of the inflammatory cells show cytotoxic CD8? T cells in close contact with neurons [24]. Third, Yo- and Hu-specific CD8? cytotoxic T cells are present in the blood of PNS patients, and they can kill target cells in an HLA-restricted manner [25, 26]. Taken together, these data suggest that PNS associated with well-characterized onconeural antibodies resulting from a T cell immune attack against neuronal antigens, probably the same that are targeted by antibodies. This hypothesis awaits confirmation in an animal model. Group Ib: Cancer-related onconeural antibodies Antibodies of this group share with well-characterized onconeural antibodies the recognition of tumor antigens also expressed in the CNS. However, there is no evidence that the immune response (antibodies or associated T cell response) is in any way pathogenically related with the PNS. At present only two antibodies would fit in this group, and both recognize antigens (ZIC and SOX families) expressed in SCLC [27, 28]. We do not have evidence of intrathecal synthesis of SOX or ZIC antibodies in patients with PNS. Moreover, the frequency of these antibodies is not higher in patients with PNS than in those with SCLC alone, with the exception of SOX1 antibodies in patients with paraneoplastic Lambert-Eaton myasthenic syndrome (LEMS) (see below), a syndrome caused by antibodies against VGCC [28]. The ZIC genes encode proteins that contain five zincfinger domains. Antibodies against ZIC2 were initially detected by serological analysis of cDNA expression libraries derived from SCLC lines [29]. Antibodies to ZIC4 were subsequently identified in a patient with subacute, severe cerebellar ataxia without cancer [30]. Antibodies against ZIC proteins were found in 15% of patients with PCD and SCLC, an incidence similar (16%) to that of patients with isolated SCLC, and in 29% of patients with different PNS and SCLC [27]. Unlike patients with PCD, many of those with other PNS and ZIC antibodies also had

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Hu or CV2 antibodies [27]. Positive sera of PCD patients reacted with ZIC2, and some also with ZIC1 and ZIC4, suggesting that the immunoreactivity of ZIC antibodies is directed primarily against the conserved zinc-finger domains of ZIC proteins [31]. Although intrathecal synthesis of ZIC4 antibodies was detected in the first reported case with subacute cerebellar ataxia without cancer, similar studies have not been carried out in patients with PCD and SCLC. Like ZIC antibodies, SOX antibodies were initially detected by serological analysis of cDNA expression libraries derived from SCLC lines [29]. In patients with PNS, SOX antibodies had been first immunohistochemically described with the term ‘‘anti-glial nuclear antibody (AGNA)’’, defined by the immunoreaction with the nuclei of the Bergmann glia of the cerebellum [32]. We found a robust association between AGNA and the presence of an underlying SCLC. However, the frequency of AGNA found was higher than expected, for the simple presence of SCLC, in paraneoplastic LEMS (43 vs. 11%) [32]. Because AGNA reactivity is widely expressed in the developing nervous system, we screened a fetal brain library with AGNA positive sera and identified the AGNA antigen as SOX1. SOX1 belongs to a large family of developmental transcription factors that share a highly conserved DNAbinding domain known as the HMG-box, defined by 79 aminoacids [33]. The human SOX gene family is classified in groups from A to H. SOX1 belongs to group B1 along with SOX2 and SOX3 [34]. Patients with SOX1 antibodies may also react with SOX2 and SOX3 proteins, suggesting that the immunoreactivity is directed against epitopes of the conserved HGM box. However, some sera react only against SOX1, indicating that assays that use SOX1 as antigen are the most sensitive to detected antibodies against SOX proteins of the group B1 [35]. Using this strategy, SOX1 antibodies were detected in 64% of patients with LEMS and SCLC but in none of the 50 with idiopathic LEMS [28]. These data indicate that the detection of SOX1 antibodies in patients with LEMS predicts the presence of SCLC and may be used to follow more closely those LEMS patients with no evidence of cancer at the initial work-up. Patients with SCLC and PNS do not have intrathecal synthesis of SOX1 antibodies, indicating that SOX autoimmunity is not involved in the pathogenesis of the PNS. SOX antibodies are found in 36% of patients with isolated SCLC and no difference in survival was observed between SOX positive and SOX negative SCLC patients [36]. Group Ic: Non-paraneoplastic antibodies associated with CNS syndromes Antibodies to GAD, adenylate kinase 5 (AK5), and Homer 3 are representative of this group. GAD antibodies were initially identified in the serum and CSF of patients with

