ARTICLE OPEN ACCESS Clinical and Laboratory Features in Anti-NF155 Autoimmune Nodopathy Lorena Martı́n-Aguilar, MD, PhD,* Cinta Lleixà, MSc,* Elba Pascual-Goñi, MD, PhD, Marta Caballero-Ávila, MD, Correspondence Dr. Querol Laura Martı́nez-Martı́nez, MD, PhD, Jordi Dı́az-Manera, MD, PhD, Ricard Rojas-Garcı́a, MD, PhD, lquerol@santpau.cat Elena Cortés-Vicente, MD, PhD, Janina Turon-Sans, MD, Noemi de Luna, PhD, Xavier Suárez-Calvet, PhD, Eduard Gallardo, PhD, Yusuf Rajabally, MD, Sangeeta Scotton, MD, Bart C. Jacobs, MD, PhD, Adája Baars, MD, Andrea Cortese, MD, PhD, Elisa Vegezzi, MD, Romana Höftberger, MD, Fritz Zimprich, MD, Cornelia Roesler, MD, Eduardo Nobile-Orazio, MD, PhD, Giuseppe Liberatore, MD, Fu Liong Hiew, MD, Alicia Martı́nez-Piñeiro, MD, Alejandra Carvajal, MD, Raquel Piñar-Morales, MD, Mercedes Usón-Martı́n, MD, Olalla Albertı́, MD, Maria Ángeles López-Pérez, MD, Fabian Márquez, MD, Julio Pardo-Fernández, MD, PhD, Laura Muñoz-Delgado, MD, Macarena Cabrera-Serrano, MD, PhD, Nicolau Ortiz, MD, PhD, Manuel Bartolomé, MD, Özgür Duman, MD, Vera Bril, MD, Darwin Segura-Chávez, MD, Kalliopi Pitarokoili, MD, Claudia Steen, MD, Isabel Illa, MD, PhD, and Luis Querol, MD, PhD Neurol Neuroimmunol Neuroinflamm 2022;9:e1098. doi:10.1212/NXI.0000000000001098 Abstract MORE ONLINE Background and Objectives Class of Evidence To study the clinical and laboratory features of antineurofascin-155 (NF155)–positive auto- Criteria for rating immune nodopathy (AN). therapeutic and diagnostic studies Methods NPub.org/coe Patients with anti-NF155 antibodies detected on routine immunologic testing were included. Clinical characteristics, treatment response, and functional scales (modified Rankin Scale [mRS] and Inflammatory Rasch-built Overall Disability Scale [I-RODS]) were retrospectively collected at baseline and at the follow-up. Autoantibody and neurofilament light (NfL) chain levels were analyzed at baseline and at the follow-up. Results Forty NF155+ patients with AN were included. Mean age at onset was 42.4 years. Patients presented with a progressive (75%), sensory motor (87.5%), and symmetric distal- predominant weakness in upper (97.2%) and lower extremities (94.5%), with tremor and ataxia (75%). Patients received a median of 3 (2–4) different treatments in 46 months of median follow-up. Response to IV immunoglobulin (86.8%) or steroids (72.2%) was poor in most patients, whereas 77.3% responded to rituximab. HLA-DRB1*15 was detected in 91.3% of patients. IgG4 anti-NF155 antibodies were predominant in all patients; anti- NF155 titers correlated with mRS within the same patient (r = 0.41, p = 0.004). Serum NfL (sNfL) levels were higher in anti-NF155+ AN than in healthy controls (36.47 vs 7.56 pg/ mL, p < 0.001) and correlated with anti-NF155 titers (r = 0.43, p = 0.001), with I-RODS at baseline (r = −0.88, p < 0.001) and with maximum I-RODS achieved (r = −0.58, p = 0.01). Anti-NF155 titers and sNfL levels decreased in all rituximab-treated patients. *These authors contributed equally to this work as co-first authors. From the Neuromuscular Diseases Unit (L.M.-A., C.L., E.P.-G., M.C.-Á., R.R.-G., E.C.-V., J.T.-S., E.N.-O., I.I., L.Q.), Department of Neurology, Hospital de La Santa Creu I Sant Pau, Universitat Autònoma de Barcelona; Biomedical Research Institute Sant Pau (IIB Sant Pau) (L.M.-A., C.L., E.P.-G., M.C.-Á., J.D.-M., E.C.-V., X.S.-C., E.G.); Immunology Department (L.M.-M.), Hospital de La Santa Creu I Sant Pau, Universitat Autònoma de Barcelona, Spain; Centro para La Investigación Biomédica en Red en Enfermedades Raras (CIBERER) (J.D.-M., R.R.-G., E.C.-V., N.L., X.S.-C., E.G., I.I., L.Q.); University Hospital Birmingham (Y.R., S.S.), UK; Erasmus Medical Center (B.C.J., A.B.), Rotterdam, the Netherlands; IRCCS Mondino Foundation (Andrea Cortese, E.V.), Pavia, Italy; Department of Neurology (R.H., F.Z.), Medical University of Vienna; Paracelsus Medical University (C.R.), Salzburg, Austria; IRCCS Humanitas Research Hospital (G.L.), Milan University, Rozzano, Italy; Kuala Lumpur General Hospital (F.L.H.), Jalan Pahang, Kuala Lumpur, Malaysia; Hospital Universitari Germans Trias I Pujol (A.M.-P.), Badalona; Hospital Universitario Virgen de Las Nieves (Alejandra Carvajal), Granada, Spain; Hospital Universitario Clı́nico San Cecilio (R.P.-M.), Granada; Hospital Son Llàtzer (M.U.-M.), Palma de Mallorca; Hospital San Jorge (O.A.), Huesca, España; Hospital San Pedro (M.Á.L.-P.), Logroño; Hospital Universitari Josep Trueta (F.M.), Girona; Hospital Clı́nico Universitario de Santiago (J.P.-F.), Santiago de Compostela; Hospital Universitario Virgen Del Rocı́o (L.M.-D., M.C.-S.), Sevilla; Hospital Universitari Sant Joan (N.O.), Reus; Complejo Asistencial de Ávila (M.B.), Ávila, Spain; Akdeniz University (Ö.D.), Antalya, Turkey; Toronto General Hospital (V.B.), University Health Network, University of Toronto, Canada; Instituto Nacional de Ciencias Neurológicas. Lima (D.S.-C.), Perú; St. Josef-Hospital (K.P.), Ruhr-University Bochum; and Sant Joseph Hospital (C.S.), Berlin, Germany. Go to Neurology.org/NN for full disclosures. Funding information is provided at the end of the article. The Article Processing Charge was funded by authors. This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND), which permits downloading and sharing the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1 Glossary CBA = cell-based assay; CIDP = chronic inflammatory demyelinating polyradiculoneuropathy; CMAP = compound muscle action potential; CNTN1 = contactin-1; EMG = electromyography; GBS = Guillain-Barré syndrome; HC = Healthy control; HLA = human leukocyte antigen; ICC = immunocytochemistry; I-RODS = Inflammatory Rasch-built Overall Disability Scale; IVIg = IV immunoglobulin; mRS = modified Rankin Scale; NCS = nerve conduction study; NF140 = neurofascin-140; NF155 = neurofascin- 155; NF186 = neurofascin-186; OD = optical density; PLEX = plasma exchange; sNfL = serum neurofilament light chain. Discussion Anti-NF155 AN presents a distinct clinical profile and good response to rituximab. Autoantibody titers and sNfL are useful to monitor disease status in these patients. The use of untagged-NF155 plasmids minimizes the detection of false anti-NF155+ cases. Classification of Evidence This study provides Class IV evidence that anti-NF155 antibodies associate with a specific phenotype and response to rituximab. Chronic inflammatory demyelinating polyradiculoneuropathy identified during routine testing for nodal/paranodal anti- (CIDP) is a clinically and pathologically diverse autoimmune bodies. The samples were obtained between May 2010 and syndrome of the peripheral nervous system, causing significant December 2020. These patients were selected for further disability.1,2 Disease-specific antibodies targeting proteins at the characterization between May 2020 and December 2020. De- node and paranode of Ranvier, such as neurofascin 155 mographic and clinical data at onset and during follow-up were (NF155),3 nodal neurofascins (NF186 and NF140),4 contactin- collected in a coded database. This study was conducted 1 (CNTN1),5 or CNTN-1/caspr-1,6,7 have been described in according to a protocol approved by the Ethics Committee of small subsets of patients with CIDP sharing immunopathologic the Hospital de la Santa Creu i Sant Pau. All patients gave mechanisms, clinical features, and treatment response and dif- written informed consent to participate in the study. fering from those of typical CIDP.8,9 This has led to the ap- pearance of the autoimmune nodopathy (AN) diagnostic category Data and Sample Collection in the recent