Diseases affecting the CNS

CNS autoimmune disorders without defined antibodies

Multiple sclerosis.

Type of antibodies seen. An increased frequency of non-organ-specific autoreactive antibodies, including antinuclear, antithyroid, and antiphospholipid antibodies, occurs in multiple sclerosis patients (Table 1) (162; 38; 48; 102). Increased titers of antithyroglobulin and antiperoxidase antibodies are detected in a significant percentage of multiple sclerosis patients, too (38; 102). The presence of these antibodies is probably an epiphenomenon of a disseminated immunological dysfunction without any usefulness for multiple sclerosis diagnosis.

Increased titers of myelin-specific antibodies in serum and spinal fluid are frequently observed in multiple sclerosis patients (Table 1). Many multiple sclerosis patients show a persistent autoantibody response to myelin basic protein or myelin oligodendrocyte glycoprotein (28; 182; 145). An antibody response to myelin basic protein or myelin oligodendrocyte glycoprotein also occurs in patients with other neurologic inflammatory or even noninflammatory disorders, and in healthy family members, at a frequency not significantly different than in multiple sclerosis (131). Testing antimyelin basic protein or antimyelin oligodendrocyte glycoprotein is not useful for diagnosing multiple sclerosis, a diagnosis that is still based on clinical and MRI findings (126).

Table 1. Antibodies Associated with Multiple Sclerosis

Antibody	Serum	CSF	Reference
Myelin basic protein	+	+	(28; 145)
Myelin oligodendrocyte glycoprotein	-	+	(182)
Myelin-associated glycoprotein	+/-	+	(11)
Antinuclear antibody	+	+	(162)
Antiphospholipid antibody	+	+	(49)
Antithyroid antibody	+	-	(38; 102; 26)

Pathogenic significance. Multiple sclerosis is an immune-mediated disorder presumably caused by T-cell-mediated inflammation, leading to demyelination and damage to oligodendrocytes. A general immune dysregulation seems to exist in multiple sclerosis patients, and, therefore, these patients may have exaggerated responses to different antigens and autoantigens (112). This dysregulation probably accounts for non-organ-specific or organ-specific, but not myelin-specific, antibodies. However, humoral mechanisms and, more specifically, antibody-mediated demyelination may contribute to disease progression in at least a subgroup of multiple sclerosis patients (39; 50; 08; 25).

Antibodies to interferons in interferon beta–treated multiple sclerosis. Interferon-beta treatment can elicit anti–interferon beta antibodies, more so in multiple sclerosis patients than in other indications (90). Anti-interferon antibodies can be measured by binding assays such as ELISA and, rarely, by radioimmunoprecipitation or column chromatography assays. The antibodies measured by these assays are termed “binding antibodies.” They can also be measured by evaluating how much of the in vitro interferon bioactivity is blocked by the serum to be tested (presumably due to anti-interferon antibodies). These are termed “neutralizing antibodies.” Methods to measure neutralizing antibodies include the virus-induced cytopathic effect assay, which remains the gold standard, and assays of induction of myxovirus A protein or RNA (41; 127; 61). A new assay uses luciferase (40; 84). This assay is faster, simpler, and less expensive than cytopathic effect assay and myxovirus A protein induction test. Unfortunately, different companies and different laboratories provide results that are difficult to compare because of differences in units and in principles used in measurement.

The Neutralizing Antibodies in Multiple Sclerosis (NABIMS) working group is attempting to clarify these issues. Simplification of techniques, agreement on which antigen to use in assays, and unitage should be the basis for agreement in the field. It has now been agreed that results should be reported as "10-fold reduction units" (TRU) as per Kawade and Grossberg (60).

When high levels of neutralizing antibodies are present, bioavailability of the drug is compromised, ie, the drug ceases to act by binding to its receptor. Bioavailability of interferons can be measured directly using the in vivo induction of myxovirus A protein mRNA. In this test, myxovirus A protein mRNA is measured in lymphocytes isolated from peripheral blood before and 12 hours after a test injection of interferon (115). The term “high antibody levels” should be reserved for the levels in which bioavailability is abrogated. As a rule of thumb, this level of inhibition is attained between 100 and 400 TRUs. The loss of clinical effect of the injected interferon due to neutralizing antibodies may vary from product to product; lower neutralizing antibody titers block interferon beta 1a products more easily than interferon beta 1b products (16). Practical application of these tests will be slow to appear and will vary between countries, as different conclusions on their use and indications have been reached by an expert committee of the European Neurological Association and the TTA committee of the American Association of Neurology (53; 64). The NABIMS committee has been attempting to bridge this gap, but with limited success (125).

Neuropsychiatric systemic lupus erythematosus. Neuropsychiatric disturbances occur in more than 50% of patients with systemic lupus erythematosus (165). Systemic lupus erythematosus is a B-cell mediated disorder accompanied by a reduction of T-cell function. A variety of autoantibodies directed against neural antigens appear in patients with systemic lupus erythematosus. These include antineuronal, antineurofilament, antiribosomal P, and antiganglioside antibodies (59; 77). Serum antiribosomal P antibodies and anti-N-methyl-D-aspartate receptor (anti-NMDAR)-NR2 subunit antibodies are more frequently seen in patients with neuropsychiatric systemic lupus erythematosus than in regular systemic lupus erythematosus patients and increase during the active phase of the disease (179; 77). They are not specific for this disease, however, and not useful for its diagnosis.

Rasmussen encephalitis. Rasmussen encephalitis is a rare but severe disorder leading to unilateral hemispheric atrophy associated with hemiparesis, cognitive decline, and intractable childhood epilepsy. It is believed to be an autoimmune disease mediated by antibodies and cytotoxic T cells. Antibodies against the subunit 3 of the ionotropic glutamate receptor are found in 6% to 10% of patients. Antibodies against the presynaptic protein Munc18-1, the NMDAR-e2 subunit, and the alpha7 nicotinic AChR, the neuronal AChR alpha-7 subunit, and NMDA receptors 2A to 2D, specifically glutamate receptor epsilon2, have been reported (177; 07). None of the autoantibodies, however, appear in more than a small number of patients with Rasmussen encephalitis, and responses to plasma exchange are unpredictable. Therefore, the role of CNS autoantibodies in the pathogenesis of Rasmussen encephalitis is still unclear. Cytotoxic T lymphocytes may also play a major part in the pathogenesis, and the inflammatory or destructive pathology often coexists with malformative or dysplastic features in cortical architecture, raising questions about the possible relationships between the two pathologies (154; 166).

Antibody-mediated nervous system demyelinating disorders

Neuromyelitis optica spectrum disorders (Devic disease). Neuromyelitis optica, originally described as Devic disease and seen then as an autonomous entity, was initially considered a “form of multiple sclerosis.” The discovery of the presence of a specific antibody against aquaporin-4 in a proportion of patients with optic neuritis and transverse myelitis has permitted refocusing the pathogenesis towards an antibody-mediated process, probably directed against aquaporin-4 on astrocytes. Antibodies to aquaporin-4 can be measured with over 40 different immunoassays, including cell-based assay (cells transfected with the antigen of interest), tissue-based assay (immunohistochemical), and protein-based (ELISA, western blotting) techniques (72). According to a meta-analysis of 30 studies, the approximated sensitivity for cell-based assay was 0.76 (95% CI 0.67-0.82), for tissue-based assay was 0.59 (95% CI 0.50-0.67), and for ELISA was 0.65 (95% CI 0.53-0.75) (138). The mean specificity of cell-based assay was 0.99 (95% CI 0.97-0.99), of tissue-based assay was 0.98 (95% CI 0.97-0.99), and of ELISA was 0.97 (95% CI 0.96-0.99). Aquaporin-4 detection in serum with immunoassays is a great tool for diagnosing patients with neuromyelitis optica due to the high specificity, allowing the clinician to differentiate this disease from other neurologic conditions that resemble neuromyelitis optica. The immunoassay that shows the best diagnostic performance is cell-based assay, but the other immunoassays are specific enough to be clinically useful for a correct diagnosis in patients with neuromyelitis optica, as well as to clarify the cases in which the rest of the clinical, paraclinical, and imaging studies are not enough to diagnose definite neuromyelitis optica. Neuromyelitis optica and CNS Sjogren disease overlap, with a similar clinical and MRI profile (76). CNS Sjogren predominantly affects young, non-white women. Neuromyelitis optica-IgG is less likely to be positive, and salivary gland inflammation is very frequent.

Anti-aquaporin-4 antibodies are involved in neuromyelitis optica pathogenesis because they damage the astrocytes, and the disease can be transferred to animals with anti-aquaporin-4 antibodies (139).

