Paraneoplastic Calmodulin Kinase-Like Vesicle-Associated Protein (CAMKV) Autoimmune Encephalitis.

OBJECTIVES: To report an autoimmune paraneoplastic encephalitis characterized by immunoglobulin G (IgG) antibody targeting synaptic protein calmodulin kinase-like vesicle-associated (CAMKV). METHODS: Serum and cerebrospinal fluid (CSF) samples harboring unclassified antibodies on murine brain-based indirect immunofluorescence assay (IFA) were screened by human protein microarray. In 5 patients with identical cerebral IFA staining, CAMKV was identified as top-ranking candidate antigen. Western blots, confocal microscopy, immune-absorption, and mass spectrometry were performed to substantiate CAMKV specificity. Recombinant CAMKV-specific assays (cell-based [fixed and live] and Western blot) provided additional confirmation. RESULTS: Of 5 CAMKV-IgG positive patients, 3 were women (median symptom-onset age was 59 years; range, 53-74). Encephalitis-onset was subacute (4) or acute (1) and manifested with: altered mental status (all), seizures (4), hyperkinetic movements (4), psychiatric features (3), memory loss (2), and insomnia (2). Paraclinical testing revealed CSF lymphocytic pleocytosis (all 4 tested), electrographic seizures (3 of 4 tested), and striking MRI abnormalities in all (mesial temporal lobe T2 hyperintensities [all patients], caudate head T2 hyperintensities [3], and cortical diffusion weighted hyperintensities [2]). None had post-gadolinium enhancement. Cancers were uterine adenocarcinoma (3 patients: poorly differentiated or neuroendocrine-differentiated in 2, both demonstrated CAMKV immunoreactivity), bladder urothelial carcinoma (1), and non-Hodgkin lymphoma (1). Two patients developed encephalitis following immune checkpoint inhibitor cancer therapy (atezolizumab [1], pembrolizumab [1]). All treated patients (4) demonstrated an initial response to immunotherapy (corticosteroids [4], IVIG [2]), though 3 died from cancer. INTERPRETATION: CAMKV-IgG is a biomarker of immunotherapy-responsive paraneoplastic encephalitis with temporal and extratemporal features and uterine cancer as a prominent oncologic association. ANN NEUROL 2024.

Evolution of methods to detect paraneoplastic antibodies.

An adaptive immune response in less than 1% of people who develop cancer produces antibodies against neuronal proteins. These antibodies can be associated with paraneoplastic syndromes, and their accurate detection should instigate a search for a specific cancer. Over the years, multiple systems, from indirect immunofluorescence to live cell-based assays, have been developed to identify these antibodies. As the specific antigens were identified, high throughput, multi-antigen substrates such as line blots and ELISAs were developed for clinical laboratories. However, the evolution of assays required to identify antibodies to membrane targets has shone a light on the importance of antigen conformation for antibody detection. This chapter discusses the early antibody assays used to detect antibodies to nuclear and cytosolic targets and how new approaches are required to detect antibodies to membrane targets. The chapter presents recent data that support international recommendations against the sole use of line blots for antibody detection and highlights a new antigen-specific approach that appears promising for the detection of submembrane targets.

Paraneoplastic neuropathies and peripheral nerve hyperexcitability disorders.

Peripheral neuropathy is a common referral for patients to the neurologic clinics. Paraneoplastic neuropathies account for a small but high morbidity and mortality subgroup. Symptoms include weakness, sensory loss, sweating irregularity, blood pressure instability, severe constipation, and neuropathic pain. Neuropathy is the first presenting symptom of malignancy among many patients. The molecular and cellular oncogenic immune targets reside within cell bodies, axons, cytoplasms, or surface membranes of neural tissues. A more favorable immune treatment outcome occurs in those where the targets reside on the cell surface. Patients with antibodies binding cell surface antigens commonly have neural hyperexcitability with pain, cramps, fasciculations, and hyperhidrotic attacks (CASPR2, LGI1, and others). The antigenic targets are also commonly expressed in the central nervous system, with presenting symptoms being myelopathy, encephalopathy, and seizures with neuropathy, often masked. Pain and autonomic components typically relate to small nerve fiber involvement (nociceptive, adrenergic, enteric, and sudomotor), sometimes without nerve fiber loss but rather hyperexcitability. The specific antibodies discovered help direct cancer investigations. Among the primary axonal paraneoplastic neuropathies, pathognomonic clinical features do not exist, and testing for multiple antibodies simultaneously provides the best sensitivity in testing (AGNA1-SOX1; amphiphysin; ANNA-1-HU; ANNA-3-DACH1; CASPR2; CRMP5; LGI1; PCA2-MAP1B, and others). Performing confirmatory antibody testing using adjunct methods improves specificity. Antibody-mediated demyelinating paraneoplastic neuropathies are limited to MAG-IgM (IgM-MGUS, Waldenström's, and myeloma), with the others associated with cytokine elevations (VEGF, IL6) caused by osteosclerotic myeloma, plasmacytoma (POEMS), and rarely angiofollicular lymphoma (Castleman's). Paraneoplastic disorders have clinical overlap with other idiopathic antibody disorders, including IgG4 demyelinating nodopathies (NF155 and Contactin-1). This review summarizes the paraneoplastic neuropathies, including those with peripheral nerve hyperexcitability.