SPS [3, 37]. This syndrome often occurs in association with other autoimmune diseases, mainly type 1 diabetes mellitus (DM1) [38]. This is important, because GAD antibodies are present in about 80% of newly diagnosed DM1 patients, although the levels are usually 100-fold higher in SPS than in DM1 [37]. GAD antibodies were also reported in a subgroup of patients with late onset isolated cerebellar ataxia [39], and a few patients with epilepsy, limbic encephalitis, downbeat nystagmus, palatal tremor, and brainstem dysfunction [40]. In a study focused in 50 patients with high levels of GAD antibodies ([2,000 U/ml by radioimmunoassay), 22 had SPS, 17 cerebellar ataxia, and 11 other neurological disorders (epilepsy 4, PNS 4, limbic encephalitis 2, myasthenia gravis 1) [40]. The frequency of increased intrathecal synthesis of GAD antibodies was 85% in SPS, 100% in cerebellar ataxia, and 86% in other neurological disorders. However, similar serum levels of GAD antibodies were found in 11 patients with DM1 without neurological manifestations. Although the presence of high GAD antibody levels probably suggests that the underlying neurological syndrome may be immunomediated, the observed high frequency of specific intrathecal synthesis support that this analysis should be done to confirm that the GAD autoimmunity is related to the neurological syndrome. This evaluation is particularly important when there is a concomitant DM1 or other autoimmune endocrine syndrome where GAD antibodies could just reflect the presence of the associated endocrine disorder. GAD antibodies have been described in a few patients with paraneoplastic SPS associated with thymoma and other solid tumors [41], but their occurrence in patients with classical PNS is less known. Two patients with paraneoplastic encephalomyelitis and PCD had intrathecal synthesis of GAD antibodies, and the tumors expressed GAD. This finding suggests that the immunity raised against the neoexpression of GAD by the tumor may cause a PNS as occurs with classical onconeuronal antibodies [40]. Overall, these data indicate that in patients without DM1 who develop neurological syndromes suggesting a PNS, detection of GAD antibodies does not exclude a paraneoplastic etiology, and therefore cancer screening is warranted. In patients with limbic encephalitis, the significance of GAD antibodies is unclear for two reasons: [42] the small number of cases reported despite the fact that GAD autoimmunity is relatively common, and the frequent presence of antibodies against cell surface antigens [43], one of them, the GABA(B) receptor, recently reported (see below) [44]. Antibodies to AK5 were identified in two patients with idiopathic LE refractory to immunotherapy [45]. AK5 is specifically expressed in the brain, located in the neuronal cytosolic fraction, and has critical neuronal-specific metabolic functions. The absence of theses antibodies in patients with other PNS, or breast cancer or SCLC without

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PNS, suggests that AK5 antibodies are associated with an immune response not related to cancer. As occurs with other immune responses to intracellular antigens, the LE of patients with AK5 antibodies is poorly responsive to treatment. Antibodies to Homer 3 were identified in a patient with cerebellar ataxia of subacute onset and CSF pleocytosis [46]. No cancer has been found after more than 5 years of followup. Homer 3 is predominantly expressed in the dendritic spines of the Purkinje cells. Homer 3 specifically binds to the C-termini of mGluR1 and to inositol 1,4,5 triphosphate receptors. This complex is physiologically important for postsynaptic calcium responses to mGluR1 stimulation. The frequency of Homer 3 antibodies in patients with idiopathic subacute cerebellar ataxia is unknown, but they seem to be associated with this clinical profile because they were not detected in other autoimmune cerebellar ataxias, including PCD and lung cancer or Hodgkin disease, and chronic sporadic cerebellar ataxia with or without GAD antibodies [46].

Group II: Antibodies against neuronal surface antigens Group IIa: Markers of the CNS syndrome The antibodies of this group share several characteristics (Table 2). They associate with characteristic CNS
Table 2 Antibodies against neuronal surface antigens Antibody VGKC Tumors (%) Small cell lung cancer (SCLC), thymoma (31) Neurological syndrome Limbic encephalitis Morvan’s syndrome Creutzfeldt-Jakob disease like syndrome[54] Encephalitis with initial psychiatric features followed by catatonia, dystonia, aphasia and hypoventilation Limbic encephalitis Atypical psychosis Limbic encephalitis

syndromes but their detection does not indicate that the disorder is paraneoplastic. Indeed, the frequency of an underlying tumor varies among the antibody type and is never higher that 70%. The antibodies recognize antigenic epitopes located in the external surface of the neuronal membrane, usually at pre- or post-synaptic sites. Moreover, a pathogenic role of the antibodies is suggested by the response of symptoms to immunotherapy and the correlation between antibody titers and neurological outcome. Antibodies to voltage-gated potassium channels (VGKC) have been associated with non-paraneoplastic limbic encephalitis [47] and case reports of Morvan syndrome [48] or isolated epilepsy [49]. The VGKC antibodyassociated LE is more prevalent in men and usually presents as a classical picture of LE. Rapid eye movement sleep behavior disorder develops at the onset of LE [50]. Hyponatremia is a frequent finding, whereas CSF analysis shows mild pleocytosis in only 41% of the patients (median white cell count 6, range 5–16 cells) [51]. Serum VGKC antibody levels are high without intrathecal synthesis of the antibody [51]. Probably, the clinical spectrum of CNS disorders associated with VGKC antibodies is wider than previously expected. A recent study showed that 10% of patients had neurological symptoms different from those of LE or diffuse encephalitis and 31% had an underlying tumor, usually lung cancer or thymoma [52].