Myelin oligodendrocyte glycoprotein-related disorder (MOGAD). MOGAD, just like NMOSD, was originally described as multiple sclerosis, but since the identification of this specific antibody, the disease is thought to be antibody-mediated. This disease has different clinical characteristics, treatment response, and prognosis compared to multiple sclerosis and NMOSD. Common presentations of MOGAD include recurrent optic neuritis, transverse myelitis, and acute disseminated encephalomyelitis. In the pediatric population, MOG IgG is often associated with acute disseminated encephalomyelitis presentation preceded by infection; this population is also more likely to have a transient titer elevation suggestive of a monophasic process rather than a recurrent one (178). The prognosis of MOGAD is generally more favorable than that of NMOSD and is often steroid-responsive. Live cell-based assays are more sensitive than fixed cell assays (176).

Glial fibrillary acidic protein astrocytopathy. Glial fibrillary acidic protein autoimmunity typically presents with acute or subacute meningoencephalomyelitis with prodromal flu-like symptoms, for which tumors and T-cell dysfunction are frequent triggers. Most patients follow a monophasic course with good outcomes and are generally steroid-responsive (58).

Stiff-person syndrome.

Clinical manifestations. Stiff-person syndrome is a rare disease associated with muscle stiffness, rigidity, and painful spasms involving axial and limb muscles (30). Its “plus” variant is progressive encephalomyelitis with rigidity and myoclonus, where rigidity is associated with severe myoclonus (34). Stiff-person syndrome can occur in association with autoimmune disorders such as insulin-dependent diabetes mellitus (30% of the patients), hyperthyroidism, pernicious anemia, and vitiligo. In some patients, stiff-person syndrome develops as a paraneoplastic neurologic disorder associated with breast or small-cell lung cancer (67).

Type of antibodies. Autoantibodies against glutamic acid decarboxylase (GAD) are detected in up to 85% of stiff-person syndrome patients, and a proportion of such antibodies are synthesized intrathecally; autoantibodies against GABA receptor-associated protein (GABARAP) occur in 65% of stiff-person syndrome patients and autoantibodies against amphiphysin in 5%. Autoantibodies against gephyrin have been described in a single patient (29). Autoantibodies against amphiphysin or gephyrin occur in association with the paraneoplastic variant and can occasionally also be seen in patients with small cell lung cancer with or without associated different paraneoplastic syndrome (142). GAD, an enzyme involved in the synthesis of the inhibitory neurotransmitter GABA, exists as two isoforms, GAD65 and GAD67, according to their molecular weights. In stiff-person syndrome, levels of anti-GAD in serum are usually highly raised and reactive with GAD65 and almost equally so with GAD67. They can be detected by immunoblotting using purified GAD from rat brain. In type 1 diabetes, on the other hand, anti-GAD are usually at low to moderate levels and mostly specific for GAD65 and react predominantly with highly conformational epitopes; therefore, they are rarely detected by immunoblotting (05). In non-paraneoplastic stiff-person syndrome/progressive encephalomyelitis with rigidity and myoclonus, the prevalence of anti-GAD65 antibodies is 82%; that of anti-GAD67 antibodies is 55%. The sensitivity for anti-GAD65 in stiff-person syndrome/progressive encephalomyelitis with rigidity and myoclonus diagnosis is 82%; its specificity is 99% (22 stiff-person syndrome/progressive encephalomyelitis with rigidity and myoclonus patients; 100 healthy controls) (99).

Anti-GABARAP antibody titers are significantly elevated in up to 70% of stiff-person syndrome patients, with more than twice the mean band intensity of the controls at immunoblot. The sensitivity is 62%; the specificity is 90% (27 stiff-person syndrome patients; 25 other neurologic disease patients) (128). Anti-GAD65 antibodies have also been reported in some 17 patients with subacute or chronic cerebellar ataxia, in almost all without an associated tumor (140).

Stiff-person syndrome is more seldomly associated with anti-amphiphysin antibodies—about 10% of anti-GAD-associated stiff-person syndrome (103). It almost always occurs in females without type 1 diabetes and with cancer (usually breast cancer, more rarely small cell lung cancer). Anti-amphiphysin antibodies are frequently coexpressed with other paraneoplastic antibodies (or, more rarely, anti-GAD65 antibodies) and have been associated with paraneoplastic neurologic disorders, especially sensory neuronopathy, encephalopathy, and myelopathy. They are, therefore, not useful for stiff-person syndrome diagnosis, but their detection mandates a search for cancer. Their sensitivity for the association with cancer is 80% (63 cancer patients) (121). Their specificity is also extremely high, almost 100%, as their association with a non-paraneoplastic neurologic disease has only been reported in a single paper describing three cases of childhood refractory epilepsy so far (95).

Pathogenic significance. An autoimmune pathogenesis of stiff-person syndrome is supported by the presence of autoantibodies, the association with other autoimmune diseases, and the clinical response to immunotherapy. All autoantigens known to date are synaptic proteins involved in inhibitory synaptic transmission (67). Glutamic acid decarboxylase is the rate-limiting enzyme for vesicular GABA synthesis and is also involved in vesicular GABA transport. GABARAP and gephyrin are postsynaptic proteins involved in assembling GABA A receptors and/or glycine receptors, respectively. Amphiphysin is an important binding partner for dynamin and a key protein for vesicular endocytosis in presynaptic nerve endings. The injection of stiff-person syndrome patients' IgG fraction in rats, including anti-amphiphysin antibodies, resulted in a dose-dependent stiffness, with spasms resembling human stiff-person syndrome (151).

Diseases affecting muscles and the peripheral nervous system

Myasthenia gravis

Clinical manifestations. Myasthenia gravis is characterized by fluctuating muscle weakness and weakness on exercise initially involving ocular musculature and generally followed by limb and respiratory muscle involvement. About 10% of Caucasians with myasthenia gravis (versus 30% of Asians) have a tumor of the thymus gland. The rest have follicular hyperplasia (mostly in young people) or a normal atrophic thymus (elderly).

Type of antibodies. Myasthenia gravis is associated with antibodies that react with the nicotinic muscle AChR. Anti-AChR antibodies are found in 85% of patients with generalized myasthenia gravis and in up to 65% of patients with ocular myasthenia gravis (111). Practically all myasthenia gravis patients with thymoma have anti-AChR antibodies. The sensitivity of the radioimmunoassay test is very high in generalized myasthenia gravis (above 85%), and it is lower in ocular myasthenia gravis (71% in 223 patients) (117). In myasthenia gravis and thymoma, it is practically 100%. Specificity of the test approaches 100% in all types of myasthenia gravis, regardless of the topography of the deficit, so the finding of a positive test will be sufficient to support the diagnosis and will have so many clinical consequences that it is imperative to try and use the best technique available. With the ELISA technique, sensitivity is 73%, and specificity is 95% (90 myasthenia gravis patients; 40 healthy controls) (111). In addition, in ELISA, cut-off levels are more difficult to determine because of the difficulty of washing wells, the smaller volumes involved, and reduced reliability. Radioimmunoassay has become the reference for measuring antibodies to the AChR.

Some myasthenia gravis patients have antibodies that bind to the skeletal and heart muscle, which are known as striational antibodies (134). These antibodies are directed against the muscle proteins titin and ryanodine receptor. They are found in about 50% of myasthenia gravis patients and up to 97% of myasthenia gravis patients with thymoma. Anti-striational antibodies and CT scan of the anterior mediastinum show a similar sensitivity for thymoma myasthenia gravis. In 146 consecutive myasthenia gravis patients, chest CT scan had a 73% sensitivity and a 90% specificity, anti-titin antibodies a 95% sensitivity and a 76% specificity, and anti-ryanodine receptor antibodies a 73% sensitivity and a 90% specificity (133). Anti-titin antibodies occur more frequently in patients with late-onset myasthenia gravis, and the frequency of titin autoantibodies in late-onset myasthenia gravis not associated with thymoma is approximately 55% (152). The presence of titin/ryanodine receptor antibodies in a young patient with myasthenia gravis strongly suggests the presence of a thymoma. The absence of these antibodies strongly excludes thymoma. Unfortunately, only testing for titin antibodies, and not anti-ryanodine receptor antibodies, is commercially available.

Between 10% and 20% of acquired myasthenia gravis patients are seronegative for anti-AChR antibodies but still have an autoimmune disorder. A certain proportion of these patients have antibodies against muscle-specific tyrosine kinase (MuSK). These antibodies are not found in anti-AChR antibody-positive patients (172). A multinational study of 633 seronegative myasthenia gravis patients found 79 anti-MuSK-positive patients, with a 13% sensitivity (164). Specificity tested in 162 healthy controls was 98%; specificity tested in 128 other neurologic disease patients was 94%. Radioimmunoassay was the gold standard in anti-MuSK antibody detection, but it yields only an approximate 6% positivity. Cell-based assays are increasingly used in routine diagnosis to detect myasthenia gravis autoantibodies as they seem able to detect antibodies in sera found to be seronegative with the conventional radioimmunoassay. The very high specificities indicated above refer to sera tested with cell-based assay (164).