Neurological autoimmunity in patients with non-pulmonary neuroendocrine neoplasms: clinical manifestations and neural autoantibody profiles.

BACKGROUND AND PURPOSE: Paraneoplastic neurological autoimmunity is well described with small-cell lung cancer, but information is limited for other neuroendocrine neoplasms (NENs). METHODS: Adult patients with histopathologically confirmed non-pulmonary NENs, neurological autoimmunity within 5 years of NEN diagnosis, and neural antibody testing performed at the Mayo Clinic Neuroimmunology Laboratory (January 2008 to March 2023) were retrospectively identified. Control sera were available from patients with NENs without neurological autoimmunity (116). RESULTS: Thirty-four patients were identified (median age 68 years, range 31-87). The most common primary tumor sites were pancreas (nine), skin (Merkel cell, eight), small bowel/duodenum (seven), and unknown (seven). Five patients received immune checkpoint inhibitor (ICI) therapy before symptom onset; symptoms preceded cancer diagnosis in 62.1% of non-ICI-treated patients. The most frequent neurological phenotypes (non-ICI-treated) were movement disorders (12; cerebellar ataxia in 10), dysautonomia (six), peripheral neuropathy (eight), encephalitis (four), and neuromuscular junction disorders (four). Neural antibodies were detected in 55.9% of patients studied (most common specificities: P/Q-type voltage-gated calcium channel [seven], muscle-type acetylcholine receptor [three], anti-neuronal nuclear antibody type 1 [three], and neuronal intermediate filaments [two]), but in only 6.9% of controls. Amongst patients receiving cancer or immunosuppressive therapy, 51.6% had partial or complete recovery. Outcomes were unfavorable in 48.3% (non-ICI-treated) and neural autoantibody positivity was associated with poor neurological outcome. DISCUSSION: Neurological autoimmunity associated with non-pulmonary NENs is often multifocal and can be treatment responsive, underscoring the importance of rapid recognition and early treatment.

Nerve transcriptomes in autoimmune and genetic demyelinating neuropathies: Pathogenic pathway assessment of nerve demyelination.

The pathogenesis of autoimmune demyelinating neuropathies is poorly understood compared to inherited demyelinating forms. We performed whole transcriptome (RNA-Seq) using nerve biopsy tissues of patients with different autoimmune and inherited demyelinating neuropathies (CIDP n = 10, POEMS n = 18, DADS n = 3, CMT1 n = 3) versus healthy controls (n = 6). A limited number of differentially expressed genes compared to healthy controls were identified (POEMS = 125, DADS = 15, CMT = 14, CIDP = 5). Divergent pathogenic pathways including inflammatory, demyelinating and neurite regeneration such as with the triggering receptor expressed on myeloid cells (TREM1) part of the immunoglobulin superfamily and RhoGD1 are found. Shared and discordant pathogenic injury are discovered between autoimmune and inherited forms.

CDR2 and CDR2L line blot performance in PCA-1/anti-Yo paraneoplastic autoimmunity.

Background: Purkinje cytoplasmic autoantibody type 1 (PCA-1)/anti-Yo autoimmunity is a common high-risk paraneoplastic neurological disorder, traditionally attributed antigenically to cerebellar degeneration-related protein 2 (CDR2), predominantly affecting women with gynecologic or breast adenocarcinoma. Single-modality CDR2 testing may produce false-positive results. We assessed the performance characteristics of the more recently purported major PCA-1/Yo antigen, CDR2-like (CDR2L), side by side with CDR2, in a line blot format. Methods: CDR2 and CDR2L were tested in six specimen groups (serum and cerebrospinal fluid (CSF)). Group 1, PCA-1/Yo mouse brain indirect immunofluorescence assay (IFA) positives; Group 2, PCA-1/Yo IFA mimics; Group 3, suspected CDR2 line blot false positives; Group 4, consecutive patient samples tested for neural antibodies over 1 year; Group 5, healthy subject serums; and Group 6, polyclonal (non-specific) immunoglobulin G (IgG)-positive serums. Results: Group 1: Of 64 samples tested, all but two were CDR2 positive (both CSF samples) and all were CDR2L positive. In individual patients, CDR2L values were always higher than CDR2. The two "CDR2L-only" positives were CSF samples with low titer PCA-1/Yo by IFA with serum negativity but with typical clinical phenotype. Group 2: All 51 PCA-1/Yo mimics were CDR2/CDR2L negative. Group 3: Nine samples [six of 1289 (0.47%) serums and three of 700 CSF samples (0.43%) were PCA-1/Yo IFA negative/CDR2 positive; two of the six available (serums from the same patient) were also CDR2L positive; the other four CDR2L negative had low CDR2 values (17-22). Group 4: Twenty-two patients had unexpected CDR2 or CDR2L positivity; none had tissue IFA positivity. Eleven of the 2,132 serum (0.5%) and three of the 677 CSF (0.4%) samples were CDR2 positive; median value was 19 (range, 11-48). Seven of the 2,132 serum (0.3%) and three of the 677 CSF (0.4%) samples were CDR2L positive; median value was 18 (range, 11-96). Group 5: All 151 healthy serum samples were negative. Group 6: One of the 46 polyclonal serum samples was CDR2L positive. Optimum overall performance was accomplished by requiring both CDR2 and CDR2L positivity in serum (sensitivity, 100%; and specificity, 99.9%) and positivity for CDR2L in CSF (sensitivity, 100%; and specificity, 99.6%). Conclusion: CDR2L provides additional PCA-1/anti-Yo sensitivity in CSF, and dual positivity with CDR2 provides additional specificity assurance in serum. Combining antigen-specific and tissue-based assays optimizes PCA-1/anti-Yo testing.