CSF pleocytosis (%)/IT synthesis 41/no

Method of detection RIAa

Comments Male predominance; associated rapid eye movement (REM) sleep behavior disorder; frequent hiponatremia ([70%) Female predominance; MRI normal in 45%, frequency of tumor higher in patients [18 years old. Female predominance; frequent relapses (60%) Seizures in 86%. Concurrent GAD antibodies in 3 patients Only one patient published. Series published in abstract form [65]

NMDA receptor

Ovarian teratoma (9–56)

91/yes

Cell based assay

AMPA receptor GABAB receptor

SCLC, breast, thymoma (70) SCLC (47)

90/yes 80/yes

Cell based assay Cell based assay

Glycine receptor

Lung cancer (3)

Progressive encephalomyelitis with rigidity, stiff person syndrome

Unknown

Cell based assay

a

Cut-off value: 100 pM

However, values between 100 and 400 pM may be seen in 5% of patients without autoimmune neurological disorder [47] VGKC voltage-gated potassium channel

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Unlike other antibodies to cell surface antigens described below, the target antigen of VGKC antibodies has never been directly precipitated by patients’ antibodies. Moreover, antibodies do not react with cells transfected with the putative antigen (e.g., Kv1 subunit of the VGKC) suggesting that the radioimmunoassay used for antibody detection could in fact be showing antibodies against antigens that coprecipitate with the VGKC, but not against the VGKC themselves. For example, the antibodies of patients with Morvan’s syndrome and thymoma are directed against contactin-associated protein 2 (CASPR2) a VGKC–associated protein [53]. Whether patients with LE and VGKC antibodies have antibodies to similar proteins is presently unknown. Until recently, only 50% of patients with LE and SCLC were considered antibody positive, usually harbouring Hu or, less frequently, other onconeural antibodies [55]. Recent studies indicate that most cases previously considered ‘‘seronegative’’ have, in fact, antibodies against neuronal surface antigens [56]. Two of these antibodies have been identified: the GluR1/2 subunits of the AMPA receptor, and the B1 subunit of the GABAB receptor [44, 57]. These two antibodies have in common the association with idiopathic or paraneoplastic LE. Seven of 15 (47%) patients with LE associated with GABAB receptor antibodies had an underlying tumor, usually an SCLC [44]. In LE associated with AMPAR antibodies, the frequency of cancer was 64%, with SCLC being the most common type, followed by thymomas and breast cancer [57]. Some clinical and immunological clues may suggest the type of antibody associated with LE. AMPA receptor antibodies have been almost exclusively identified in women and the LE has a tendency to relapse [57]. Patients with LE and GABABR antibodies present with early and prominent seizures and frequently have other concurrent autoantibodies, particularly against GAD [44]. This overlap is intriguing, as it suggests that some patients with LE attributed to GAD antibodies may have GABAB receptor antibodies as a more plausible cause of the disorder. A severe but treatment-responsive encephalitis has been associated with antibodies to NR1, a crucial subunit of the NMDA receptors [58]. Most patients are children or young women who do not develop the classical picture of LE [59, 60]. They are initially seen or admitted to psychiatric wards for acute anxiety, behavioral change, or psychosis followed in a few days by seizures, decline of consciousness, aphasia, and abnormal movements. Patients may develop hypoventilation and autonomic imbalance that requires admission to intensive care units [59]. The frequency of ovarian teratomas is higher (56%) in women older than 18 years than girls under the age of 14 years (9%). High titers of antibodies significantly associated with the