Using this test, a further 15% of seronegative myasthenia gravis patients tested positive for anti-low-density lipoprotein receptor-related protein 4 (anti-LPR4) antibodies. In addition, some patients were double-positive (12% anti-AChR/anti-MuSK, 20% anti-MuSK/anti-LPR4). Finally, 180 randomly chosen myasthenia gravis samples remaining seronegative after the anti-MuSK cell-based assay screening were also tested by cell-based assay for autoantibodies against clustered AChR (α, β, γ, δ, and ε AChR subunits and rapsyn) (92). Of these, seven (4%) were found to be positive. Data on specificity are not available.

Pathogenic significance. Anti-AChR antibodies bind to the receptor and affect neuromuscular transmission by different mechanisms. The most relevant is to promote endocytosis and accelerated degradation of AChR by cross-linking to the receptor, resulting in a faster turnover and reduced receptor number. They also block ACh binding to AChR sites by steric hindrance, and, if they are IgM or of the 1, 2, or 3 IgG isotype, they can attract complement and macrophages, resulting in the destruction of the postjunctional folds. Excellent response to plasma exchange and IVIg in patients with myasthenia gravis confirms the antibody-mediated pathogenesis of myasthenia gravis. The observation that the disease can be transferred passively in mice by injection of IgG obtained from sera of myasthenia gravis patients further supports autoimmune pathogenesis (163). MuSK is a receptor tyrosine kinase that, once stimulated by agrin, a protein synthesized by motor neurons, promotes the clustering of postsynaptic proteins, including AChR, at the neuromuscular junction during synapse formation. LRP4 is a receptor for agrin and for MuSK. Anti-MuSK or anti-LPR4 antibodies reduce agrin-induced clustering of AChR in vitro, but it is not yet clear how they affect the neuromuscular junction in vivo (65).

Lambert-Eaton myasthenic syndrome

Clinical manifestations. Lambert-Eaton myasthenic syndrome is characterized by proximal muscle weakness, distal paresthesias, areflexia, autonomic dysfunction, constipation, and dry mouth. Lambert-Eaton myasthenic syndrome is paraneoplastic in about 50% of patients, most frequently associated with small cell lung cancer, and non-paraneoplastic in the other 50% (114; 181).

Type of antibodies. Lambert-Eaton myasthenic syndrome is associated with highly specific antibodies against voltage-gated calcium channels (VGCC) located at motor nerve terminals (P/Q-type) that are involved in the release of acetylcholine. These antibodies may be present in up to 90% of Lambert-Eaton myasthenic syndrome patients (101; 104). Small cell lung cancer may be less common in the 10% of Lambert-Eaton myasthenic syndrome patients seronegative for anti-VGCC antibodies (104). Sensitivity of anti-VGCC antibodies for Lambert-Eaton myasthenic syndrome diagnosis is 92%; specificity is 98%. Their sensitivity for small cell lung cancer is 21%, with an 83% specificity (6842 neurologic patients tested in Mayo Clinic, 236 with anti-VGCC antibodies, 50 of whom with lung cancer, 173 healthy controls) (188). Their sensitivity for the association with small cell lung cancer in Lambert-Eaton myasthenic syndrome patients is 70%, with an 88% specificity (110 Lambert-Eaton myasthenic syndrome patients, 93 anti-VGCC positive patients) (104). Anti-VGCC antibodies are also occasionally found in patients with paraneoplastic cerebellar degeneration, usually in association with Lambert-Eaton myasthenic syndrome (98; 174). Anti-VGCC antibodies are detected with radioimmunoassay using 125I-labelled synthetic ω-conotoxin identical to Conus genus snail toxin, which binds to both P- and Q-type VGCC. Diagnostic assays using other toxins or other techniques (ELISA, western blotting) are less sensitive.

Some 10% to 15% of patients with Lambert-Eaton myasthenic syndrome have no detectable anti-P/Q type VGCC antibodies. Passive transfer of seronegative Lambert-Eaton myasthenic syndrome sera to mice seemed to reproduce the typical electrophysiological changes seen in mice passively transferred with seropositive sera. Several studies reported antibodies to synaptotagmin, a synaptic vesicle protein partly exposed at the surface during exocytosis, to muscarinic AChRm1, to ELKS/RAB6-interacting/CAST family member 1 (ERC1) protein, or to SOX proteins (belonging to the Sry-like high mobility group superfamily of developmental transcription factors) (161). There is no satisfactory evidence for pathogenicity of any of these antibodies, although some might be relevant for detecting an underlying tumor. Anti-SOX antibodies have a 67% sensitivity and a 95% specificity to discriminate between Lambert-Eaton myasthenic syndrome with or without small cell lung cancer (43 Lambert-Eaton myasthenic syndrome with small cell lung cancer patients; 43 Lambert-Eaton myasthenic syndrome without small cell lung cancer patients) (160). ELISA and western blotting have the same reliability for anti-SOX protein antibody detection.

Pathogenic significance. The antibody-mediated loss of VGCC leads to a decrease in calcium-mediated quantal release of ACh from motor nerve terminals resulting in abnormal transmission at the neuromuscular junction. Passive transfer of human autoantibodies to mice induces disease (81). The dry mouth and constipation commonly seen in Lambert-Eaton myasthenic syndrome are due to the presence of P/Q channels in some smooth muscles (175).

Acquired neuromyotonia or Isaac disease

Clinical manifestations. Neuromyotonia is characterized by muscle twitching, myokymia, continuous muscle fiber hyperactivity, increased sweating, and other autonomic symptoms (105). Electrophysiologically, there is evidence of spontaneous muscle action potentials that persist after nerve block. These potentials disappear with inhibition of neuromuscular transmission by curare, suggesting their origin in distal nerves.

Type of antibodies. Antibodies in patients with acquired neuromyotonia are directed towards the presynaptic voltage-gated potassium channel (VGKC), especially the dendrotoxin-sensitive fast potassium channels located along the distal motor nerve or at the nerve terminal (09). Radio-iodinated alpha-dendrotoxin, which binds with high affinity to three different VGKC subunits, Kv1.1, 1.2, and 1.6, is used to label VGKC extracted from mammalian brain. Most anti-VGKC antibodies bind to associated VGKC-complex proteins and not directly to Kv subunits (70). Contactin-associated protein 2 (Caspr2) co-localizes with Kv1.1 and 1.2 at juxtaparanodes of the nodes of Ranvier. It is expressed on the surface of hippocampal neurons, and it is essential for VGKC clustering in vivo. Leucine-rich glioma inactivated 1 (LGi1) associates specifically with Kv1.1 subunits in CNS presynaptic terminals, and it is a key hippocampal protein that is secreted in the synaptic space.

Their clinical significance is different: peripheral nerve hyperexcitability and limbic encephalitis without thymoma for LGi1; peripheral nerve hyperexcitability, Morvan syndrome, and frequent thymoma for CASPR2. Most anti-VGKC antibodies of neuromyotonia patients bind to Caspr2. Anti-Caspr2 antibody–positive patients might, however, have neuromyotonia, Morvan syndrome, or limbic encephalitis. The sensitivity of anti-Caspr2 antibodies for neuromyotonia is 37%, and it is 53% for an associated tumor (mostly thymoma) (70; 71). Anti-VGCK complex antibodies are detected using a cell-based assay radioimmunoassay and anti-Caspr2 and anti-LGi1 antibodies with a tissue-based assay (70; 71).

Pathogenic significance. Neuromyotonia is an antibody-mediated potassium channel disorder (channelopathy). These antibodies decrease the density of voltage-gated potassium channel and suppress the voltage-gated outward potassium current, resulting in hyperexcitability of the peripheral nerve and increased motor action potentials (09; 89). The autoimmune etiology of neuromyotonia is proven by the passive transfer of disease in mice following injection of IgG purified from the plasma of patients with neuromyotonia (147).

Acute inflammatory demyelinating polyradiculoneuropathy or Guillain-Barré syndrome

Clinical manifestations. Acute inflammatory demyelinating polyradiculoneuropathy is a postinfectious autoimmune disease characterized by acute monophasic illness with ascending muscle weakness, paresthesia, areflexia, and oftentimes autonomic dysfunction. Electrophysiological studies show slowing of motor nerve conduction. Its variant, Miller-Fisher syndrome, typically presents with areflexia, ataxia, and ophthalmoplegia. Acute motor axonal neuropathy and acute motor sensory axonal neuropathy are acute inflammatory demyelinating polyradiculoneuropathy variants that occur more commonly in China and in Japan and have a high association with preceding diarrheal illness and Campylobacter jejuni infection. Electrophysiological studies show a motor or motor-sensory axonal neuropathy. Pure acute sensory ataxic neuropathy is another acute inflammatory demyelinating polyradiculoneuropathy variant defined by the presence of sensory ataxia, absence of ophthalmoplegia, and no or minimal muscle weakness.