Identification of SKOR2 IgG as a novel biomarker of paraneoplastic neurologic syndrome.

Introduction: The development of new autoantigen discovery techniques, like programmable phage immunoprecipitation sequencing (PhIP-Seq), has accelerated the discovery of neural-specific autoantibodies. Herein, we report the identification of a novel biomarker for paraneoplastic neurologic syndrome (PNS), Sloan-Kettering-Virus-Family-Transcriptional-Corepressor-2 (SKOR2)-IgG, utilizing PhIP-Seq. We have also performed a thorough clinical validation using normal, healthy, and disease/cancer control samples. Methods: Stored samples with unclassified staining at the junction of the Purkinje cell and the granule cell layers were analyzed by PhIP-Seq for putative autoantigen identification. The autoantigen was confirmed by recombinant antigen-expressing cell-based assay (CBA), Western blotting, and tissue immunofluorescence assay colocalization. Results: PhIP-Seq data revealed SKOR2 as the candidate autoantigen. The target antigen was confirmed by a recombinant SKOR-2-expressing, and cell lysate Western blot. Furthermore, IgG from both patient samples colocalized with a commercial SKOR2-specific IgG on cryosections of the mouse brain. Both SKOR2 IgG-positive patients had central nervous system involvement, one presenting with encephalitis and seizures (Patient 1) and the other with cognitive dysfunction, spastic ataxia, dysarthria, dysphagia, and pseudobulbar affect (Patient 2). They had a refractory progressive course and were diagnosed with adenocarcinoma (Patient 1: lung, Patient 2: gallbladder). Sera from adenocarcinoma patients without PNS (n=30) tested for SKOR2-IgG were negative. Discussion: SKOR2 IgG represents a novel biomarker for PNS associated with adenocarcinoma. Identification of additional SKOR2 IgG-positive cases will help categorize the associated neurological phenotype and the risk of underlying malignancy.

Serological fingerprints link antiviral activity of therapeutic antibodies to affinity and concentration.

The effectiveness of therapeutic monoclonal antibodies (mAbs) against variants of the SARS-CoV-2 virus is highly variable. As target recognition of mAbs relies on tight binding affinity, we assessed the affinities of five therapeutic mAbs to the receptor binding domain (RBD) of wild type (A), Delta (B.1.617.2), and Omicron BA.1 SARS-CoV-2 (B.1.1.529.1) spike using microfluidic diffusional sizing (MDS). Four therapeutic mAbs showed strongly reduced affinity to Omicron BA.1 RBD, whereas one (sotrovimab) was less impacted. These affinity reductions correlate with reduced antiviral activities suggesting that affinity could serve as a rapid indicator for activity before time-consuming virus neutralization assays are performed. We also compared the same mAbs to serological fingerprints (affinity and concentration) obtained by MDS of antibodies in sera of 65 convalescent individuals. The affinities of the therapeutic mAbs to wild type and Delta RBD were similar to the serum antibody response, indicating high antiviral activities. For Omicron BA.1 RBD, only sotrovimab retained affinities within the range of the serum antibody response, in agreement with high antiviral activity. These results suggest that serological fingerprints provide a route to evaluating affinity and antiviral activity of mAb drugs and could guide the development of new therapeutics.

Identification of Caveolae-Associated Protein 4 Autoantibodies as a Biomarker of Immune-Mediated Rippling Muscle Disease in Adults.