update of the European Academy of Neurology/ Data were collected retrospectively by patients’ neurologists Peripheral Nerve Society CIDP diagnostic guidelines.10 in 24 different centers by chart review. European Academy of Neurology/Peripheral Nerve Society diagnostic criteria for Previous case series describe the association of anti-NF155 an- CIDP19 were assessed, and patients were classified as having tibodies with predominantly distal motor involvement, ataxia definite, probable, or possible CIDP. Demographic data (age and low-frequency tremor with cerebellar features,3,11 marked and gender) and clinical features (initial diagnosis, time to nerve conduction abnormalities,12 and DRB1*15 human leu- nadir, the presence of weakness or sensory deficits, presence kocyte antigen (HLA) Class II alleles.13 Moreover, these patients of ataxia, and tremor) were collected. Clinical presentation respond poorly to IV immunoglobulin (IVIg) and usually well to was defined as sensorimotor, pure motor, or pure sensory/ rituximab.14 Anti-NF155 antibodies, almost always of the IgG4 ataxic. The results of routine nerve conduction studies isotype,15 are pathogenic according to passive transfer experi- (NCS), CSF examination, and treatments were also col- ments in animal models16 and pathologic studies detecting IgG4 lected. As an electrophysiologic marker of axonal damage, deposition and axoglial junction dissection at the paranode (in we used the lowest (left or right) median nerve compound the absence of classical macrophage-mediated demyelination).17 muscle action potential (CMAP) negative peak amplitude Recently, high serum neurofilament light chain (sNfL) levels and, when available, the presence of spontaneous activity in were described in a subset of patients with anti-NF155+ AN.18 the electromyography (EMG) at the tibialis anterior muscle. CSF protein levels higher than 0.45 g/L were considered Our work describes the clinical, immunologic, biomarker, relevant.20 Disability scores were collected at nadir and at the treatment response and prognostic features of the largest anti- follow-up, including the modified Rankin Scale (mRS)21 and NF155+ AN cohort so far. the Inflammatory Rasch-built Overall Disability Scale (I- RODS) scores22 (from 0 to 100; 100 indicating no disabil- ity), when available. Response to therapy was defined as a good response, partial response, or no response as classified Methods by their primary neurologists after chart review of the neu- Protocol Approvals, Registrations, and rologic examination. For rituximab-treated patients, mRS Patient Consents was prospectively collected pretreatment and, at least, once In this multicenter retrospective observational study, we in- posttreatment; infusion protocol and adverse events (in- cluded all sera reacting against NF155 transfected cells and fusion reactions and infections) were also collected. Serum 2 Neurology: Neuroimmunology & Neuroinflammation | Volume 9, Number 1 | January 2022 Neurology.org/NN samples were obtained at diverse time points during routine Data Availability autoantibody testing and stored at −80°C until needed. Anonymized data not published within this article will be made available by request from any qualified investigator. Anti-NF155 Antibody Detection and Titration Serum antibodies against NF155 were analyzed in the same laboratory using a cell-based assay (CBA) with human Results recombinant NF155-transfected HEK293 cells as pre- viously described.3,23 The DDK-myc-tagged RC228652 Anti-NF155 Autoantibody Screening NF155 plasmid (OriGene, Rockville, MD) was used for We detected 44 sera with a positive staining in the screening initial anti-NF155 detection, and the untagged EX-Z7183- NF155 CBA and negative staining in the NF140/NF186 M02 NF155 plasmid (GeneCopoeia, Rockville, MD) was CBA. After performing a confirmatory study with anti-NF155 used for false positive detection in those patients with ELISA, 40 patients were confirmed true positives with ELISA discrepant results in the CBA and the ELISA. ELISA was and were selected for further characterization. The other 4 used as a confirmatory test and for isotype identification patients were classified as false positives in the CBA (9.1%). and titration, as previously described.3 Optical density We used an untagged neurofascin-155 plasmid and confirmed (OD) was measured at 450 nm with a Multiskan ELISA that those 4 patients were negative when the myc-DDK tag reader. Blank OD was subtracted to NF155 OD to control was removed (eFigure 1, links.lww.com/NXI/A641). for unspecific background signal. The samples were con- Clinical Features of Anti-NF155 Patients sidered positive by ELISA when they had a DOD higher With AN than average healthy control (HC) DOD plus 2 SD. Titer Thirty-nine patients with anti-NF155+ fulfilled the CIDP di- variation within the same patient was expressed as the agnostic criteria; in 1 patient, antibodies were detected post- percentage of titer change compared with pretreatment mortem (supplementary results, links.lww.com/NXI/A641). levels. All samples were tested under the same conditions. Nine patients were previously reported in other series.3,14,15,25,26 Serum NfL Measurements The initial diagnosis was CIDP for most patients (80%), but 5 sNfL levels were measured in all available anti-NF155 AN patients were initially diagnosed with Guillain-Barré syndrome patient samples and compared with 78 HCs, using the Simoa (GBS). Patients with anti-NF155+ AN had a median age at NF-light kit in the SR-X Immunoassay Simoa analyzer onset of 42.4 years and were predominantly men (72.5%). The (Quanterix Corp, Boston, MA), as previously described.24 most frequent clinical presentation was sensory motor (87.5%), The samples were analyzed in duplicates following the man- and most patients had a progressive (75%) and chronic (67.5%) ufacturer’s instructions and standard procedures. All NfL clinical course. Most patients had a symmetric weakness with values were within the linear ranges of the assay. The intra- distal predominance in upper (97.2%) and lower extremities assay and interassay coefficients of variation at intermediate (94.5%). The sensory deficit was symmetric and more frequent level (15.25 pg/mL) were 3.9% and 9.5%, respectively. in lower (97.5%) than in upper extremities (67.5%). Seventy-five percent of patients had tremor and ataxia (of which, 5 had only HLA Genotyping ataxia, 5 tremor, and 25 a combination of both). Tremor was Genomic DNA from the peripheral blood from patients with classified as intention tremor or action tremor in 18 patients anti-NF155+ with AN was extracted following standard pro- (60%). Thirty percent of patients had cranial nerve involvement: tocols. HLA-DRB1 and HLA-DQB1 genotypes were analyzed bilateral facial palsy was the most frequent (70%), and 2 patients as previously described.13 had bilateral optic neuritis confirmed by evoked potentials27 with normal brain and spine MRI and negative MOG and Statistical Analysis antiaquaporin-4 antibodies. Further information about disease A descriptive data analysis was performed. Descriptive characteristics is detailed in Table 1. statistics are shown as mean (±SD) or median (inter- quartile range) in continuous variables and as frequencies Regarding nerve conduction studies, 38 patients fulfilled (percentages) in categorical variables. Comparisons be- definite electrodiagnostic European Academy of Neurology/ tween patients with anti-NF155+ AN and HC were per- Peripheral Nerve Society criteria for CIDP,19 1 patient was formed by the Wilcoxon rank-sum test. The Kruskal-Wallis defined as possible CIDP, and 1 patient did not have nerve test was used to compare groups. Wilcoxon-Matched Pairs conduction studies performed because diagnosis was con- Signed Rank test was used to compare baseline anti-NF155 firmed postmortem. We collected 33 (82.5%) NCS in which titters and sNfL levels at different time points. We used only 26 (65%) had needle EMG available. Median amplitude the Spearman coefficient to assess correlation between of distal CMAPs of different nerves are shown in eTable 1 variables. (links.lww.com/NXI/A641). Seventeen of 26 patients (65.4%) had spontaneous activity on EMG. CSF was exam- Statistical significance for all analyses was set at 0.05 (2-sided). ined in 37 (92.5%) patients; most patients had less than 5 cells All statistical analyses were performed with GraphPad Prism in CSF (72.2%), and all patients had high CSF protein levels v8 and SPSS Statistics version 23 (IBM Corp). with a median of 2 g/L (0.95–3.67). Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 9, Number 1 | January 2022 3 Table 1 Demographic and Clinical Characteristics of Table 1 Demographic and Clinical Characteristics of Patients With NF155+ AN Patients With NF155+ AN (continued) Baseline characteristics Baseline characteristics Age at onset (mean ± SD) 42.40 ± 19.48 Superficial sensation 22 (81.5%) Age at diagnosis (mean ± SD) 43.25 ± 19.30 Leg sensory deficit 39 (97.5%) Sex (male; n, %) 29 (72.5%) Symmetric 38 (95%) Initial diagnosis (n, %) Modality CIDP 32 (80%) Vibration 37 (92.5%) GBS 5 (12.5%) Pinprick 32 (80%) Sensory neuropathy 1 (2.5%) Superficial sensation 31 (77.5%) Demyelinating neuropathy 1 (2.5%) Reflexes (n, %) Cervical myelopathy 1 (2.5%) Absent 30 (75%) CIDP clinical course (n, %) Decreased 10 (25%) Progressive 30 (75%) Ataxia (n, %) 30 (75%) Relapsing-remitting 10 (25%) Tremor (n, %) 30 (75%) Time to nadir (n, %) Cranial nerve involvement (n, %) 12 (30%) Acute (<1 mo) 2 (5%) Bilateral facial palsy 7 Subacute (1–2 mo) 11 (27.5%) Ophthalmoparesis 3 Chronic (>2 mo) 27 (67.5%) Optic nerve 2 Clinical presentation (n, %) Clinical scales Sensory motor 35 (87.5%) mRS (median, IQR) Pure sensory/ataxic 4 (10%) Sampling (n = 27) 3 (2–4) Pure motor 1 (2.5%) Maximum (n = 37) 4 (2–4) Weakness (n, %) Final (n = 37) 2 (1–3) Upper extremity weakness 35 (87.5%) I-RODS (median, IQR) Symmetric 33 (94.3%) Sampling (n = 14) 49 (38–68) Proximal and distal 15 (42.9%) Maximum (n = 17) 40 (29–57) Distal 19 (54.3%) Final (n = 22) 59 (54–88) Proximal 1 (2.9%) Abbreviations: AN = autoimmune nodopathy; CIDP = chronic inflammatory demyelinating polyradiculoneuropathy; GBS = Guillain-Barré syndrome; I- Lower extremity weakness 37 (92.5%) RODS = Inflammatory Rasch-built Overall Disability Scale; IQR = interquartile range; mRS = modified Rankin Scale. Symmetric 34 (91.9%) Proximal and distal 17 (45.9%) Treatment Response and Clinical Follow-up Distal 18 (48.6%) The median number of treatments received was 3 (2–4). Proximal 2 (5.4%) Most patients were treated with IVIg (95%) and/or cortico- steroids (90%), and approximately half of patients (46.2%) Sensory deficit (n, %) were treated with plasma exchange (PLEX) with a median Arm sensory deficit 27 (67.5%) number of sessions of 6 (5–9). Twenty-three patients Symmetric 26 (96.3%) (57.5%) were treated with rituximab, and 1 patient was in- cluded in a blinded clinical trial of rituximab vs placebo. Of Modality those patients treated with rituximab (n = 23), 13 were also Vibration 16 (59.3%) treated with plasma exchange before starting rituximab, and Pinprick 16 (59.3%) 10 patients were treated with rituximab alone. Nine patients were treated with azathioprine, and 8 patients received other 4 Neurology: Neuroimmunology & Neuroinflammation | Volume 9, Number 1 | January 2022 Neurology.org/NN weekly + 2 monthly 375 mg/m2 doses, followed by 1 + 1 Table 2 Treatment and Clinical Response (separated 2 weeks) 1,000 mg doses (27.3%) and 4 weekly 375 No. of mg/m2 doses (27.3%). Five (21.7%) patients had a relapse, a patients Response median of 21 (4.5–59.5) months after induction with ritux- Treatment (n, %) (n, %) Dose/Protocol imab; 11 (47.8%) patients received rituximab reinfusions. Four IVIg 38 (95%) Yes: 5 (13.1%) 2g/kg per course (17%) patients had adverse effects related to rituximab: 2 mild Partial: 9 infusion reactions, 1 pneumonia, and 1 disseminated varicella (23.7%) No: 24 (63.2%) infection. Treatment frequencies, doses, and responses to treatment are further detailed in Table 2. Steroids 36 (90%) Yes: 10 (27.8%) 1 mg/kg/d: 23 (63.9%) Partial: 16 MP iv pulse: 4 (11.1%) (44.4%) MP iv pulse + mg/kg/d: 5 The clinical scales at baseline, at nadir, and after treatment are No: 10 (27.8%) (13.9%) Others: 4 (11.1%) described in Table 1. The median follow-up time was 46 (20–81) months. Patients with facial diplegia had lower PLEX 18 (46.2%) Yes: 7 (38.9%) No of sessions (median, Partial: 6 IQR): 6 (5–9) maximum and final I-RODS than patients who did not have (33.3%) facial involvement (median maximum I-RODS 22 vs 47, p = No: 5 (27.8%) 0.003 and median final I-RODS 43 vs 61, p = 0.035 (eTable 3, Rituximaba,b 23 (57.5%) Yes: 17 (77.3%) 4 + 2: 8 (36.4%) links.lww.com/NXI/A641). Three patients died during Partial: 3 4: 6 (27.3%) (13.6%) 1 + 1: 6 (27.3%) follow-up: 1 because of CIDP disease course, 1 because of No: 2 (9.1%) Others: 2 (9.1%) aspiration pneumonia, and 1 because of a disseminated vari- Azathioprine 9 (22.5%) Yes: 1 (11.1%) — cella infection. Patients who received rituximab had higher Partial: 4 median mRS and lower I-RODS at nadir, although differences (44.4%) were not statistically significant (4 [3–4] vs 3 [2–4], p = 0.5; No: 4 (44.4%) 40 [29–49] vs 47 [9–77], p = 0.78; Table 3). They received a Mycophenolate 3 (7.5%) Partial: 1 — higher number of previous treatments than those patients (33.3%) No: 2 (66.7%) who did not received rituximab (4 [3–5] vs 2 [2–3], p = 0.03), including PLEX, but they did not differ at the final mRS or Methotrexate 3 (7.5%) Partial: 1 — (33.3%) I-RODS from those patients not treated with rituximab de- No: 2 (66.7%) spite being more drug resistant (Table 3). There were no Cyclosporine 1 (2.5%) No: 1 (100%) — differences between patients treated with PLEX and rituximab (n = 13) or with rituximab alone (n = 10), regarding response Interferon beta 1 (2.5%) No: 1 (100%) — 1a treatment, relapses, or reinfusions needed. Abbreviations: IVIg = IV immunoglobulin; MP = methylprednisolone; PLEX = Baseline Immunologic Characteristics plasma exchange. All sera with an anti-NF155+ CBA were also positive by 4 + 2: 375 mg/m2 every week for 4 consecutive weeks and then monthly for the next 2 months; 1 + 1: 2 1 g doses separated by 2 weeks; 4: 375 mg/m2 ELISA; anti-NF155 titers ranged from 1:300 to 1:72,300. every week for 4 consecutive weeks. a One patient included in a blinded clinical trial of rituximab vs placebo. Autoantibodies were predominantly of the IgG4 subclass in all b Improvement in mRS after rituximab treatment is detailed in eTable 1 patients. In addition, we evaluated NF155 positivity in 4 CSF (links.lww.com/NXI/A641). from patients with anti-NF155+ AN and 3 of them tested positive for NF155 antibodies. We were able to perform subclass analysis and titration in 2 CSF samples: both were treatments (mycophenolate, methotrexate, cyclosporine, or IgG4 and their titers significantly lower than in sera (1:160 in interferon beta1a). both CSF samples and 1:24,300 and 1:72,300 in sera). Only 5 of 38 (13.1%) patients had a good response to IVIg, 10 Regarding the HLA genotyping, DRB1*15 alleles (DRB1*15: of 36 (27.8%) patients had