Type of antibodies. Anti-GM1, -GD1a, -GM1b, and -GalNAc-GD1a IgG antibodies can be found in acute inflammatory demyelinating polyradiculoneuropathy, acute motor axonal neuropathy, and acute motor sensory axonal neuropathy (184; 185; 110; 44; 82; 109). They have a high specificity (97% to 100%) but a very low sensitivity, ranging from 15% to 56% according to the different techniques: ELISA 15% (2/13 patients) to 34% (26/78 patients) and immunoblotting 56% (28/50 patients) (04; 47; 14). Reactivity against gangliosides sharing disialosyl epitopes has been reported in pure acute sensory ataxic neuropathy, but, again, the sensitivity is low (58%, 7/12 patients) (132). Immunoblotting has a higher sensitivity because it simultaneously detects multiple anti-ganglioside antibodies. In addition, acute inflammatory demyelinating polyradiculoneuropathy patient sera often react with ganglioside complexes consisting of two different gangliosides (namely, GM1 and GalNAc-GD1a), but not with each constituent ganglioside. Some antibodies can recognize epitopes comprising more than one molecule, which exist in the complex environment of the cell membrane and is not easily reproduced using traditional antibody detection assays (54). Screening for one or more heteromeric complexes (of gangliosides GM1, GA1, GD1a, GD1b, GQ1b, LM1, and the glycosphingolipid SPGP) might slightly increase the sensitivity but brings about a high specificity for the clinical variants (up to 98% for acute motor axonal neuropathy tested with GM1 or GD1b complexes) (266 acute inflammatory demyelinating polyradiculoneuropathy patients, 579 controls) (63). Antiganglioside antibody testing is not useful for the diagnosis of acute inflammatory demyelinating polyradiculoneuropathy, acute motor axonal neuropathy, acute motor sensory axonal neuropathy, or acute sensory ataxic neuropathy, which is better based on clinical and EMG findings. Nonetheless, testing for antiganglioside antibody complexes might be useful for identifying clinical variants and managing prognosis and treatment.

Antibodies to GQ1b ganglioside are detected with ELISA in almost all Miller-Fisher syndrome patients, with a 96% sensitivity (24 Miller-Fisher syndrome patients) and a 100% specificity (23). They are a useful biological marker of the disease (180).

Pathogenic significance. Lipo-oligosaccharides extracted from Campylobacter jejuni (CF90-26) isolated from an acute motor axonal neuropathy patient with anti-GM1 IgG antibody were identical to those of GM1 ganglioside (186). An acute motor axonal neuropathy model (flaccid limb weakness of acute onset with a monophasic course and with high anti-GM1 IgG antibody titers) was established by sensitization of Japanese white rabbits with a bovine brain ganglioside mixture that included GM1 (187). Pathologic findings corresponded well with those of human acute motor axonal neuropathy. These observations led to the concept that molecular mimicry could play a role in the pathogenesis of the disease.

The anti-GQ1b antibody in Miller-Fisher syndrome recognizes epitopes expressed in the paranodal region of oculomotor and cerebellar molecular layer neurons and dorsal root ganglion cells. The human oculomotor nerve contains more GQ1b, which may explain the association of ophthalmoplegia and anti-GQ1b antibodies in Miller-Fisher syndrome (23). These antibodies block neuromuscular transmission via pre- and postsynaptic mechanisms and might play an important pathophysiological role in Miller-Fisher syndrome (124; 18).

Chronic inflammatory demyelinating polyradiculoneuropathies

Clinical manifestations. Chronic inflammatory demyelinating polyradiculoneuropathy starts with a slowly progressive progression of symptoms (over 8 weeks or more), symmetric proximal and distal weakness with sensory loss, and hyporeflexia. It is associated with elevated CSF protein and EMG evidence of demyelinating neuropathy.

Chronic inflammatory demyelinating polyradiculoneuropathy associated with paraproteinemia may differ from chronic inflammatory demyelinating polyradiculoneuropathy not associated with paraproteinemia. The more frequent neuropathy is that associated with IgM monoclonal gammopathy of unknown significance (MGUS), in which antibodies are specific against myelin-associated glycoprotein. The prevalence of this neuropathy is around 8% in patients with MGUS, which has, in turn, a high prevalence (1% below 60 years of age; 3% above) (107). It is a demyelinating sensory neuropathy with more pronounced and predominantly distal conduction velocity slowing and lower frequency of conduction block compared with chronic inflammatory demyelinating polyradiculoneuropathy, and it has been categorized as distal acquired demyelinating symmetric neuropathy (79). It usually affects elderly men, presents with ataxia, distal sensorimotor involvement, and tremor, and has a poor response to immunotherapy (97; 24). The neuropathy associated with either IgG or IgA MGUS and no myelin-associated glycoprotein antibody is usually similar to chronic inflammatory demyelinating polyradiculoneuropathy without paraproteinemia.

Multifocal motor neuropathy predominantly affects men and is characterized by asymmetric motor weakness in the upper extremities, fasciculations, and cramps in the absence of significant sensory abnormalities or muscle atrophy (118). EMG studies show the presence of motor conduction block in two or more motor nerves that are not at common sites of entrapment; in contrast, sensory nerve conduction studies are normal (113).

Types of antibodies. Antibodies against peripheral myelin protein 22 (PMP22), myelin protein 0 (P0), or neurofascin155 (NF155) are detected in some patients with chronic inflammatory demyelinating polyradiculoneuropathy. The specificity is good: 90% for anti-PMP22 antibodies (17 chronic inflammatory demyelinating polyradiculoneuropathy patients; 30 other neuropathy patients), 100% for anti-P0 antibodies (21 chronic inflammatory demyelinating polyradiculoneuropathy patients, 15 normal controls), and 100% for anti-NF155 IgG4 antibodies (191 chronic inflammatory demyelinating polyradiculoneuropathy patients; 99 controls with other polyneuropathies or multiple sclerosis or healthy controls) (45; 183; Kadoya et 2016). However, the sensitivity is low: 41% for anti-PMP22 (17 chronic inflammatory demyelinating polyradiculoneuropathy patients), 29% for anti-P0 antibodies (21 chronic inflammatory demyelinating polyradiculoneuropathy patients), and 8% for anti-NF155 IgG4 antibodies (191 chronic inflammatory demyelinating polyradiculoneuropathy patients).

By contrast, most patients with distal acquired demyelinating symmetric neuropathy and IgM MGUS have anti-myelin-associated glycoprotein antibodies (107; 150). In these patients, the M-protein reacts with the epitope sulfate-3-glycuronate of myelin-associated glycoprotein and the two glycosphingolipids, SGPG and SGLPG, of peripheral nerve myelin sheath (107; 180). In MGUS patients with polyneuropathy, the sensitivity of anti-myelin-associated glycoprotein antibodies is 97%, with a specificity of 86% (117 MGUS patients; 102 healthy controls) at a titer of 1000 BTU (21). The specificity is 100% at 10,000 BTU. ELISA is a reliable technique. Using human myelin-associated glycoprotein, its reliability is comparable to that of western blotting (75). IgM anti-sulfatide antibodies are associated with IgM MGUS with anti-myelin-associated glycoprotein (MAG) antibodies. They have been associated with chronic painful axonal sensory polyneuropathy, with higher disability, and with favorable response to immunomodulating treatment (22). Increased titers of IgM anti-sulfatide antibodies have been found in 6.8% of patients with neuropathy or related syndromes, and the vast majority (85%) also had an IgM monoclonal gammopathy and increased titers of IgM antibodies to MAG. In these patients, reactivity to sulfatide was most often restricted to the same light chain of the M-protein and corresponded to that of anti-MAG antibodies, suggesting that this reactivity was related to the same antibodies. The clinical and electrophysiological features of patients with both anti-sulfatide and anti-MAG antibodies did not differ from those of patients with only anti-MAG antibodies. In conclusion, testing for IgM anti-sulfatide only rarely provides information that may have some implications on the diagnostic assessment of patients with neuropathy (570 neuropathy patients) (52).

IgM anti-GM1 ganglioside antibodies are detected in approximately 50% to 85% of patients with multifocal motor neuropathy; GM2 and GD1a antibodies occur less frequently (158; 119; 106). Conventional ELISA is still a reliable technique. With combinatorial glycoarray methods, in which antibodies react to heteromeric complexes of two or more glycolipids, out of the 55 possible single glycolipids and their 1:1 complexes, antibodies to the GM1:galactocerebroside complex were the most significantly associated with multifocal motor neuropathy, with a 100% sensitivity and a 93% specificity (with ELISA, 75% sensitivity and 85% specificity) (33 multifocal motor neuropathy patients; 30 other neurologic disease patients; 27 healthy controls) (46). ELISA testing for IgM reactivity to the gangliosides, either GM1 or GM2 or galactocerebroside, or to a 1:10 mixture of GM1 and galactocerebroside yielded a 48% sensitivity and a 93% specificity for anti-GM1 IgM and a 75% sensitivity and a 85% specificity for anti-GM1/galactocerebroside antibodies (40 multifocal motor neuropathy patients; 142 other neuropathy patients) (108).