Importance: Immune-mediated rippling muscle disease (iRMD) is a rare myopathy characterized by wavelike muscle contractions (rippling) and percussion- or stretch-induced muscle mounding. A serological biomarker of this disease is lacking. Objective: To describe a novel autoantibody biomarker of iRMD and report associated clinicopathological characteristics. Design, Setting, and Participants: This retrospective cohort study evaluated archived sera from 10 adult patients at tertiary care centers at the Mayo Clinic, Rochester, Minnesota, and Brigham & Women's Hospital, Boston, Massachusetts, who were diagnosed with iRMD by neuromuscular specialists in 2000 and 2021, based on the presence of electrically silent percussion- or stretch-induced muscle rippling and percussion-induced rapid muscle contraction with or without muscle mounding and an autoimmune basis. Sera were evaluated for a common biomarker using phage immunoprecipitation sequencing. Myopathology consistent with iRMD was documented in most patients. The median (range) follow-up was 18 (1-30) months. Exposures: Diagnosis of iRMD. Main Outcomes and Measures: Detection of a common autoantibody in serum of patients sharing similar clinical and myopathological features. Results: Seven male individuals and 3 female individuals with iRMD were identified (median [range] age at onset, 60 [18-76] years). An IgG autoantibody specific for caveolae-associated protein 4 (cavin-4) was identified in serum of patients with iRMD using human proteome phage immunoprecipitation sequencing. Immunoassays using recombinant cavin-4 confirmed cavin-4 IgG seropositivity in 8 of 10 patients with iRMD. Results for healthy and disease-control individuals (n = 241, including myasthenia gravis and immune-mediated myopathies) were cavin-4 IgG seronegative. Six of the 8 individuals with cavin-4 IgG were male, and the median (range) age was 60 (18-76) years. Initial symptoms included rippling of lower limb muscles in 5 of 8 individuals or all limb muscles in 2 of 8 sparing bulbar muscles, fatigue in 9 of 10, mild proximal weakness in 3 of 8, and isolated myalgia in 1 of 8, followed by development of diffuse rippling. All patients had percussion-induced muscle rippling and half had percussion- or stretch-induced muscle mounding. Four of the 10 patients had proximal weakness. Plasma creatine kinase was elevated in all but 1 patient. Six of the 10 patients underwent malignancy screening; cancer was detected prospectively in only 1. Muscle biopsy was performed in 7 of the 8 patients with cavin-4 IgG; 6 of 6 specimens analyzed immunohistochemically revealed a mosaic pattern of sarcolemmal cavin-4 immunoreactivity. Three of 6 patients whose results were seropositive and who received immunotherapy had complete resolution of symptoms, 1 had mild improvement, and 2 had no change. Conclusions and Relevance: The findings indicate that cavin-4 IgG may be the first specific serological autoantibody biomarker identified in iRMD. Depletion of cavin-4 expression in muscle biopsies of patients with iRMD suggests the potential role of this autoantigen in disease pathogenesis.

LRP4-IgG service line testing in seronegative myasthenia gravis and controls.

BACKGROUND: LRP4 is a post-synaptic membrane protein that promotes acetylcholine (AChR) clustering on the crest of post-synaptic neuromuscular folds. Autoantibodies against LRP4 are suggested to account for myasthenia gravis (MG) patients negative for antibodies to AChR. OBJECTIVES: To report a clinical experience with service-line LRP4-IgG cell-based testing in electrodiagnostically confirmed MG patients and controls. METHODS: We identified all Mayo patients undergoing MG evaluations with send out LRP4-IgG antibody testing by cell-based assay, having clinical-electrodiagnostic (EDX) testing. To be included, muscle acetylcholine receptor binding (AChR-Bi) and muscle-specific tyrosine kinase (MuSK) antibodies had to be absent prior to LRP4-IgG testing. Follow-up AChR-Bi antibody testing was reviewed. Also tested for LRP4-IgGs were 119 healthy subjects. RESULTS: Identified were 25 generalized MG, 24 ocular MG, and 55 patients initially considered to have MG prior to negative EDX testing. No seronegative patients with EDX confirmed MG had LRP4-IgG positivity but five non-MG patients did: Guillain-Barre syndrome with fatigue (N = 1); multiple cranial neuropathies (N = 1); functional neurologic disorders (N = 3). Of healthy subjects, 4% (5/119) were LRP4-IgG positive (N = 5) or had a borderline result (N = 1). Of MG patients with repeat AChR-Bi testing, 40% (10/25) seroconverted (5 with ocular MG and 5 with generalized MG) (median AChR IgG value: 0.34 nmol/L, range 0.2-20.9 nmol/L, median followup 26 months, range 2-72 months). CONCLUSION: Clinical review of LRP4-IgG commercial cell-based testing suggests lack of diagnostic utility in seronegative EDX-confirmed MG. The clinical utility of LRP4-IgG testing is not substantiated in service line testing. In contrast, repeat testing for AChR-Bi antibodies is shown clinically useful.

Cancer and immune-mediated necrotizing myopathy: a longitudinal referral case-controlled outcomes evaluation.