presence of a tumor. Despite the severity of the syndrome, often requiring prolonged intensive care support, 75% of patients fully recover or have mild residual deficits. Antibody titers (mainly those in the CSF) vary according the clinical outcome [59]. Patients whose tumors are treated promptly (usually along with immunotherapy) have faster and more complete neurological recovery, without relapses, than those who are not treated or only receive immunotherapy either because no tumor is found or it is initially missed [59]. Application of patients’ NMDA or AMPA receptor antibodies into cultures of rat hippocampal neurons significantly decreases the number of specific receptor clusters in synapses, an effect that is reversed when patients’ antibodies are removed or replaced by control human IgG. Moreover, recent studies showed a correlation between antibody titers and the decrease of clusters of NMDA receptors in cultured neurons, as well as in the hippocampus of rats infused with patients’ antibodies [61]. Overall, these data and the detection of deposits of IgG in the brain of autopsied patients with NMDA receptor encephalitis strongly suggest that these antibodies are probably responsible for the neurological syndrome [62]. Antibodies against the glycine receptor have been reported in a patient with progressive encephalomyelitis with rigidity [63]. This syndrome is considered a variant of stiff-person syndrome characterized by subacute onset of limb and trunk rigidity, muscle spasms, brainstem dysfunction, hyperekplexia, and encephalopathy [64]. This is a severe encephalopathy that may be reversed with immunotherapy. Preliminary work suggests that glycine receptor antibodies could be associated with this rare disorder; the antibodies have also been identified in some patients with isolated hyperekplexia, and a few cases with SPS [65]. Group IIb: Markers of a CNS paraneoplastic syndrome There are two antibodies that can be placed in this group because their presence in a patient with cerebellar ataxia suggest a paraneoplastic origin. Antibodies against the metabotropic glutamate receptor type 1 (mGLuR1) have been described in only two patients with cerebellar ataxia 2 and 9 years after the diagnosis of Hodgkin disease [66]. Intrathecal injection of the IgG of one of the patients into rats caused a transient cerebellar ataxia, suggesting a pathogenic role of the antibody [66, 67]. The long delay between the tumor diagnosis and the cerebellar ataxia suggests mGluR1 antibodies are linked to the neurological syndrome rather than to the tumor. This impression must be confirmed with the description of more patients with mGluR1 antibodies, cerebellar ataxia, and no cancer. If more cases are described, this antibody will likely be reclassified in group Ib.

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Antibodies to P/Q type voltage-gated calcium channels (VGCC) are associated with Lambert-Eaton myasthenic syndrome (LEMS) and are responsible for the syndrome [68]. The clinical observation that some LEMS patients also have cerebellar ataxia and the demonstration of P/Q type VGCC in Purkinje cells raised the question of whether these antibodies could be involved in cases of isolated cerebellar ataxia. Increased levels of P/Q type VGCC antibodies were found in 16 (41%) of 39 patients with paraneoplastic cerebellar degeneration (PCD) and lung cancer. Only seven of the 16 patients had an associated LEMS [69]. In contrast, patients with non-paraneoplastic cerebellar ataxia usually do not harbor VGCC antibodies or do so at low levels [70]. Therefore, the detection of P/Q type VGCC antibodies in a patient with cerebellar ataxia, associated or not with LEMS, strongly indicates the presence of an underlying lung cancer. The pathogenic role of VGCC antibodies in PCD is unclear despite the observation of loss of P/Q type VGCC in postmortem studies of cerebellums of patients with PCD and LEMS [71]. Unlike other syndromes associated with neuronal surface receptor antibodies, the cerebellar ataxia does not improve with immunotherapy either because the Purkinje cells are irreversibly damaged at early stage of the syndrome, or because the treatments used are unable to remove the pathogenic antibodies.

Experience with the recently described antibodies, with exception of those against NMDAR, is still relatively small. Therefore, their inclusion in one particular group of the proposed classification must we viewed with caution until more cases are described. Good clinical-immunological correlations are crucial to define the clinical syndrome that most likely associates with a particular antibody. Researchers in this field must provide good clinical descriptions of the case series associated with the antibody they study. This approach will help clinicians to identify the clinical syndromes and to make a rational decision on which antibodies to request.
Acknowledgments This study was supported in part by grant PS09/ 00193 Fondo de Investigaciones Sanitarias, Madrid, Spain. Conflict of interest statement None.

References
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Final comments In a few years the number of antineuronal antibodies described in CNS disorders has increased dramatically. There are two important features that differentiate most of the novel antibodies from previous antineuronal antibodies: first, they are directed against cell surface or synaptic proteins, and second, the target antigens are relevant receptors involved in wellknown mechanisms of synaptic transmission or plasticity. Screening by immunohistochemistry on rat brain sections may detect most of these antibodies, but these studies do not confer specificity. This is based on direct immunoprecipitation of the antigen and, once it is characterized, the development of cell based assays specifically expressing the antigen. Indirect immunoprecipitation techniques, such as those used for the VGKC antibodies, although may be useful as a diagnostic test do not necessarily provide the main target antigen. Future studies must address the sensitivity and specificity of immunohistochemistry to screen these novel antibodies in a way similar to what has been done with anti-aquaporin-4 (NMO-IgG) antibodies [72] and to confirm by the specific cell based assays only the positive samples. This approach, if feasible, would be more practical and cheaper than to initially analyze all the samples by the different cell based assays.

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