a good response to steroids, and 7 of 01 or DRB1*15:02) were present in 21 of 23 patients with anti- 18 (38.9%) had a good response to PLEX. On the contrary, 17 NF155+ AN (91.3%). Most frequent allele was DRB1*15:01 of 23 (77.3%) patients had a good response to rituximab and 13 (n = 13; 72.2%). No clinical differences were observed between of 23 (56.5%) patients have an improvement of ≥2 points in patients with DRB1*15:01 and DRB1*15:02, except for a trend mRS after rituximab treatment. Rituximab-treated patients in to younger age in patients with DRB1*15:02 (45.2 ± 20.5 vs which mRS remained stable had lower median follow-up time, 30.6 ± 14.6, p = 0.14). In contrast, HLA-DRB1*15 is found in although differences were not statistically significant (eTable 2, 17% of Spanish population, 12% of North Italy population, links.lww.com/NXI/A641). Of the 4 rituximab-treated patients 25% of Southern France population, and 20% of English without detectable improvement in the mRS score, 2 patients population.28 were classified as nonresponders by their primary physicians (median follow-up time of 37 months) and 2 patients were Baseline Serum NfL Levels classified as partial responders (median follow-up time of 6 Serum NfL levels were determined in all samples available months). Most frequent infusion protocol (36.4%) was 4 (36/40) at baseline. Anti-NF155 + AN patients had Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 9, Number 1 | January 2022 5 Table 3 Rituximab Treatmenta Patients treated with rituximab (n = 23) Patients not treated with rituximab (n = 16) p Value Age at onset (mean ± SD) 44.1 ± 20.7 39.25 ± 18.6 0.51 Age at diagnosis (mean ± SD) 45.2 ± 20.7 39.8 ± 18.12 0.44 Sex (male; n, %) 17 (77.3%) 11 (68.8%) 0.41 Baseline NfL levels (median, IQR) (n = 36) 47.69 (18.87–154.29) 14.43 (7.58–64.68) 0.08 NF155 titers (median, IQR) 1/24,300 (1/8,100–1/24,300) 1/8,100 (1/2,700–1/24,300) 0.19 No. of previous treatments (median, IQR) 4 (3–5) 2 (2–3) 0.03 PLEX (n, %) 14 (63.6%) 4 (25%) 0.02 mRS (median, IQR) Baseline (n = 27) 3 (2–4) 3 (1–4) 0.94 Maximum (n = 37) 4 (3–4) 3 (2–4) 0.53 Final (n = 37) 2 (1–2) 2 (1–3) 0.62 R-ODS (median, IQR) Baseline (n = 13) 45 (40–60) 61 (18–86) 0.83 Maximum (n = 16) 40 (29–49) 47 (9–77) 0.78 Final (n = 21) 58 (51–88) 60 (55–88) 0.69 Abbreviations: IQR = interquartile range; I-RODS = Inflammatory Rasch-built Overall Disability Scale; MP = methylprednisolone; mRS = modified Rankin Scale; NF155 = neurofascin-155; PLEX = plasma exchange. a We exclude 1 patient included in a blinded clinical trial of rituximab vs placebo. significantly higher sNfL levels than HC (36.47 pg/mL without spontaneous activity (67.33 vs 25.12 pg/mL, p = 0.1), vs 7.56 pg/mL, p < 0.001, Figure 1 and Table 4) at baseline. the differences were not statistically significant. sNfL levels correlated with age in HC (r = 0.73, p < 0.001) but not in patients with anti-NF155 + AN (r = 0.26, Relationship Between NF155 Titers, sNfL p = 0.12). The samples collected pretreatment (n = 12) Levels, and Treatment Response to Rituximab: have higher sNfL levels than those collected after treat- Kinetics ment had been started (n = 24) (65.84 vs 18.41 pg/mL; In rituximab-treated patients with anti-NF155+ AN in which p = 0.002). follow-up samples at regular time points were available (n = 7), antibody titers decreased during follow-up. This decline Relationship Between NF155 Titers, sNfL was significant as early as 3 months after administration of Levels, and Clinical Status rituximab (mean decrease of 66.7%, Figure 2). At 1 year, a Absolute anti-NF155 titers did not correlate with clinical mean titer reduction of 98.6% in rituximab-treated patients status across patients, but they did when we evaluated was achieved. In patients not treated with rituximab in which follow-up NF155 titers using baseline titers as the reference follow-up sample at 1 year (n = 6) was available, no signif- (r = 0.41, p = 0.004; eFigures 2 and 3, links.lww.com/NXI/ icant decrease of antibodies was observed (2 patients had a A641). Baseline sNfL levels negatively correlated with median decrease of 94%, 2 patients remained stable, and 2 I-RODS at blood sampling (r = −0.88, p < 0.001) and with patients increased their NF155 titers) (Figure 3). sNfL levels maximum I-RODS achieved (r = −0.58, p = 0.01) (eFigure 4, were higher in rituximab-treated patients compared with links.lww.com/NXI/A641). However, correlation between the those not treated with rituximab (47.69 vs 14.43 pg/mL, p = sNfL levels and the final I-RODS (r = −0.36; p = 0.1) did 0.08, Table 3), but differences were not statistically signifi- not reach statistical significance. sNfL levels correlated with cant. sNfL levels decreased at 1 year in rituximab-treated NF155 titers at baseline (n = 36; r = 0.43, p = 0.001) and patients (median of 37.98 pg/mL at baseline vs 11.72 pg/mL at every time point available (n = 105; r = 0.34, p < 0.001). at 1 year, p = 0.04). In patients not treated with rituximab, median baseline sNfL levels were normal and no changes Baseline sNfL levels did not correlate with lowest CMAP in were observed at 1 year (7.62 vs 6.95 pg/mL, p = 0.16). nerve conduction studies in any of the nerves tested. Although Clinical status improved at 1 year in both groups, but only patients showing spontaneous activity in the needle EMG of the rituximab-treated group improved significantly (median the tibialis anterior showed higher sNfL levels than patients of mRS 4 [3–4] at baseline vs 2 [1–2] at the 1-year follow-up, 6 Neurology: Neuroimmunology & Neuroinflammation | Volume 9, Number 1 | January 2022 Neurology.org/NN Figure 1 Baseline Serum NfL in Anti-NF155 + Patients With AN and Healthy Controls Patients with anti-NF155 + AN had significantly higher sNfL levels than HC. The line in the center represents the median value, and the whiskers indicate the interquartile range. AN = autoimmune nodopathy; CIDP = chronic inflammatory demyelinating polyradiculoneuropathy; HC = healthy control; NF155 = neurofascin-155; sNfL = serum neurofilament light chain. p = 0.004, in rituximab-treated AN patients and 3 [2–4] at plasmids should be preferentially used and that a second baseline vs 2 [1–4] at 1 year, p = 0.25, in patients not treated test (ELISA or teased-nerve immunohistochemistry) is with rituximab). always recommended. Previous case series and systematic reviews suggested that pa- Discussion tients with anti-NF155+ AN respond poorly to IVIg or that IVIg Our study describes the clinical, laboratory, treatment re- response is less frequent than in seronegative CIDP.3,30,31,33 This sponse and prognostic features of the largest anti-NF155+ has been described in other IgG4-mediated diseases such as anti- AN cohort published so far.3,4,15,29-31 It confirms that pa- muscle-specific tyrosine kinase-positive myasthenia gravis.34 tients with AN with autoantibodies against NF155 present There are different hypotheses on why this happens in IgG4- at a younger age (including a significant proportion of mediated diseases, although none has been validated. On the one patients below 30 years)5 with a specific clinical phenotype hand, complement and cell-mediated cytotoxicity do not happen with distal weakness, tremor, and ataxia. The presence of in IgG4 diseases, and thus, any effect that IVIg may have over these features in a patient fulfilling the CIDP criteria should complement effector mechanisms or cytotoxic cells may be lost. immediately prompt anti-NF155 antibody testing, as rec- On the other hand, IgG4 is secreted exclusively by IL10+ reg- ommended in the recently published revision