OBJECTIVES: To investigate immune-mediated necrotizing myopathy (IMNM) association with cancer and its clinical implications. METHODS: IMNM cases were identified 1 January 2000 to 31 December 2020 matching sex and age controls (4:1). RESULTS: A total of 152 patients with IMNM were identified and among serologically tested, 60% (83/140) were HMGCR-IgG+, 14% (20/140) were SRP-IgG+ and 26% (37/140) were seronegative. Cancer rates were not significantly different between serological subgroups; 18.1% (15/83) HMGCR-IgG+, 25% (5/20) SRP-IgG+ and 30% (11/37) seronegative (P = 0.34). Cancer screening was performed within 12 months from IMNM diagnosis in 88% (134/152) (whole-body CT plus FDG-PET CT in 53, CT alone in 72 and FDG-PET alone in 9). FDG-PET/CT was positive in 73% (25/34) of cancers. Increasing age was the only risk associated with cancer (P = 0.02). The odds of developing cancer at ±3 or ±5 years from IMNM diagnosis was not higher than controls (OR = 0.49; CI: 0.325-0.76). Lifetime IMNM diagnosis of cancer was less compared with controls (OR = 0.5 CI: 0.33-0.78, P = 0.002). Most patients responded to treatment (137/147, P < 0.001). Death and treatment response did not significantly differ between cancer [23% (8/34); 88% (29/33)] and non-cancer patients [19% (23/118); 92% (108/118)]. In total, 13% (20/152) of patients died during follow-up compared with 14% (41/290) of medicine and 16% (46/290) of neurology controls (P = 0.8). Seropositives had greater life expectancy than seronegatives (P = 0.01). CONCLUSIONS: Greater cancer risk is not observed in IMNM vs controls. Cancer screening in IMNM should be individualized based on age-personal and family history, including consideration of FDG-PET/CT. Immune-treatment response did not differ with cancer.

CASPR2-IgG-associated autoimmune seizures.

OBJECTIVE: This study was undertaken to report clinical presentations and outcomes of CASPR2-IgG-associated seizures. METHODS: Mayo Clinic Neuroimmunology database was queried to identify CASPR2-IgG-seropositive (CASPR2-IgG+) patients evaluated at our institution (2009-2019). RESULTS: Of the 53 CASPR2-IgG+ patients (titer ≥ 1:10), 20 had seizures (38%). All seizure patients were male, with median onset age of 68 years. Eighteen (90%) had seizures at initial presentation. One patient was found to have malignancy (colon adenocarcinoma). Two patients had coexisting LGI1-IgG. Twelve patients had archived sera, which on titration had CASPR2-IgG titers ≥ 1:100. Fifteen patients (75%) met criteria for autoimmune encephalitis. Patients most commonly presented with focal onset, nonmotor seizures with impaired awareness (n = 14, 70%). Eleven patients also had focal motor and/or sensory seizures as one of the seizure semiologies. The majority of patients (n = 11, 55%) developed generalized tonic-clonic seizures during their disease course. Seizure clusters occurred in 12 patients. In addition to seizures, patients developed cognitive disturbance (n = 16, 80%), episodic emotional lability (n = 13, 65%), paroxysmal dizziness (n = 9, 45%), episodic ataxia (n = 6, 30%), and chronic ataxia (n = 9, 45%). Only three patients (15%) had coexisting peripheral nervous system involvement. Frontotemporal or temporal ictal and/or interictal electroencephalographic abnormalities were present among nine patients, and three had multifocal epileptiform abnormalities. Eight patients (40%) had medial temporal T2/fluid-attenuated inversion recovery hyperintensity on brain magnetic resonance imaging. Elevated cerebrospinal fluid protein and/or lymphocytic pleocytosis was present in most cases (13/14, 93%). Thirteen patients reached seizure freedom following initiation of antiseizure medication (ASM; n = 4) or a combination of immunotherapy and ASM (n = 9). Median duration of follow-up was 25 months (range = 2-136 months). SIGNIFICANCE: CASPR2-IgG evaluation should be considered among older male patients with new onset focal seizures and impaired awareness often occurring in clusters with/without features of encephalitis. Coexisting neurological manifestations, including episodic emotional lability, ataxia, and paroxysmal dizziness, also aid in the diagnosis.

Characterisation of TRIM46 autoantibody-associated paraneoplastic neurological syndrome.

OBJECTIVES: To report the expanded neurological presentations and oncological associations of tripartite motif-containing protein 46 (TRIM46)-IgG seropositive patients. METHODS: Archived sera/cerebrospinal fluid (CSF) were evaluated by tissue-based immunofluorescence assay to identify patients with identical axon initial segment (AIS)-specific staining pattern. Phage immunoprecipitation sequencing (PhIP-Seq) was used to identify the putative autoantigen. RESULTS: IgG in serum (17) and/or CSF (16) from 25 patients yielded unique AIS-specific staining on murine central nervous system (CNS) tissue. An autoantibody specific for TRIM46 was identified by PhIP-Seq, and autoantigen specificity was confirmed by transfected COS7 cell-based assay. Clinical information was available for 22 TRIM46-IgG seropositive patients. Fifteen were female (68%). Median age was 67 years (range 25-87). Fifteen (68%) patients presented with subacute cerebellar syndrome (six isolated; nine with CNS accompaniments: encephalopathy (three), brainstem signs (two), myelopathy (two), parkinsonism (one)). Other phenotypes included limbic encephalitis (three), encephalopathy with/without seizures (two), myelopathy (two). Eighteen (82%) had cancer: neuroendocrine carcinomas (9; pancreatic (3), small-cell lung (4), oesophagus (1), endometrium (1)), adenocarcinomas (6; lung (2), ovarian (2), endometrial (1), breast (1)), sarcoma (2) and gastrointestinal tumour (1). Neurological symptoms in three followed immune checkpoint inhibitor (ICI) administration. CONCLUSIONS: This study supports TRIM46-IgG being a biomarker of paraneoplastic CNS disorders and expands the neurological phenotypes, oncological and ICI-related adverse event associations.