Pathogenic significance. The pathogenic role of autoantibodies in chronic inflammatory demyelinating polyradiculoneuropathy is suggested by indirect evidence. Anti-P0 antibodies induce conduction block and demyelination after intraneural injection in animals and have been detected in a subgroup of patients who responded well to plasma exchange (183). Systemic transfusion of chickens with monoclonal human anti-myelin-associated glycoprotein IgM antibody produced peripheral demyelination highly characteristic of the human syndrome (157). The pathogenic role of IgM anti-GM1 antibodies in multifocal motor neuropathy is supported only by indirect evidence, such as the high antibody titer and the correlation between antibody titer and weakness, disability, or axonal loss severity (20).

Myopathies

Clinical manifestations. The inflammatory myopathies are acquired diseases characterized by inflammatory infiltrates in the skeletal muscle. Three major diseases can be identified: polymyositis, dermatomyositis, and inclusion-body myositis. Dermatomyositis is a complement-mediated microangiopathy affecting skin and muscle. Polymyositis and inclusion-body myositis are dominated by muscular manifestations (symmetric proximal muscle weakness associated with muscle cell destruction). In dermatomyositis, cutaneous signs accompany or precede muscular involvement. Together with the muscular manifestations, there may be cutaneous, joint, gastrointestinal, pulmonary, heart, and renal manifestations.

Types of antibodies. Antibodies to nuclear and cytoplasmic antigens are detected in patients with idiopathic inflammatory myopathies. They have been divided into myositis-specific autoantibodies and myositis-associated autoantibodies. The latter also occur in autoimmune diseases without myositis (62). Among myositis-specific autoantibodies, the most common is the anti-Jo-1 (anti-histidyl tRNA synthetase) antibody, found in 30% to 40% of polymyositis patients. Other myositis-specific antibodies are anti-Mi-2, anti-p155/140, anti-small ubiquitin-like modifier activating enzyme, anti-signal recognition particle antibodies, and others (17; 51; 130). They can be found in other connective tissue diseases and, despite the name, they have a low specificity for myositis. The diagnosis of myositis is based on clinical, EMG, and histologic (bioptic) criteria.

Pathogenic significance. The pathogenic significance of myositis-specific autoantibodies is not known.

Diseases affecting the central and peripheral nervous systems

Paraneoplastic neurologic disorders

Paraneoplastic neurologic disorders are rare and associated with cancer but not caused by direct invasion, metastasis, or a direct result of cancer therapy treatment, cerebrovascular disease, coagulopathy, infection, or toxic or metabolic causes (36). They are probably caused by the immune response to tumor-derived antigens. These syndromes affect only 0.01% of patients with cancer, with the exception of Lambert-Eaton myasthenic syndrome, which is paraneoplastic in 50% to 60% of cases and affects 1% to 3% of patients with small cell lung cancer (32). The clinical diagnosis of a paraneoplastic neurologic disorder or the detection of a paraneoplastic antibody, however, can be extremely important in the diagnostic workup. In fact, the clinical symptoms and the paraneoplastic neurologic disorder–associated antibody finding might precede the detection of a tumor by several months in almost 80% of patients, and positron emission tomography might detect a tumor or tumor recurrence in 90% of antibody-positive paraneoplastic neurologic disorder patients (96; 68). These antibodies are sometimes highly specific for a particular cancer and can help identify it at a stage before it is clinically overt, potentially leading to early successful therapy. Paraneoplastic neurologic disorders are rapidly progressive in many cases, leaving patients severely debilitated within weeks to months (56). Paraneoplastic neurologic disorders usually do not respond well to immunotherapies. However, treatment of the underlying tumor may lead to improvement of symptoms. Patients with autoantibodies have a relatively favorable prognosis compared to patients with identical tumors that are not associated with paraneoplastic disorders or antibodies.

Clinical manifestations. Several distinct paraneoplastic neurologic disorders have been described depending on the clinical presentation.

Limbic encephalitis. Limbic encephalitis usually presents with cognitive dysfunction, seizures, and psychiatric symptoms (91). The diagnosis is made by brain MRI, showing unilateral or bilateral limbic abnormalities, and by finding paraneoplastic antibodies (Tables 2 and 3).

Anti-NMDAR encephalitis. Anti-NMDAR encephalitis presents with prominent psychiatric symptoms or, less frequently, with short-term memory loss. Seizures, progressive unresponsiveness, central hypoventilation, autonomic instability, and orofacial or limb dyskinesias follow thereafter. Although patients may die of status epilepticus if untreated, most patients recover after immunotherapy and tumor removal (86; 55).

Anti-AMPAR encephalitis. Anti-AMPAR encephalitis presents with prominent psychiatric symptoms, followed by the symptoms of limbic encephalitis (86; 55).

Patients with brainstem encephalitis with antineuronal nuclear antibody type 1 (ANNA-1) antibodies usually present with predominant involvement of the medulla that may result in central hypoventilation (141). In contrast, patients with ANNA-2 antibodies develop opsoclonus and various degrees of extrapyramidal rigidity, ataxia, and postural instability. These patients may also have predominant symptoms of jaw dystonia and laryngospasm, at times associated with severe lethal respiratory distress (123). In patients with antiparaneoplastic neurologic Ma protein type 2 (Ma-2) antibodies, brainstem encephalitis affects the rostral brainstem portion with hypokinetic and oculomotor symptoms.

LGI1 encephalitis. Patients with anti-LGI1 encephalitis can present with encephalopathy and faciobrachial dystonic seizures with sudden, brief, lateralized tonic contractions mainly involving the upper limb and the ipsilateral face (occasionally the leg), and hand dystonia. They can be unilateral, or bilateral asynchronous and can recur several times per day. Anti-LGI1 encephalitis is associated with antibodies that have a low-risk association with thymomas (159).

Opsoclonus-myoclonus-ataxia syndrome. Opsoclonus-myoclonus-ataxia syndrome is one of the most common pediatric paraneoplastic syndromes. These patients usually present with subacute ataxia manifesting as gait imbalance, staggering, falls, or a refusal to walk. Autoantibodies have been implicated (ANNA-1, ANNA-1) and are associated with neuroblastoma in just 2% to 3% of patients and rarely with ovarian teratomas (137).

Progressive encephalomyelitis with rigidity and myoclonus. Progressive encephalomyelitis with rigidity and myoclonus is a hypokinetic disorder presenting with limb and truncal rigidity, painful spasms, brainstem signs, and hyperreflexia. This syndrome is most commonly associated with antibodies to GAD, glycineR, NMDAR, and dipeptidyl-peptidase-like protein 6 (DPPX) (27). About 20% of progressive encephalomyelitis with rigidity and myoclonus cases are paraneoplastic and associated with small-cell lung cancer, lymphomas, melanomas, breast cancer, or thymomas.

Paraneoplastic narcolepsy and cataplexy. Paraneoplastic narcolepsy and cataplexy are seen in young men with anti-Ma2 antibody encephalitis and testicular germ cell tumors. Immunoglobulin-like cell adhesion molecule 5 (IgLON5) has also been associated with sleep-disordered breathing and parasomnias (42).

Paraneoplastic movement disorders. Paraneoplastic movement disorders occur in two other well-differentiated clinical settings. Orobuccolingual and often trunk, abdomen, and extremity movements occur in 80% of patients with anti-NMDAR encephalitis (116). Paraneoplastic chorea is usually associated with anti-collapsin response–mediator brain protein type 5 (CRMP5) antibodies and small cell lung cancer. Most patients present other neurologic symptoms, and brain MRI usually reveals high T2-signal abnormalities in the caudate and putamen (169).

Myelopathy. Myelopathy is often associated with other neurologic syndromes (paraneoplastic encephalomyelitis). However, a study described 31 patients who developed a subacute, usually severe, isolated myelopathy with cerebrospinal fluid pleocytosis and spinal MRI abnormalities (43). Immunotherapy and cancer treatments were not very effective.

Paraneoplastic cerebellar degeneration. Paraneoplastic cerebellar degeneration presents with severe cerebellar ataxia, but other parts of the nervous system may also be involved (146).

Paraneoplastic subacute sensory neuronopathy. Paraneoplastic subacute sensory neuronopathy presents with subacute, painful, predominantly sensory neuropathy with relative preservation of muscle strength (19). Later, pain is replaced by numbness, limb ataxia, and pseudoathetotic movements of the hands due to proprioceptive deficit.

Autonomic neuropathy. Autonomic neuropathy often presents with acute or chronic gastrointestinal pseudo-obstruction with weight loss, persistent constipation, and abdominal distension. Orthostatic hypotension or sexual disturbance are also symptoms of autonomic neuropathy.