of the EAN/ ulatory B-cells, and these cells, interestingly, have significantly PNS CIDP diagnostic guidelines.10 Other associated fea- lower expression of the inhibitory immunoglobulin receptor tures, which may suggest the presence of anti-NF155 AN FCGRIIb on their surface (and this could decrease the ability of and prompt antinodal/paranodal autoantibody testing, are IgG4-producing cells to be inhibited by IgG).35 Our study has the presence of cranial nerve palsies, particularly facial also found that most patients with anti-NF155+ AN do not palsy, high CSF protein content, and poor response to IVIg. respond appropriately to IVIg (or, to a lower extent, cortico- An important implication of our study for the testing rec- steroids) according to their physicians. On the contrary, most ommendations in diagnostic guidelines is that almost 10% patients respond to rituximab even when they are refractory to of the patients testing positive for anti-NF155 in CBA IVIg and corticosteroid therapy (this also happens in other IgG4- performed with the myc-DDK tagged NF155 plasmid are mediated diseases36,37). More than 50% of patients in our cohort false positives. This agrees with our previous observation were treated with rituximab after a poor response to other that demonstrated that a positive test in the NF155 CBA therapies, and more than 75% had a good response. This im- could be due to antibodies targeting the myc-DDK tag and provement agrees with prospectively collected follow-up mRS not NF155 itself.32 This implies that untagged-NF155 scores that show that most patients improved at least 1 point Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 9, Number 1 | January 2022 7 antibody titers can be a good biomarker for disease activity and Table 4 Baseline Serum NfL in Anti-NF155 + Patients With treatment response when assessed in individual patients and AN and Healthy Controls represented as changes relative to baseline levels. Indeed, in NF155 + patients those patients treated with rituximab, IgG4 anti-NF155 de- with AN HC p Value creased more than a 90% relative to baseline titers or even Age at sampling, 47.87 ± 20.16 48 ± 18.1 0.78 became negative in a few patients. This suggests that the mean ± SD reappearance or a significant increase in the pathogenic auto- Sex, n, % male 28 (71.8%) 31 (39.7%) 0.001 antibody may precede a relapse and, thus, could guide treat- ment reinfusions. Again, this use of the autoantibodies needs to sNfL (pg/mL) <0.001 be validated prospectively, but the temporal evolution of the n 36 78 autoantibody titers, paralleling the sNfL levels and the clinical status in the few patients in which prospective follow-up was Median 36.47 7.56 available, is promising. Max 536.64 56.82 Min 3.44 2.30 We identified IgG4 anti-NF155 antibodies in the CSF of 3 of 4 patients in which a CSF sample was available. Intrathecal Abbreviations: AN = autoimmune nodopathy; HC = healthy control; NF155 = antipan neurofascin has been previously described,41 and neurofascin-155; sNfL = serum neurofilament light chain. anti-NF155 antibodies in CSF have been described in 2 patients with combined central and peripheral de- (82.6%), and a significant proportion (56.5%) improved 2 or myelination42 but not in anti-NF155 AN so far. The high more points, despite being resistant to other therapies. Indeed, protein content in CSF, the absence of oligoclonal bands, despite rituximab-treated patients had used a higher number of and the presence of higher anti-NF155 titers in serum than different drugs and showed higher disability and sNfL levels at in CSF suggests that anti-NF155 antibodies appear in the baseline, their final prognosis did not differ from patients who CSF because of blood-brain barrier disruption and not be- responded to first-line therapies. The great benefit that rituximab cause of intrathecal synthesis. The presence of these auto- provides to these patients supports that it could be used as an antibodies in the CSF could help explaining the cerebellar earlier therapeutic option, although careful assessment of the risk- features in patients with anti-NF155+ AN, but larger cohorts benefit balance and vaccination status needs to be performed including patients with and without tremor in which CSF is before rituximab treatment, as one patient died due to an in- analyzed are needed. fection aggravated by immune-suppresion. Approximately 50% of the patients in our cohort received PLEX. This is the result of 2 Our study also showed that sNfL levels were higher in patients facts: first, patients presented with an aggressive neuropathy that with anti-NF155+ AN than in HC. High sNfL levels have also did not respond to first-line therapies and second, because rit- been recently described in CIDP, particularly in a small subset uximab effect is not clearly seen until the third month of disease of patients with anti-NF155+ AN who showed higher sNfL and PLEX is used to eliminate as much autoantibody as possible levels than seronegative CIDP.18 In our study, we found a before the use of rituximab to shorten the recovery period. Our study did not find differences in treatment response, relapses, or reinfusions between the group of rituximab-treated patients with Figure 2 Clinical Status, NF155 Titers, and sNfL Levels After anti-NF155 AN pretreated with PLEX and the group nontreated Rituximab Treatment Induction: Kinetics with PLEX, but larger, dedicated studies are needed to clarify the utility of this therapeutic strategy. Anti-NF155 antibodies are pathogenic according to in vitro and in vivo models.16 As such, we hypothesized that their titers should correlate with disease severity. We found that IgG4 anti- NF155 antibody titers correlate with clinical status within the same patient, but not across patients. This is something that has been described in other IgG4 autoimmune diseases treated with rituximab, such as anti-muscle-specific tyrosine kinase myasthenia gravis,37,38 and in other polyneuropathies as IgM antimyelin-associated glycoprotein neuropathy.39,40 Several factors may explain why autoantibody titers do not correlate with clinical activity across patients: autoantibody affinity for Rituximab treated anti-NF-155 patients with follow-up samples at regular time points show improvement in the mRS scale, a decrease in NF155 titers, and a their target antigen and diverse biases arising from the retro- decrease in sNfL levels starting on the third month of treatment infusion. The spective nature of the study (diverse time points, diverse line in the center represents the median value, and the whiskers indicate the interquartile range. mRS = modified Rankin Scale; NF155 = neurofascin-155; treatment regimens, and diverse baseline severities and ages) NF155 = neurofascin-155; sNfL = serum neurofilament light chain. among others. However, our study proves that anti-NF155 8 Neurology: Neuroimmunology & Neuroinflammation | Volume 9, Number 1 | January 2022 Neurology.org/NN Figure 3 Rituximab Treatment Response: Clinical Status, NF155 Titers, and sNfL Levels Clinical improvement is present in patients treated with first-line therapies or rituximab but only the rituximab-treated group improved significantly. Anti- NF155 titers and sNfL levels decreased only in rituximab-treated group. The line in the center represents the median value, and the whiskers indicate the interquartile range. IVIg = IV immunoglobulin; mRS= modified Rankin Scale; NF155= neurofascin-155; sNfL= serum neurofilament light chain. strong correlation between baseline sNfL levels and initial the tissue status and anti-NF155 titers that