Autoimmune/Paraneoplastic Encephalitis Antibody Biomarkers: Frequency, Age, and Sex Associations.

OBJECTIVE: To determine the frequency of detection and the age and sex associations of autoimmune/paraneoplastic encephalitis antibody biomarkers (AE-Abs). METHODS: There were 42,032 patients tested in the Mayo Clinic Neuroimmunology Laboratory between January 2018 and December 2019 for AE-Abs in serum or cerebrospinal fluid (CSF), including NMDA-R-IgG, AMPA-R-IgG, GABAB-R-IgG, CASPR2-IgG, LGI1-IgG, GAD65-IgG, CRMP5-IgG, amphiphysin-IgG, PCA1/2/Tr-IgGs, ANNA1/2/3-IgGs, GFAP-IgG, mGluR1-IgG, DPPX-IgG, and MOG-IgG1. Results were examined to determine frequency of antibody positivity. Age and sex associations were examined by multivariable logistic regression. RESULTS: Adult serum analysis (22,472 patients; 56% female) revealed that 814 (3.6%) were positive: NMDA-R-IgG (24.6%) > GAD65-IgG (21.5%) > LGI1-IgG (20.5%) > others. Of children (5649; 50% female), 251 (4.4%) were positive: NMDA-R-IgG (53.1%) > MOG-IgG1 (32%) > GAD65-IgG (7.1%) > others. Adult CSF analysis (18,745 patients; 54% female) revealed that 796 (4.2%) were positive: NMDA-R-IgG (39.7%) > GAD65-IgG (28.5%) > LGI1-IgG (11.4%) > others. Of children (5136; 50% female), 282 (5.5%) were positive: NMDA-R-IgG (88.1%) > GAD65-IgG (8.7%) > others. Age younger than 20 years was associated with NMDA-R-IgG and MOG-IgG1 (odds ratio [OR], 8.11 and 7.84, respectively; P<.001). Age older than 65 years was associated with GABAB-R-IgG, LGI1-IgG, CASPR2-IgG, and ANNA1-IgG (OR, 7.33, 14.98, 3.67, and 14.53; P<.001). Women accounted for 60% of NMDA-R-IgG (CSF) and 78% of GAD65-IgG (CSF and serum) cohorts (OR, 1.32 [P=.002] and 2.23 [P<.001], respectively). Men accounted for 62% of the LGI1-IgG cohort (OR, 1.87; P<.001). Age and sex interacted for NMDA-R-IgG, particularly in female patients younger than 20 years (OR, 7.72; P<.001). CONCLUSION: The most frequent AE-Abs detected were NMDA-R-IgG, GAD65-IgG, LGI1-IgG, and MOG-IgG1. Age and sex associations may suggest paraneoplastic, or aging influences on neurologic autoimmunity.

Immune-mediated necrotizing myopathy: Unusual presentations of a treatable disease.

INTRODUCTION/AIMS: Immune-mediated necrotizing myopathy (IMNM) is an immune-mediated myopathy typically presenting with progressive subacute weakness and characteristic, but nonspecific, myopathological findings. Atypical cases however can mimic other inherited or acquired myopathies, depriving patients of treatment. We describe a cohort of such patients. METHODS: We retrospectively identified IMNM patients who either previously carried a diagnosis of an inherited myopathy established on clinicopathological grounds or whose muscle biopsies displayed atypical features suggestive of a different myopathy. RESULTS: Among 131 IMNM patients, seven previously unreported patients (5%) met one of the above criteria. Three patients were diagnosed with limb-girdle muscular dystrophy on the basis of a chronic progressive course of weakness and family history of myopathy or cardiomyopathy. The other four patients displayed atypical histological features (two prominent mitochondrial abnormalities, one myofibrillar pathology, and one granulomatous inflammation). Immunostaining of biopsies from 12 additional IMNM patients did not identify myofibrillar pathology. The patient with granulomatous inflammation was known to have pulmonary sarcoidosis. Genetic testing for inherited myopathies was unrevealing. Antibodies against 3-hydroxy-3-methylglutaryl-CoA reductase or signal recognition particle were identified in 5 and 1 patients, respectively. Four patients presented with slowly progressive weakness over 3-13 y, while weakness was subacute over ≤6 mo in three patients. All patients responded to immunomodulatory therapy. DISCUSSION: Atypical clinical and histological features can occur in IMNM patients, causing delays in diagnosis and treatment. Clinicians should, therefore, consider IMNM in the differential diagnosis of unexplained proximal myopathies in spite of atypical clinical and myopathological findings.

Pure Motor Onset and IgM-Gammopathy Occurrence in Multifocal Acquired Demyelinating Sensory and Motor Neuropathy.