PNS and immune checkpoint inhibitors. With the more widespread use of immune checkpoint inhibitors in cancer therapy, there have been multiple reports of paraneoplastic neurologic syndromes thought to be directly related to the use of these therapies. These therapies target immunosuppressive or immunoregulatory molecules, such as cytotoxic T lymphocyte-associated antigen 4 (CTLA4) and programmed death-1 (PD1) or its ligand (PDL1), which enhance endogenous immune responses and anticancer immunity, leading to unwanted outcomes affecting other organ systems. Neurologic complications of immune checkpoint inhibitor therapies, including either development or worsening of existing neurologic autoimmunity, can be seen in approximately 4.2% of patients receiving monotherapy and 14% of those receiving combination CTLA4 and PD1/PDL1 inhibitors (144; 37). This group reported on the neural autoantibody profiles and outcome predictors of a large cohort of immune checkpoint inhibitor-treated patients who subsequently had autoimmune neurologic complications. Neurologic outcome was poor in one third of the patients and was associated with pre-immune checkpoint inhibitor treatment characteristics (older age, female sex, lung cancer, and presence of systemic immune checkpoint inhibitor-related autoimmunity) and clinical phenotype (CNS involvement and severity of clinical symptoms at onset). After commencing immune checkpoint inhibitors, patients with preexistent paraneoplastic CNS autoimmunity and neural autoantibody seropositivity had particularly severe neurologic deterioration that was poorly responsive to protracted immunotherapy. Therefore, the authors recommended screening for autoantibodies in patients with tumors that are traditionally associated with paraneoplastic syndromes, even if patients are neurologically asymptomatic when immune checkpoint inhibitors are going to be part of their cancer treatment regimen (144).

Types of antibodies. Broadly speaking, autoantibodies that are potentially paraneoplastic can be grouped into two categories: those that bind to the cell surface or synaptic antigens and those that bind to intracellular or cytoplasmic antigens (Tables 2 and 3) (86; 55). Antibodies with intracellular antigens are typically not considered directly pathogenic and could involve cytotoxic CD8 T cells, which recognize intracellular antigens expressed on major histocompatibility complex class 1 cell surface molecules that mark the cell for destruction by these cytotoxic T lymphocytes. Intracellular antibodies have a much higher frequency of cancer (greater than  80%). An exception to this rule is anti-GAD65 antibodies, which are less commonly associated with cancer (42). The frequency of an underlying tumor varies depending on the antibody type, and the associated neurologic symptoms may respond to immunotherapy. All these antibodies, particularly those associated with small-cell lung cancer (ANNA-1, anti-CRMP5, and anti-amphiphysin), can be found in patients with cancer but without paraneoplastic neurologic disorders (100).

Table 2. Antibodies Associated With Intracellular or Cytoplasmic Antigens (Onconeural Antigens)

Antibody	Predominant tumor	Syndrome
PCA-1 (previously anti-Yo)	Ovary, breast cancer	Paraneoplastic cerebellar degeneration
ANNA-1 (previously anti-Hu)	Small cell lung cancer, breast or ovarian cancer, neuroblastoma, prostate cancer	Paraneoplastic sensory neuronopathy, encephalomyelitis, brainstem encephalitis, paraneoplastic cerebellar degeneration
ANNA-2 (previously anti-Ri)	Small cell lung cancer, breast or gynecological and bladder cancers	Brainstem encephalitis, ataxia with or without opsoclonus-myoclonus sometimes with jaw dystonia and severe laryngospasm
ANNA-3	Lung cancer	Sensory neuronopathy, encephalomyelitis, paraneoplastic cerebellar degeneration
Anti-Ma-1	Lung and other cancers	Brainstem encephalitis, paraneoplastic cerebellar degeneration
Anti-Ma-2	Testicular and lung cancer, or intestinal neuroendocrine tumors without paraneoplastic disorders	Limbic encephalitis, brainstem encephalitis, paraneoplastic cerebellar degeneration, narcolepsy-cataplexy
Anti-recoverin	Small cell lung cancer, melanoma, gynecological cancers	Retinopathy
Anti-CRMP5 (previously anti-CV2)	Small cell lung cancer, thymoma	Encephalomyelitis, paraneoplastic cerebellar degeneration, limbic encephalitis, sensory neuronopathy, chorea, isolated myelopathy, optic neuropathy
Anti-amphiphysin	Breast cancer and small cell lung cancer, thymoma	Stiff-person syndrome, limbic encephalitis, paraneoplastic encephalomyelitis, isolated myelopathy
Anti-GAD	Breast cancer and insulin-dependent diabetes mellitus or other autoimmune diseases	Stiff-person syndrome, limbic encephalitis, cerebellar ataxia
Anti-SOX	Small cell lung cancer	Lambert-Eaton myasthenic syndrome
Anti-ZIC	Small cell lung cancer	Paraneoplastic cerebellar degeneration
Anti-TRIM46	Small cell lung cancer, neuroendocrine carcinoma, pancreatic cancer, GI cancers, adenocarcinoma, sarcoma	Cerebellar syndrome, encephalomyelitis, seizures, parkinsonism
Anti-SKOR	Adenocarcinoma	Encephalitis, seizures, cerebellar syndrome

Table 3. Antibodies Against Cell Surface or Synaptic Antigens

Antibody	Syndrome	Associated cancers
Anti-VGCC	Lambert-Eaton myasthenic syndrome, paraneoplastic cerebellar degeneration	Small cell lung cancer, none
Anti-GABABR	Limbic encephalitis	Small cell lung cancer
Anti-AMPAR	Limbic encephalitis	Breast cancer
Anti-Caspr2	Limbic encephalitis, Morvan syndrome, neuromyotonia, painful neuropathy	Thymoma
Anti-LGi1	Limbic encephalitis with hyponatremia and myoclonic jerks	Thymoma, small cell lung cancer
Anti-NMDAR (NR1/NR2 heteromers)	Encephalitis with central hypoventilation, autonomic instability, and orofacial and/or limb dyskinesias	Ovarian teratoma
Anti-DNER	Hodgkin lymphoma	Paraneoplastic cerebellar degeneration
Anti-mGluR-1	Paraneoplastic cerebellar degeneration	Hodgkin lymphoma
Anti-GluR-5	Limbic encephalitis	Hodgkin lymphoma
Anti-ACHR α3 subunit	Autonomic neuropathy, neuromyotonia	Small cell lung cancer (50%); bladder, rectum, and other cancers

Antibodies targeting intracellular antigens are more likely associated with cancers. Well-characterized neurologic syndromes, cancer-specific and paraneoplastic neurologic disorder-associated intracellular antibodies include the following:

	• PCA-1. PCA-1 is usually associated with ovary, breast, or other gynecological malignancies. Its detection is highly specific for a tumor, particularly for gynecological malignancies (100% specificity and 93% specificity for gynecological malignancies; 100% sensitivity and 91% specificity for ovary/fallopian tube/uterus tumor; 18% sensitivity and 86% specificity for tumor) (101 PCA-1-positive patients, 68 with tumor) (121). It is also 99% specific for a paraneoplastic neurologic disorder in cancer patients (107 PCA-1-positive cancer patients) and 90% specific for a subacute cerebellar degeneration syndrome (55 PCA-1-positive patients) (120; 56).
	• ANNA-1. ANNA-1 is not specific for a paraneoplastic neurologic disorder and can be detected in patients with a wide range of neurologic symptoms, from limbic encephalitis to subacute cerebellar degeneration or to subacute sensory neuronopathy. It is, however, quite specific for a tumor, namely small cell lung cancer (68% specificity for tumor; 72% specificity for small cell lung cancer) (217 ANNA-1-positive patients) (121). It is also 84% specific for a paraneoplastic neurologic disorder in cancer patients (196 small cell lung cancer patients without paraneoplastic neurologic disorder) (56).
	• ANNA-2. The specificity for tumor is high, 98% (breast cancer or small cell lung cancer) (17 ANNA-2-positive patients) (121). The specificity for a paraneoplastic neurologic disorder in cancer patients is 96% (181 ovarian cancer patients without paraneoplastic neurologic disorder), and the sensitivity for brainstem encephalitis with ataxia and opsoclonus/myoclonus is 71% (28 ANNA-2-positive patients) (122; 56).
	• ANNA-3. The specificity for tumors is high, 99% (small cell lung cancer or others, mainly an intrathoracic neoplasm) (10 ANNA-3-positive patients) and for a paraneoplastic neurologic disorder in cancer patients is 100% (58 cancer patients without paraneoplastic neurologic disorder) (56; 121). It is not associated with a specific neurologic syndrome.
	• Anti-Ma-2. The specificity for tumors is 96% (55 anti-Ma-2-positive patients) (56). Among tumors it is 87% sensitive and 67% specific for germinal testis tumor in males; it is associated with various tumors if associated with other reactivities (against Ma-1 or Ma-3 protein) (29 anti-Ma protein–positive patients) (135). It is 100% specific for a paraneoplastic neurologic disorder in cancer patients (350 cancer patients without paraneoplastic neurologic disorder) (56). It is usually associated with limbic or brainstem encephalitis, and patients with anti-Ma-2 alone and testis tumors have a better outcome after surgery and immunosuppressive treatment than patients with reactivities to other Ma proteins.
	• Anti-retinal antibodies. Antibodies against different retinal antigens (enolase, carbonic anhydrase II, recoverin, and transducin) can be found in cancer-related retinopathy patients. Associated cancers are usually breast (31%), lung (16%), melanoma (16%), hematological malignancies (lymphoma, leukemias, myelomas) (15%), gynecological neoplasms (9%), prostate (7%), and colon (6%) (01). Anti-retinal antibodies have a low 63% sensitivity and 58% specificity for cancer compared to patients with no cancer-related retinopathy and a higher 81% specificity compared to healthy controls (52 cancer-related retinopathy patients; 141 patients with no cancer-related retinopathy; 79 healthy controls) (03). Anti-recoverin antibodies can be predictive for cancer because they have a high 99% specificity for lung cancer compared with controls (other lung diseases and healthy controls). The very low sensitivity (17%) does not allow one to consider them as a valuable marker of lung cancer (99 small cell lung cancer patients; 44 other lung disease patients; 50 healthy controls) (13). Anti-recoverin or anti-enolase antibodies in women with retinopathy have a strong association with breast cancer (anti-recoverin antibodies: sensitivity 5%, specificity 100%; anti-enolase antibodies: sensitivity 32%, specificity 82%) (111 women with vision loss; 78 healthy controls) (02).
	• Anti-CRMP5. The specificity for tumors is 60% (65% small cell lung cancer patients; 208 anti-CRMP5-positive patients) and for a paraneoplastic neurologic disorder in cancer patients is 90% (74 small cell lung cancer patients without paraneoplastic neurologic disorder) (56; 121). It is not associated with a specific neurologic syndrome.
	• Anti-amphiphysin. The specificity for tumors is high, 98% (48% small cell lung cancer, 38% breast cancer, 26 anti-amphiphysin-positive patients), and for a paraneoplastic neurologic disorder in cancer patients is 99% (146 small cell lung cancer patients without paraneoplastic neurologic disorder) (56; 121). It is not associated with a specific neurologic syndrome.
	• PCA-2. Nine patients with PCA-2 and neurologic symptoms have been reported (74). The specificity for tumors is high, 90% (53% small cell lung cancer, 43 PCA-2-positive patients) and for a paraneoplastic neurologic disorder in cancer patients is 98% (58 cancer patients without paraneoplastic neurologic disorder) (56; 121). It is not associated with a specific neurologic syndrome (paraneoplastic cerebellar degeneration, limbic encephalitis, Lambert-Eaton myasthenic syndrome, autonomic neuropathy, and others).

Intracellular antigen-specific antibodies are associated with neurologic syndromes with possible cancer association.

• Anti-SOX antibodies. They are detected in 64% of patients with Lambert-Eaton myasthenic syndrome and small-cell lung cancer but very rarely in idiopathic Lambert-Eaton myasthenic syndrome. They have a 95% specificity to discriminate between Lambert-Eaton myasthenic syndrome with or without small cell lung cancer (160).

• Anti-ZIC antibodies (zinc fingers of the cerebellum). They have been found in 15% of patients with paraneoplastic cerebellar degeneration and small cell lung cancer, but also in 16% of patients with small cell lung cancer alone and in 29% of patients with a different paraneoplastic neurologic disorder and small cell lung cancer. Despite the low sensitivity (16%), they are 100% specific for small cell lung cancer (74 small cell lung cancer patients; 34 other cancer patients; 20 healthy controls) and for small cell lung cancer and paraneoplastic neurologic disorder (167 small cell lung cancer with paraneoplastic neurologic disorder patients; 48 other tumors and paraneoplastic neurologic disorder patients) (12).

Antibodies against surface or synaptic antigens.

	• Anti-VGCC antibodies. Anti-VGCC antibodies are occasionally found in patients with paraneoplastic cerebellar degeneration, usually in association with Lambert-Eaton myasthenic syndrome (98; 174). Although their sensitivity for small cell lung cancer in paraneoplastic cerebellar degeneration is low (12% to 41%), they have an 88% to 100% specificity (66 paraneoplastic cerebellar degeneration patients with small cell lung cancer; 27 paraneoplastic cerebellar degeneration patients with other tumors) (174) (39 paraneoplastic cerebellar degeneration patients; 21 small cell lung cancer patients; seven paraneoplastic cerebellar degeneration patients with other tumors) (57).
	• Anti-GABABR antibodies. Anti-GABABR antibodies have been detected in some patients with paraneoplastic encephalitis with the classic limbic encephalitic symptoms and MRI findings (15). Their sensitivity for encephalitis/encephalopathy is low (4% to 8%), and their sensitivity for an associated cancer (usually small cell lung cancer) is also low, ranging from 33% to 41% (126 suspected paraneoplastic neurologic disorder or autoimmune encephalitis/encephalopathy patients, 10 anti-GABABR positive) (35) (410 suspected paraneoplastic neurologic disorder or autoimmune encephalitis/encephalopathy patients, 17 anti-GABABR positive) (85).
	• Anti-AMPAR antibodies. Anti-AMPAR antibodies are associated with limbic encephalitis, which may present with prominent psychiatric symptoms. Their sensitivity for encephalitis/encephalopathy is low (2%), and their sensitivity for an associated cancer (usually ovarian cancer) ranges from 33% to 70% (126 suspected paraneoplastic neurologic disorder or autoimmune encephalitis/encephalopathy patients, three anti-AMPAR positive) (35) (10 anti-AMPAR positive) (83).
	• Anti-Caspr2 antibodies. Anti-Caspr2 antibodies are associated with a broader spectrum of symptoms, including Morvan syndrome, neuromyotonia and painful neuropathy, and, more rarely, limbic encephalitis (70; 71). Tumors, especially thymomas, are mostly associated with anti-Caspr2 antibody-positive neuromyotonia or Morvan syndrome, whereas tumors appear to be uncommon in anti-Caspr2 antibody-positive limbic encephalitis. Among patients with CNS or peripheral nerve involvement, anti-Caspr2 antibodies have a 82% sensitivity and an 83% specificity for an associated thymoma (82 CNS or peripheral nerve patients) (171).
	• Anti-LGi1 antibodies. Anti-LGi1 antibodies are associated with limbic encephalitis, often with hyponatremia. They have an 89% sensitivity for limbic encephalitis (70).
	• Anti-NMDAR antibodies. Anti-NMDAR antibodies are associated with the most frequent and best-characterized autoimmune encephalitis, which is clinically different from limbic encephalitis. The syndrome usually develops with a sequential presentation of symptoms, including a prodrome of headache and fever followed by behavioral changes, psychosis, catatonia, decreased level of consciousness, dyskinesias, and autonomic instability, which may require ventilatory support. Seizures can occur at any stage but most commonly occur early. Thirty percent of patients show a unique electroencephalographic pattern named “extreme delta brush” (143). Anti-NMDAR antibody encephalitis usually affects young women (aged 18 to 40 years) with ovarian teratoma. Within encephalitis patients, anti-NMDAR antibodies have a 59% sensitivity for a tumor and a 93% sensitivity for a teratoma (98 anti-NMDAR antibody encephalitis patients) (31).
	• Anti-DNER (previously anti-Tr) antibodies. Anti-DNER antibodies are associated with paraneoplastic cerebellar degeneration and Hodgkin lymphoma. In patients with subacute and severe cerebellar ataxia, anti-DNER antibodies have a 91% sensitivity and a 100% specificity for Hodgkin lymphoma (11 subacute and severe cerebellar ataxia patients; 215 other neurologic disease patients; 31 healthy controls) (33). Anti-DNER antibodies are also absent in patients with paraneoplastic cerebellar degeneration and non-Hodgkin lymphoma (33).
	• Anti-mGluR-1 antibodies. Anti-mGluR-1 antibodies have been found in five patients with pure cerebellar symptoms. Three of the patients had a tumor, and two had Hodgkin lymphoma (73). It is of interest that the passive transfer of patient anti-mGluR-1 IgG into the CSF of mice induced severe, transient ataxia (148).
	• Anti-mGluR-5 antibodies. Anti-mGluR-5 antibodies are associated with encephalitis in patients with Hodgkin lymphoma (Ophelia syndrome) (87). The syndrome is highly similar to limbic encephalitis, but MRI may show involvement beyond the limbic system. So far, they have been reported in less than 10 patients with neurologic symptoms, all with Hodgkin lymphoma (94).
	• Anti-nicotinic AChR α3 subunit antibodies. Anti-nicotinic AChR alpha3 subunit antibodies are associated with autonomic neuropathy or neuromyotonia, usually with anti-VGKC antibodies in the latter disease (57). Their sensitivity for autonomic neuropathy is 41%, for idiopathic autonomic neuropathy is 50%, and for paraneoplastic autonomic neuropathy is 28%. Their specificity for idiopathic or paraneoplastic autonomic neuropathy compared to other types of dysautonomia is high, 95%, and they are not found in patients with non-diabetic dysautonomia or degenerative dysautonomia (chronic progressive pure autonomic failure or multiple system atrophy). Their specificity for a tumor associated with dysautonomia (67% small cell lung cancer) is low, 20% (28 idiopathic autonomic neuropathy patients; 18 paraneoplastic autonomic neuropathy patients; 111 other types of dysautonomia patients) (168; 167).