inform about the I-RODS and maximum I-RODS achieved, but not with final immunologic effector mechanism, at regular intervals after I-RODS, suggesting that the final outcomes are not completely treatment, could be useful to guide treatment choices and determined by initial severity because effective therapies detect suboptimal therapeutic responses. Hypothetically, an change the course of the disease and most patients improve eventual increase in autoantibody titers or sNfL levels could significantly, regardless of the treatment used. These data differ herald a subsequent relapse and detecting the biomarker in- from those found in GBS,24 a monophasic disorder in which crease could help prevent it. However, the need of retreat- initial events determine long-term outcomes, but nonetheless ment should be assessed individually based on patient’s suggest that sNfL may be useful to monitor disease because it clinical status and not only based on the laboratory data. seems to happen in other peripheral neuropathies.43-45 HLA loci are the group of genetic factors that has most fre- Our study was not designed to correlate sNfL levels with quently been associated with autoimmune diseases, including electrophysiologic parameters because EMG and sNfL levels strong associations with other IgG4-mediated diseases.46-48 were not performed at the same time points, but we explored Previous studies have shown a strong association between a potential correlations between CMAP amplitudes, the pres- specific Class II allele, HLA-DRB1*15 (either 15:01 or 15: ence of spontaneous activity at distal, most affected, muscles, 02), and patients with anti-NF155+ AN.13 Our study shows a and sNfL levels. We failed to find strong associations. sNfL stronger association than previously reported (91.3%), con- levels did not correlate with CMAP amplitudes, but they ten- firming that this HLA allele is a constitutive risk factor that, ded to be higher in patients with spontaneous activity. Thus, associated with unknown environmental factors, may be our results, although preliminary, support the ability of sNfL to driving the appearance of the anti-NF155 autoantibodies. The monitor axonal damage. Altogether, the correlation of sNfL study of this genetic association in conjunction with geo- with disability scales and, less strongly, with the appearance of graphic distribution of the disorder, lifestyle, concomitant residual disability or spontaneous activity agrees with the rel- disorders, microbiome, or environmental triggers may yield atively frequent presence of distal muscle atrophy because of interesting pathophysiologic insights but requires significantly secondary axonal damage that some of these patients display larger cohorts of patients. and would support the use of sNfL as an early marker of potential axonal damage that could guide treatment selection to The main limitations of our study arise from the small number prevent the appearance of this permanent damage. of patients and its retrospective nature, including the retro- spective analysis of treatment efficacy using chart review. sNfL levels and anti-NF155 antibody titers decreased in all Furthermore, considering that patients were identified patients in which prospective follow-up was performed on through routine diagnostic testing, it is likely that our cohort is rituximab therapy, whereas neither sNfL or anti-N155 levels enriched in patients with tremor or a lack of response to IVIg showed comparable changes with other treatments. The ob- because of selection bias. However, since anti-NF155+ AN servation of the rituximab-treated prospectively followed account for, approximately, 5% of all patients with CIDP, with subset of patients suggests, considering the caveats of clinical 40 patients, our cohort provides the largest cohort in which a evaluation in monitoring disease activity in autoimmune comprehensive clinical, serologic, and treatment response neuropathies, that monitoring sNfL levels that inform about analysis has been performed. Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 9, Number 1 | January 2022 9 In conclusion, our study confirms that anti-NF155+ AN constitutes a defined subset of patients with characteristic Appendix Authors clinical, epidemiologic, and immunologic features that Name Location Contribution response to IVIg and steroids is often poor, whereas rit- Lorena Hospital de la Santa Creu i Acquisition of data, uximab is an effective therapy for most patients and that Martı́n- Sant Pau, IIB-Sant Pau performed the experiments, anti-NF155 antibody titers and sNfL levels could be used in Aguilar, MD, Barcelona, Spain analyzed the data, drafted combination to monitor clinical activity, ongoing axonal PhD the manuscript for intellectual content damage, and treatment response. Cinta Lleixà, Hospital de la Santa Creu i Acquisition of data, MSc Sant Pau, IIB-Sant Pau performed the experiments, Acknowledgment Barcelona, Spain drafted and revised the The authors would like to acknowledge the Department of manuscript for intellectual content Medicine at the Universitat Autònoma de Barcelona. Several authors of this publication are members of the European Elba Pascual- Hospital de la Santa Creu i Acquisition of data, revised Goñi, MD, Sant Pau, IIB-Sant Pau the manuscript for Reference Network for rare neuromuscular diseases (EURO- PhD Barcelona, Spain intellectual content NMD). Marta Hospital de la Santa Creu i Acquisition of data, revised Caballero, Sant Pau, Barcelona, Spain the manuscript for Study Funding MD intellectual content This work is supported by Fondo de Investigaciones San- Laura Hospital de la Santa Creu i Performed HLA itarias (FIS), Instituto de Salud Carlos III, Spain and Martı́nez- Sant Pau, Barcelona, Spain experiments and analyzed Martı́nez, the data, revised the FEDER under grant FIS19/0140 and personal grant MD, PhD manuscript for intellectual INT20/00080, personal grant GBS-CIDP Foundation to content CL, personal grant Rio Hortega CM19/00042 to LMA and Jordi Diaz- Hospital de la Santa Creu i Acquisition of samples and the ER20P3AC7624 project of the ACCI call of the Manera, MD, Sant Pau, Barcelona, Spain data. Revised the CIBERER network, Madrid, Spain. XSC was supported by PhD manuscript for intellectual content a “Sara Borrell” postdoctoral fellowship project “CD18/ 00195,” funded by Instituto de Salud Carlos III and co- Ricard Rojas- Hospital de la Santa Creu i Acquisition of samples and Garcı́a, MD, Sant Pau, Barcelona, Spain data, revised the funded by European Union (ERDF/ESF, “A way to make PhD manuscript for intellectual Europe”/“Investing in your future”). A.C. thanks the content Medical Research Council (MR/T001712/1), the Fonda- Elena Cortés- Hospital de la Santa Creu i Acquisition of samples and zione CARIPLO (2019-1836), the Italian Ministry of Vicente, MD, Sant Pau, IIB-Sant Pau data, revised the PhD Barcelona, Spain manuscript for intellectual Health-Ricerca Corrente, the Inherited Neuropathy Con- content sortium (INC) and Fondazione Regionale per la Ricerca Janina Turón- Hospital de la Santa Creu i Acquisition of samples and Biomedica for grant suport. Sans, MD Sant Pau, Barcelona, Spain data, revised the manuscript for intellectual Disclosure content L. Querol has provided expert testimony for Grifols, Sanofi- Noemı́ de IIB-Sant Pau Barcelona, Revised the manuscript for Genzyme, Novartis, UCB, Roche and CSL Behring and re- Luna, PhD Spain intellectual content ceived research funds from Novartis Spain, Sanofi-Genzyme Xavier IIB-Sant Pau Barcelona, Revised the manuscript for and Grifols. L. Martı́n-Aguilar