OBJECTIVES: To longitudinally investigate patients with multifocal acquired demyelinating sensory and motor (MADSAM) neuropathy, quantifying timing and location of sensory involvements in motor-onset patients, along with clinico-histopathological and electrophysiological findings to ascertain differences in patients with and without monoclonal gammopathy of uncertain significance (MGUS). METHODS: Patients with MADSAM neuropathy seen at Mayo Clinic and tested for monoclonal gammopathy and ganglioside antibodies, were retrospectively reviewed (January 1st, 2007-December 31st, 2018). RESULTS: Of 76 patients with MADSAM, 53% had pure motor, 16% pure sensory, 30% sensorimotor and 1% cranial nerve onsets. Motor-onset patients were initially diagnosed as multifocal motor neuropathy (MMN). MGUS occurred in 25% (89% IgM subtype), associating with ganglioside autoantibodies (p<0.001) and higher IgM titers (p<0.04). Median time to sensory involvements (confirmed by electrophysiology) in motor-onset patients was 18 months (range: 6-180). Compared to initial motor nerve involvements, subsequent sensory findings were within the same territory 35% (14/40), outside 20% (8/40), or both 45% (18/40). Brachial and lumbosacral plexus MRI was abnormal in 87% (34/39) and 84% (21/25), respectively, identifying hypertrophy and increased T2 signal predominantly in brachial plexus trunks (64%), divisions (69%), and cords (69%), and intrapelvic sciatic (64%) and femoral (44%) nerves. Proximal fascicular nerve biopsies (n=9) more frequently demonstrated onion-bulb pathology (p=0.001) and endoneurial inflammation (p=0.01) than distal biopsies (n=17). MRI and biopsy findings were similar amongst patient subgroups. Initial Inflammatory Neuropathy Cause and Treatment (INCAT) disability scores were higher in patients with MGUS relative to without (p=0.02). Long-term treatment responsiveness by INCAT score reduction ≥1 or motor Neuropathy Impairment Score (mNIS) >8 point reduction occurred in 75% (49/65) irrespective of MGUS or motor-onsets. Most required ongoing immunotherapy (86%). Patients with MGUS more commonly required dual-agent immunotherapy for stability (p=0.02). DISCUSSION: Pure motor-onsets are the most common MADSAM presentation. Long-term follow-up, repeat electrophysiology and nerve pathology help distinguish motor-onset MADSAM from MMN. Better long-term immunotherapy responsiveness occurs in motor-onset MADSAM compared to MMN reports. Patients having MGUS commonly require dual immunotherapy. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that most clinical, electrophysiological, and histopathological findings were similar between patients with MADSAM with and without monoclonal gammopathy of unknown significance.

Positive Predictive Value of Myelin Oligodendrocyte Glycoprotein Autoantibody Testing.

Importance: Myelin oligodendrocyte glycoprotein-IgG1-associated disorder (MOGAD) is a distinct central nervous system-demyelinating disease. Positive results on MOG-IgG1 testing by live cell-based assays can confirm a MOGAD diagnosis, but false-positive results may occur. Objective: To determine the positive predictive value (PPV) of MOG-IgG1 testing in a tertiary referral center. Design, Setting, and Participants: This diagnostic study was conducted over 2 years, from January 1, 2018, through December 31, 2019. Patients in the Mayo Clinic who were consecutively tested for MOG-IgG1 by live cell-based flow cytometry during their diagnostic workup were included. Patients without research authorization were excluded. Main Outcomes and Measures: Medical records of patients who were tested were initially reviewed by 2 investigators blinded to MOG-IgG1 serostatus, and pretest probability was classified as high or low (suggestive of MOGAD or not). Testing of MOG-IgG1 used a live-cell fluorescence-activated cell-sorting assay; an IgG binding index value of 2.5 or more with an end titer of 1:20 or more was considered positive. Cases positive for MOG-IgG1 were independently designated by 2 neurologists as true-positive or false-positive results at last follow-up, based on current international recommendations on diagnosis or identification of alternative diagnoses; consensus was reached for cases in which disagreement existed. Results: A total of 1617 patients were tested, and 357 were excluded. Among 1260 included patients tested over 2 years, the median (range) age at testing was 46 (0-98) years, and 792 patients were female (62.9%). A total of 92 of 1260 (7.3%) were positive for MOG-IgG1. Twenty-six results (28%) were designated as false positive by the 2 raters, with an overall agreement on 91 of 92 cases (99%) for true and false positivity. Alternative diagnoses included multiple sclerosis (n = 11), infarction (n = 3), B12 deficiency (n = 2), neoplasia (n = 2), genetically confirmed adrenomyeloneuropathy (n = 1), and other conditions (n = 7). The overall PPV (number of true-positive results/total positive results) was 72% (95% CI, 62%-80%) and titer dependent (PPVs: 1:1000, 100%; 1:100, 82%; 1:20-40, 51%). The median titer was higher with true-positive results (1:100 [range, 1:20-1:10000]) than false-positive results (1:40 [range, 1:20-1:100]; P < .001). The PPV was higher for children (94% [95% CI, 72%-99%]) vs adults (67% [95% CI, 56%-77%]) and patients with high pretest probability (85% [95% CI, 76%-92%]) vs low pretest probability (12% [95% CI, 3%-34%]). The specificity of MOG-IgG1 testing was 97.8%. Conclusions and Relevance: This study confirms MOG-IgG1 as a highly specific biomarker for MOGAD, but when using a cutoff of 1:20, it has a low PPV of 72%. Caution is advised in the interpretation of low titers among patients with atypical phenotypes, because ordering MOG-IgG1 in low pretest probability situations will increase the proportion of false-positive results.