Evaluation of paraneoplastic antibody testing. The gold standard of detection is tissue-based assay, immunohistochemical staining pattern on brain tissue sections, combined with confirmation by immunoblotting using recombinant purified proteins or with cell-based assay with suitable cell lines (such as HEK293 cells) that are transfected with an eukaryotic expression vector (plasmid) encoding the antigen. Rat cerebellum sections are used for ANNA-1, PCA-1, ANNA-2, anti-CRMP5, anti-Ma protein, anti-amphiphysin, anti-SOX1, and anti-GAD antibodies, and rat hippocampus sections are used for anti-NMDAR, anti-LGi1, anti-Caspr2, anti-AMPAR, and anti-GABABR antibodies. Different companies provide commercially available immunoblots for the most common onconeuronal antibodies (ANNA-1, PCA-1, ANNA-2, anti-CRMP5, anti-Ma proteins, anti-amphiphysin, anti-SOX1, and anti-GAD) or plasmids or already transfected cells for the most common surface proteins (anti-NMDAR, anti-LGI1, anti-Caspr2, anti-AMPAR, anti-GABABR). Specialized laboratories provide in-house cell-based assays to detect additional antibodies such as anti-glycine receptor or anti-mGluR1/5. Immunoprecipitation is used to detect anti-VGCC antibodies (66). Paraneoplastic neurologic disorders are rare diseases, and diagnosis should be performed in experienced centers to guarantee high diagnostic quality. For the three most common paraneoplastic antibodies (ANNA-1, PCA-1, ANNA-2) the sensitivity in established paraneoplastic neurologic disorders was 28.9% for tissue-based assay, 26.3% for western blot, and 31.5% for line blot with a specificity of 95.2% for tissue-based assay, 97.1% for western blot, and 98.1% for line blot (38 established paraneoplastic neurologic disorder patients; 44 other neurologic disease patients) (155). The combined use of the three methods brought sensitivity to 39.4%. For the diagnosis of suspected paraneoplastic neurologic disorder in cancer patients, simultaneously testing ANNA-1, PCA-1, ANNA-2, anti-amphiphysin, anti-CRMP5, and anti-Ma-2 antibodies reported a sensitivity of 11.9% for immunoprecipitation, 7% for tissue-based assay, and 6.3% for immunoblot with a specificity of 98.3% for immunoprecipitation and 99.8% for both tissue-based assay and immunoblot (555 cancer patients with suspected paraneoplastic neurologic disorder; 300 healthy controls) (153).

Pathogenic significance. Most paraneoplastic neurologic disorders are presumably immune-mediated, although the relative contribution of T and B cells remains unclear. The autoimmune hypothesis in paraneoplastic neurologic disorders is supported by the inflammatory CSF findings and T-cell infiltration in the affected part of the nervous system revealed by pathologic examination (68). Tumor expression of the same onconeuronal antigens normally expressed in the neurons triggers an immune response (36). The tumor antigen, which is identical to the neural antigen, is identified as foreign by the immune system, and an attack is mounted against the tumor. On the other hand, namely in paraneoplastic neurologic disorders involving the CNS, the antineuronal antibodies might be an epiphenomenon marking autoimmunity without a direct pathogenic effect (36). Some autoantibodies from patients with paraneoplastic disorders do not cause injury to the cultured neurons (156).

When the target antigens are cell surface or synaptic antigen receptors, the pathogenic role of the autoantibodies is reflected by the response to immunotherapy and by the correlation between antibody titers and outcome. In some cases, experimental evidence confirmed the autoantibody pathogenicity, such as for Lambert-Eaton myasthenic syndrome patients' anti-VGCC IgG that can reproduce the electrophysiologic disturbance in animals (170). In vivo infusion of anti-NMDAR encephalitis patients' CSF or IgG into rodent hippocampus caused cross-linking and internalization of the target receptors and was accompanied by a decrease in synaptic NMDAR-mediated currents (69). Anti-AMPAR antibodies also cause receptor cross-linking and internalization and result in a reversible decrease in AMPAR clusters at synapses (83). Antibodies to the GABABR inhibit receptor function but do not cause receptor internalization (86). Antibodies to nicotinic AChR α3 subunit induce internalization of these receptors and thereby impair synaptic transmission (80).

The role of antibodies directed against intracellular antigens remains questionable in the pathogenesis of paraneoplastic disorders. It has been argued that intracellular antigens can be expressed on cell surface and also that certain antibodies can get access to cytoplasm. The lack of response to plasma exchange in most cases of paraneoplastic disorders associated with antibodies directed against intracellular antigens can be explained by the slow removal of antibodies from the CNS compared to the rapid removal of circulating antibodies and by permanent damage caused by antibodies.

T cells can recognize intracellular antigens presented to them as MHC-peptide complexes and thereby kill neurons. Antigen-specific cytotoxic T cells in paraneoplastic neurologic disorders were clearly documented in patients with acute paraneoplastic cerebellar degeneration and PCA-1 or ANNA-1. Activated T cells in the blood could lyse target cells presenting PCA-1 antigen in vitro (32). For paraneoplastic neurologic disorders of the CNS in which most of the known target antigens are intracellular proteins, animal models have not provided evidence that antibodies have a role in the pathogenesis.

Morvan syndrome

Clinical manifestations. This is a very rare disease characterized by neuromyotonia associated with sleep disturbances, memory loss, confusion, hallucinations, dysautonomia, and neuropathic pain (71).

Type of antibodies. Almost all patients are men, mostly with antibodies against proteins that are complexed with the VGKC of presynaptic nerve terminals (71). They are directed against Caspr2, LGi1, or, commonly, both. Anti-LGi1 antibodies can be found in limbic encephalitis, too. Comparing patients with anti-VGKC complex antibodies and Morvan syndrome or limbic encephalitis (29 Morvan syndrome patients; 64 limbic encephalitis patients), the positivity for anti-VGKC complex antibodies has a 42% sensitivity and a 100% specificity for the association with a tumor (mostly thymoma). Comparing Morvan syndrome patients positive for anti-Caspr2, anti-LGL1, or both (n=24), the positivity for anti-Caspr2 or for both in a Morvan syndrome patient has a 52% sensitivity and a 100% specificity for the association with a tumor. Anti-VGCK complex antibodies are detected using a cell-based assay radioimmunoprecipitation assay, and anti-Caspr2 and anti-LGi1 antibodies with a tissue-based assay (70; 71).

Pathogenic significance. The pathogenic significance of anti-VGKC complex antibodies in Morvan syndrome is not known.

Clinical applications

Autoantibodies are routinely tested in many neuropathies, neuromuscular junction disorders, and myopathies and can be useful in the diagnosis and management.

In CNS disorders, the pathogenic role of autoantibodies is still emerging. Neuromyelitis optica has emerged as a specific pathophysiological entity due to the specificity of aquaporin-4 antibodies. Autoantibodies to CNS antigens can help detect a specific tumor associated with a paraneoplastic disorder and are routinely tested in suspected cases. ANNA-1, anti-VGCC, and anti-CRMP5 antibodies are often associated with lung cancer, PCA-1 and ANNA-2 with gynecologic cancers, and anti-Ma2 with testicular cancer in men and gynecologic cancer in women. The early detection and treatment of the underlying tumor may lead to clinical improvement in some cases. Antibodies to surface or synaptic CNS antigens may be associated with treatable syndromes, which are, at times, paraneoplastic. Disorders associated with anti-NMDAR, anti-GABABR, anti-AMPAR, anti-mGluR, anti-LGi1, anti-Caspr2, and anti-VGCC antibodies may respond to plasma exchange and intravenous immunoglobulin. Antibodies to cell surface or synaptic proteins are directed against conformational epitopes, and the reactivity is usually lost when the antigen is denatured so that these antibodies cannot be detected by standard immunoblot or ELISA. Rather, the detection of these antibodies requires an immunohistochemistry protocol adapted to cell surface antigens, such as the use of tissue-based assay or cell-based assay (136).

The search for more autoantibodies and the effort to better define their contribution to the disease process are ongoing. In the future, new paradigms for their detection and treatment of antibody-mediated diseases may be established.