has received speaking hono- Suárez- Spain intellectual content Calvet, PhD raria from Roche. E. Pascual-Goñi has received speaking honoraria from Roche and Biogen. J. Dı́az-Manera has Eduard IIB-Sant Pau Barcelona, Revised the manuscript for Gallardo, PhD Spain intellectual content provided expert testimony for PTC and Sanofi-Genzyme, has been external advisor for Sanofi, Sarepta and Audentes Yusuf University Hospital Acquisition of samples and Rajabally, MD Birmingham, Birmingham, data, revised the and received research funds from Sanofi-Genzyme and UK manuscript for intellectual Boehringer. G. Liberatore reported travel grants to attend content scientific meetings from CSL Behring and Kedrion. M. Sangeeta University Hospital Acquisition of samples and Cabrera-Serrano has received honoraria from Biogen for Scotton, MD Birmingham, Birmingham, data, revised the scientific translation works. K. Pitarokoili received travel UK manuscript for intellectual content grants and speakers’ honoraria from Novartis, Biogen idec, Teva, Bayer, Celgene, CSL Behring and Grifols, all not re- Bart C. Erasmus Medical Center, Acquisition of samples and Jacobs, MD, Rotterdam, The data, revised the lated to the manuscript. The other authors report no dis- PhD Netherlands manuscript for intellectual closures. Go to Neurology.org/NN for full disclosures. content Adája Baars, Erasmus Medical Center, Acquisition of samples and Publication History MD Rotterdam, The data, revised the Netherlands manuscript for intellectual Received by Neurology: Neuroimmunology & Neuroinflammation content March 2, 2021. Accepted in final form September 27, 2021. 10 Neurology: Neuroimmunology & Neuroinflammation | Volume 9, Number 1 | January 2022 Neurology.org/NN Appendix (continued) Appendix (continued) Name Location Contribution Name Location Contribution Andrea IRCCS Mondino Foundation, Acquisition of samples and Julio Pardo- Hospital Clı́nico Acquisition of samples and Cortese, MD, Pavia, Italy data, revised the Fernandez, Universitario de Santiago, data, revised the PhD manuscript for intellectual MD, PhD Santiago de Compostela, manuscript for intellectual content Spain content Elisa Vegezzi, IRCCS Mondino Foundation, Acquisition of samples and Laura Muñoz- Hospital Universitario Acquisition of samples and MD Pavia, Italy data, revised the Delgado, MD Virgen del Rocı́o, Sevilla, data, revised the manuscript for intellectual Spain manuscript for intellectual content content Romana Medical University of Acquisition of samples and Macarena Hospital Universitario Acquisition of samples and Höftberger, Vienna, Vienna, Austria data, revised the Cabrera- Virgen del Rocı́o, Sevilla, data, revised the MD manuscript for intellectual Serrano, MD, Spain manuscript for intellectual content PhD content Fritz Medical University of Acquisition of samples and Nicolau Ortiz, Hospital Universitari Sant Acquisition of samples and Zimprich, MD Vienna, Vienna, Austria data, revised the MD, PhD Joan de Reus, Tarragona, data, revised the manuscript for intellectual Barcelona manuscript for intellectual content content Cornelia Paracelsus Medical Acquisition of samples and Manuel Complejo Asistencial de Acquisition of samples and Roesler, MD University, Salzburg, Austria data, revised the Bartolomé, Ávila, Ávila, Spain data, revised the manuscript for intellectual MD manuscript for intellectual content content Eduardo Humanitas Clinical and Acquisition of samples and Özgür Akdeniz University, Antalya, Acquisition of samples and Nobile- Research Institute, Milan, data, revised the Duman, MD Turkey data, revised the Orazio, MD, Italy manuscript for intellectual manuscript for intellectual PhD content content Giuseppe Humanitas Clinical and Acquisition of samples and Vera Bril, MD Toronto General Hospital, Acquisition of samples and Liberatore, Research Institute, Milan, data, revised the University Health Network, data, revised the MD Italy manuscript for intellectual University of Toronto, manuscript for intellectual content Toronto, Canada content Hiew Fu Kuala Lumpur General Acquisition of samples and Darwin Instituto Nacional de Acquisition of samples and Liong, MD Hospital, Jalan Pahang, data, revised the Segura- Ciencias Neurológicas, data, revised the Kuala Lumpur, Malaysia manuscript for intellectual Chávez, MD Lima, Perú manuscript for intellectual content content Alicia Hospital Universitari Acquisition of samples and Kalliopi St. Josef-Hospital, Ruhr- Acquisition of samples and Martı́nez- Germans Trias i Pujol, data, revised the Pitarokoili, University Bochum, data, revised the Piñeiro, MD Badalona, Spain manuscript for intellectual MD Bochum, Germany manuscript for intellectual content content Alejandra Hospital Universitario de Acquisition of samples and Claudia Sant Joseph Hospital, Berlin, Acquisition of samples and Carvajal, MD Granada, Granada, Spain data, revised the Steen, MD Germany data, revised the manuscript for intellectual manuscript for intellectual content content Raquel Piñar- Hospital Universitario Acquisition of samples and Isabel Illa, Hospital de la Santa Creu Revised the manuscript for Morales, MD Clı́nico San Cecilio, Granada, data, revised the MD, PhD i Sant Pau, Barcelona, intellectual content Spain manuscript for intellectual Spain content Luis Querol, Hospital de la Santa Creu i Design and conceptualized Mercedes Hospital Son Llàtzer, Palma Acquisition of samples and MD, PhD Sant Pau, Barcelona, Spain the study, interpreted the Usón-Martı́n, de Mallorca, Spain data, revised the data; revised the MD manuscript for intellectual manuscript for intellectual content content Olalla Albertı́, Hospital San Jorge, Huesca, Acquisition of samples and MD Spain data, revised the manuscript for intellectual content References 1. Lehmann HC, Burke D, Kuwabara S. Chronic inflammatory demyelinating poly- Maria Hospital San Pedro, Acquisition of samples and neuropathy: update on diagnosis, immunopathogenesis and treatment. J Neurol Ángeles Logroño, Spain data, revised the Neurosurg Psychiatry. 2019;90(9):981-987. López-Pérez, manuscript for intellectual 2. Mathey EK, Park SB, Hughes RA, et al. Chronic inflammatory demyelinating poly- MD content radiculoneuropathy: from pathology to phenotype. J Neurol Neurosurg Psychiatry. 2015;86(9):973-985. Fabián Hospital Universitari Josep Acquisition of samples and 3. Querol L, Nogales-Gadea G, Rojas-Garcia R, et al. Neurofascin IgG4 antibodies in Márquez, MD Trueta, Girona, Spain data, revised the CIDP associate with disabling tremor and poor response to IVIg. Neurology. 2014: manuscript for intellectual 82(10):879-886. content 4. Delmont E, Manso C, Querol L, et al. Autoantibodies to nodal isoforms of neurofascin in chronic inflammatory demyelinating polyneuropathy. Brain. 2017;140(7):1851-1858. Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 9, Number 1 | January 2022 11 5. Querol L, Nogales-Gadea G, Rojas-Garcia R, et al. Antibodies to contactin-1 in 25. Athanasopoulos D, Motte J, Fisse AL, et al. Longitudinal study on nerve ultrasound chronic inflammatory demyelinating polyneuropathy. Ann Neurol. 2013;73(3): and corneal confocal microscopy in NF155 paranodopathy. Ann Clin Transl Neurol. 370-380. 2020;7(6):1061-1068. 6. Doppler K, Appeltshauser L, Villmann C, et al. Auto-antibodies to contactin- 26. De Simoni D, Ricken G, Winklehner M, et al. Antibodies to nodal/paranodal proteins associated protein 1 (Caspr) in two patients with painful inflammatory neuropathy. in paediatric immune-mediated neuropathy. Neurol Neuroimmunol Neuro- Brain. 2016;139(pt 10):2617-2630. inflammation. 2020;7(4):e763. 