Clinical Utility of Striational Antibodies in Paraneoplastic and Myasthenia Gravis Paraneoplastic Panels.

OBJECTIVE: To critically assess the clinical utility of striational antibodies (StrAbs) within paraneoplastic and myasthenia gravis serological evaluations. METHODS: All Mayo Clinic patients tested for StrAbs from January 1st 2012-December 31st 2018 utilizing Mayo's Unified Data Platform (UDP) were reviewed for neurological diagnosis and cancer. RESULTS: 38,502 unique paraneoplastic and 1,899 MG patients were tested. In paraneoplastic evaluations, the StrAbs positivity rate was higher in cancer vs without cancer (5% [321/6775] vs 4% [1154/31727]; p<0.0001; OR 1.35; CI=1.19-1.53) but ROC analysis indicated no diagnostic accuracy in cancer (AUC=0.505). No neurological phenotype was significantly associated with StrAbs in the paraneoplastic group. Positivity was more common in all MG cancers compared to paraneoplastic cancers (p<0.0001). In MG evaluations, the StrAbs positivity rate was higher in those with cancer vs without (46% [217/474] vs 26% [372/1425]; p<0.0001; OR 2.39, CI 1.9-2.96) with ROC analysis indicating poor diagnostic accuracy for thymic cancer (AUC 0.634, recommended cutoff=1:60, sensitivity=56%, specificity=71%), with worse accuracy for extrathymic cancers (AUC 0.543). In paraneoplastic or MG evaluations, the value of antibody positivity did not improve cancer predictions. Paraneoplastic evaluated patients were more likely with positive StrAbs to obtain computed tomography (CT) (p=0.0001) with 3% (12/468) cancer found. CONCLUSION: Despite a statistically significant association with cancer, an expansive review of performance in clinical service demonstrates that StrAbs are neither specific nor sensitive in predicting malignancy or neurological phenotypes. CT imaging is over utilized with positive StrAbs results. Removal of StrAbs from paraneoplastic or MG evaluations will improve the diagnostic characteristics of the current MG test. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that the presence of StrAbs do not accurately identify patients with malignancy or neurological phenotypes.

Clearing drug interferences in myeloma treatment using mass spectrometry.

OBJECTIVE: To explore the possibility of using a combination of a rapid MALDI-TOF-MS method (Mass-Fix) in conjunction with higher resolution LC-ESI-QTOF-MS (miRAMM) measurements to discriminate the IgG kappa M-protein from daratumumab, elotuzumab and isatuximab in myeloma patients. DESIGN & METHODS: 86 patients with an IgG kappa M-protein were spiked with therapeutic levels of the drugs and examined by Mass-Fix and miRAMM to establish the percent of cases that could be resolved by each method. The method was then applied to 21 samples from patients receiving one of the drugs. RESULTS: Mass-Fix was capable of resolving the t-mAb from M-protein for 87 percent of the spiked samples. For the cases unresolved by Mass-Fix, miRAMM was capable of resolving the remaining drug interferences. The 21 IgG kappa myeloma patients that were receiving the drugs were all resolved by Mass-Fix. CONCLUSION: This proposed algorithm allows use of a clinical available assay (Mass-Fix) while maximizing the number of cases that can accurately resolve the t-mAb from the M-protein.

Neural Antibody Testing in Patients with Suspected Autoimmune Encephalitis.

BACKGROUND: Autoimmunity is an increasingly recognized cause of encephalitis with a similar prevalence to that of infectious etiologies. Over the past decade there has been a rapidly expanding list of antibody biomarker discoveries that have aided in the identification and characterization of autoimmune encephalitis. As the number of antibody biomarkers transitioning from the research setting into clinical laboratories has accelerated, so has the demand and complexity of panel-based testing. Clinical laboratories are increasingly involved in discussions related to test utilization and providing guidance on which testing methodologies provide the best clinical performance. CONTENT: To ensure optimal clinical sensitivity and specificity, comprehensive panel-based reflexive testing based on the predominant neurological phenotypic presentation (e.g., encephalopathy) is ideal in the workup of cases of suspected autoimmune neurological disease. Predictive scores based on the clinical workup can aid in deciding when to order a test. Testing of both CSF and serum is recommended with few exceptions. Appropriate test ordering and interpretation requires an understanding of both testing methodologies and performance of antibody testing in different specimen types. SUMMARY: This review discusses important considerations in the design and selection of neural antibody testing methodologies and panels. Increased collaboration between pathologists, laboratorians, and neurologists will lead to improved utilization of complex autoimmune neurology antibody testing panels.