7. Pascual-Goñi E, Fehmi J, Lleixà C, et al. Antibodies to the Caspr1/contactin-1 27. Verghese A, Hiew FL, Chia YK, Querol L. Visual pathway demyelination in complex in chronic inflammatory demyelinating polyradiculoneuropathy. Brain. neurofascin-155 IGG4- positive combined central and peripheral demyelination. 2021:144(4):1183-1196. J Neurol Sci. 2019;405:196-197. 8. Pascual-Goñi E, Martı́n-Aguilar L, Querol L. Autoantibodies in chronic inflammatory 28. Allele Frequency Net Database. Accessed October 29, 2021. allelefrequencies.net demyelinating polyradiculoneuropathy. Curr Opin Neurol. 2019;32(5):651-657. 29. Ogata H, Yamasaki R, Hiwatashi A, et al. Characterization of IgG4 anti-neurofascin 9. Querol L, Devaux J, Rojas-Garcia R, Illa I. Autoantibodies in chronic inflammatory neu- 155 antibody-positive polyneuropathy. Ann Clin Transl Neurol. 2015;2(10):960-971. ropathies: diagnostic and therapeutic implications. Nat Rev Neurol. 2017:13(9):533-547. 30. Devaux JJ, Miura Y, Fukami Y, et al. Neurofascin-155 IgG4 in chronic inflammatory 10. Van den Bergh PYK, Doorn PA, Hadden RDM, et al. European Academy of demyelinating polyneuropathy. Neurology. 2016;86(9):800-807. Neurology/Peripheral Nerve Society Guideline on diagnosis and treatment of chronic 31. Hu W, Xin Y, He Z, Zhao Y. Association of neurofascin IgG4 and atypical chronic inflammatory demyelinating polyradiculoneuropathy: report of a Joint Task Force — inflammatory demyelinating polyneuropathy: a systematic review and meta‐analysis. Second Revision. J Peripher Nerv Syst. 2021:26(3):242-268. Brain Behav. 2018;8(10):e01115. 11. Painous C, López-Pérez MÁ, Illa I, Querol L. Head and voice tremor improving with 32. Martı́n-Aguilar L, Pascual-Goñi E, Lleixà C, et al. Antibodies against nodo-paranodal immunotherapy in an anti-NF155 positive CIDP patient. Ann Clin Transl Neurol. proteins are not present in genetic neuropathies. Neurology. 2020;95(4):e427-e433. 2018;5(4):499-501. 33. Guo X, Tang L, Huang Q, Tang X. A systematic review and meta-analysis of auto- 12. Kouton L, Boucraut J, Devaux J, et al. Electrophysiological features of chronic in- antibodies for diagnosis and prognosis in patients with chronic inflammatory de- flammatory demyelinating polyradiculoneuropathy associated with IgG4 antibodies myelinating Polyradiculoneuropathy. Front Neurosci. 2021:15:637336. targeting neurofascin 155 or contactin 1 glycoproteins. Clin Neurophysiol. 2020; 34. Oh SJ, Morgan MB, Lu L, et al. Different characteristic phenotypes according to 131(4):921-927. antibody in myasthenia gravis. J Clin Neuromuscul Dis. 2012;14(2):57-65. 13. Martinez-Martinez L, Lleixà MC, Boera-Carnicero G, et al. Anti-NF155 chronic in- 35. Van De Veen W, Stanic B, Yaman G, et al. IgG4 production is confined to human IL- flammatory demyelinating polyradiculoneuropathy strongly associates to HLA- 10-producing regulatory B cells that suppress antigen-specific immune responses. DRB15. J Neuroinflammation. 2017;14(1):1-6. J Allergy Clin Immunol. 2013;131(4):1204-1212. 14. Querol L, Rojas-Garcı́a R, Diaz-Manera J, et al. Rituximab in treatment-resistant CIDP 36. Joly P, Mouquet H, Roujeau J-C, et al. A single cycle of rituximab for the treatment of with antibodies against paranodal proteins. Neurol Neuroimmunol Neuroinflamm. severe pemphigus. N Engl J Med. 2007;357(6):545-552. 2015;2(5):e149. 37. Dı́az-Manera J, Martı́nez-Hernández E, Querol L, et al. Long-lasting treatment effect 15. Cortese A, Lombardi R, Briani C, et al. Antibodies to neurofascin, contactin-1, and of rituximab in MuSK myasthenia. Neurology. 2012;78(3):189-193. contactin-associated protein 1 in CIDP: clinical relevance of IgG isotype. Neurol 38. Marino M, Basile U, Spagni G, et al. Long-Lasting rituximab-induced reduction of Neuroimmunol Neuroinflamm. 2020;7(1):e639. specific—but not total—IgG4 in MuSK-positive myasthenia gravis. Front Immunol. 16. Manso C, Querol L, Lleixà C, et al. Anti-neurofascin-155 IgG4 antibodies prevent 2020;11:1-9. paranodal complex formation in vivo. J Clin Invest. 2019;129(6):2222-2236. 39. Svahn J, Petiot P, Antoine JC, et al. Anti-MAG antibodies in 202 patients: clinico- 17. Koike H, Kadoya M, Kaida KI, et al. Paranodal dissection in chronic inflammatory pathological and therapeutic features. J Neurol Neurosurg Psychiatry. 2018;89(5): demyelinating polyneuropathy with anti-neurofascin-155 and anti-contactin-1 anti- 499-505. bodies. J Neurol Neurosurg Psychiatry. 2017;88(6):465-473. 40. Dalakas MC, Rakocevic G, Salajegheh M, et al. Placebo-controlled trial of rituximab in 18. Fukami Y, Iijima M, Koike H, Yamada S, Hashizume A, Katsuno M. Association of IgM anti-myelin-associated glycoprotein antibody demyelinating neuropathy. Ann serum neurofilament light chain levels with clinicopathology of chronic inflammatory Neurol. 2009;65(3):286-293. demyelinating polyneuropathy, including NF155 reactive patients. J Neurol. 2021: 41. Stengel H, Vural A, Brunder AM, et al. Anti-pan-neurofascin IgG3 as a marker of 268(10):3835-3844. fulminant autoimmune neuropathy. Neurol Neuroimmunol Neuroinflamm. 2019;6(5): 19. Van den Bergh PYK, Hadden RDM, Bouche P, et al. European Federation of Neu- e603. rological Societies/Peripheral Nerve Society Guideline on management of chronic 42. Kawamura N, Yamasaki R, Yonekawa T, et al. Anti-neurofascin antibody in patients inflammatory demyelinating polyradiculoneuropathy: Report of a Joint Task Force of with combined central and peripheral demyelination. Neurology. 2013;81(8):714-722. the European Federation of Neurological Societies and the Peripher. Eur J Neurol. 43. Lieverloo GGA, Wieske L, Verhamme C, et al. Serum neurofilament light chain in 2010;17:356-363. chronic inflammatory demyelinating polyneuropathy. J Peripher Nerv Syst. 2019; 20. Breiner A, Bourque PR, Allen JA. Updated cerebrospinal fluid total protein reference 24(2):187-194. values improve chronic inflammatory demyelinating polyneuropathy diagnosis. 44. Sandelius Å, Zetterberg H, Blennow K, et al. Plasma neurofilament light chain con- Muscle Nerve. 2019;60(9):180-183. centration in the inherited peripheral neuropathies. Neurology. 2018;90(6):e518-e524. 21. Quinn TJ, Dawson J, Walters MR, Lees KR. Functional outcome measures in con- 45. Kapoor M, Foiani M, Heslegrave A, et al. Plasma neurofilament light chain concen- temporary stroke trials. Int J Stroke. 2009;4(9):200-205. tration is increased and correlates with the severity of neuropathy in hereditary 22. Van Nes SI, Vanhoutte EK, Van Doorn PA, et al. Rasch-built Overall Disability Scale (R- transthyretin amyloidosis. J Peripher Nerv Syst. 2019:24(4):314-319. ODS) for immune-mediated peripheral neuropathies. Neurology. 2011;76(9):337-345. 46. Niks EH, Kuks JBM, Roep BO, et al. Strong association of MuSK antibody-positive 23. Querol L, Siles AM, Alba-Rovira R, et al. Antibodies against peripheral nerve antigens in myasthenia gravis and HLA-DR14-DQ5. Neurology. 2006;66(11):1772-1774. chronic inflammatory demyelinating polyradiculoneuropathy. Sci Rep. 2017;7(9):14411. 47. Huijbers MG, Querol LA, Niks EH, et al. The expanding field of IgG4-mediated 24. Martı́n-Aguilar L, Camps-Renom P, Lleixà C, et al. Serum neurofilament light chain neurological autoimmune disorders. Eur J Neurol. 2015;22(8):1151-1161. predicts long-term prognosis in Guillain-Barré syndrome patients. J Neurol Neurosurg 48. Kim TJ, Lee ST, Moon J, et al. Anti-LGI1 encephalitis is associated with unique HLA Psychiatry. 2021;92:70-77. subtypes. Ann Neurol. 2017;81(2):183-192. 12 Neurology: Neuroimmunology & Neuroinflammation | Volume 9, Number 1 | January 2022 Neurology.org/NN