ARTICLE OPEN ACCESS Serum Contactin-1 in CIDP A Cross-Sectional Study Luuk Wieske, MD, PhD, Lorena Martı́n-Aguilar, MD, Janev Fehmi, MD, Cinta Lleixà, MD, Correspondence Dr. Wieske Marleen J.A. Koel-Simmelink, PhD, Madhurima Chatterjee, PhD, Zoë van Lierop, MD, Joep Killestein, MD, PhD, l.wieske@amsterdamumc.nl Camiel Verhamme, MD, PhD, Luis Querol, MD, PhD, Simon Rinaldi, MD, PhD, Charlotte E. Teunissen, PhD,* and Filip Eftimov, MD, PhD* Neurol Neuroimmunol Neuroinflamm 2021;8:e1040. doi:10.1212/NXI.0000000000001040 Abstract MORE ONLINE Objective Class of Evidence To investigate whether serum levels of contactin-1, a paranodal protein, correlate with para- Criteria for rating nodal injury as seen in patients with CIDP with antibodies targeting the paranodal region. therapeutic and diagnostic studies Methods NPub.org/coe Serum contactin-1 levels were measured in 187 patients with CIDP and 222 healthy controls. Paranodal antibodies were investigated in all patients. Results Serum contactin-1 levels were lower in patients (N = 41) with paranodal antibodies compared with patients (N = 146) without paranodal antibodies (p < 0.01) and showed good discrimi- nation between these groups (area under the curve 0.84; 95% CI: 0.76–0.93). Conclusions These findings suggest that serum contactin-1 levels have the potential to serve as a possible diagnostic biomarker of paranodal injury in CIDP. Classification of Evidence This study provides class II evidence that serum contactin-1 levels can discriminate between patients with CIDP with or without paranodal antibodies with a sensitivity of 71% (95% CI: 56%–85%) and a specificity of 97% (95% CI: 83%–100%). *These authors contributed equally to this work. From the Department of Neurology and Neurophysiology (L.W., C.V., F.E.), Amsterdam Neuroscience, Amsterdam UMC, Location AMC, Amsterdam, the Netherlands; Neuromus- cular Diseases Unit (L.M.-A., C.L., L.Q.), Department of Neurology, Hospital de La Santa Creu i Sant Pau, Universitat Autònoma de Barcelona, Spain; Department of Clinical Neurosciences (J.F., S.R.), West Wing, John Radcliffe Hospital, Oxford, United Kingdom; Neurochemistry Lab (M.J.A.K.-S., C.E.T.), Department of Clinical Chemistry, Amsterdam Neuroscience, Amsterdam UMC, Vrije Universiteit, Amsterdam, the Netherlands; and Department of Neurology (M.C., Z.L., J.K.), Amsterdam Neuroscience, Amsterdam UMC, Location VU Medical Center, Amsterdam, the Netherlands. Go to Neurology.org/NN for full disclosures. Funding information is provided at the end of the article. Statistics were performed by the principal author (L. Wieske). 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 AUC = area under the curve; CNTN1 = contactin-1; IVIg = IV immunoglobulin; ROC = receiver operating characteristic; sCNTN1 = serum levels of contactin-1. Most patients with CIDP show a good response to first-line > 15% and outliers were repeated and were excluded if the CV treatments like corticosteroids or IV immunoglobulin (IVIg). remained >15% (N =1). For sCNTN1 measurements below However, several reports have emphasized that patients with the LLOQ, a value was assigned of half of the manufacturer’s CIDP and paranodal antibodies have poor response to first-line reported LLOQ (i.e., 1.8 pg/mL). treatments. Early identification of these patients is important to guide treatment decisions and prevent long-term axonal damage. The presence of paranodal antibodies was determined at the time of sampling in Barcelona (for the Amsterdam and Bar- Contactin-1 (CNTN1) is an axonal protein that anchors par- celona cohort) and Oxford (for the Oxford cohort) using anodal myelin in complex with contactin-associated protein 1 dedicated cell-based assays and ELISAs against NF-155, NF- (Caspr1) and neurofascin-155 (NF-155).1 Pathogenic IgG4 186, CNTN1, and Caspr1.7 antibodies targeting these paranodal proteins are found in up to 10%.2 The CNTN1 (protein) exists in a soluble form making it Statistical Analysis good biomarker candidate for paranodal damage.3 Decreased A receiver operating characteristic (ROC) curve with area CNTN1 levels have been found in other demyelinating dis- under the curve (AUC) was used to investigate the discrim- orders such as MS and neuromyelitis optica.4,5 We hypothesize inatory potential of sCNTN1 for the presence or absence of that paranodal injury in CIDP leads to altered serum levels of paranodal antibodies. The Youden index was used to select contactin-1 (sCNTN1) in patients with CIDP with paranodal the optimal cutoff point for the ROC curve. Data were ana- antibodies compared to CIDP patients without. lyzed using R, version 3.6.2. Data Availability Methods The data that support the findings of this study are available from the corresponding author on reasonable request. Patients were selected from cohorts from 3 CIDP tertiary re- ferral centers in the Netherlands (Amsterdam), Spain (Barce- lona), and the United Kingdom (Oxford). The Amsterdam cohort comprised patients who were included in ongoing Results prospective CIDP cohort studies (N = 103). The Barcelona (N A total of 188 patients with CIDP and 222 healthy controls = 55) and Oxford (N = 30) cohorts comprised nonconsecutive were included. One measurement in a patient with CIDP was patients who were referred because of suspected antibody- excluded due to high CV. Clinical data for patients with CIDP mediated CIDP. All patients fulfilled the definite or probable can be found in the table. Paranodal antibodies were found in EFNS/PNS criteria.6 Samples were collected during different 41 (22%) patients, NF-155 antibodies in 18 patients, CNTN1 disease stages. In addition, 222 healthy controls were included. antibodies in 13, Caspr1 antibodies in 6, and NF-140 anti- bodies in 4. Treatment status at sampling is shown in the table. Standard Protocol Approvals, Registrations, and Patient Consents sCNTN1 in Healthy Controls The study was approved by the local medical ethical com- In 222 healthy controls (mean age 46 years; SD 14; range mittees of participating centers. All patients provided signed 19–98 years), median sCNTN1 levels were 12,470 pg/mL informed consent. (IQR 10,160–14,440 pg/mL). sCNTN1 levels were not as- sociated with age (r: −0.07 95% CI: −0.20 to 0.06). Serum Measurements of CNTN1 and Antibodies sCNTN1 in Patients With and Without Isolated serum was stored at −80°C in each center. sCNTN1 Paranodal Antibodies levels were measured centrally in the Neurochemistry Labo- The figure shows sCNTN1 levels for patients with or without ratory at Amsterdam UMC on Bio-Plex 200 system (Bio-Rad paranodal antibodies. Median sCNTN1 levels were lower in Laboratories, Veenendaal, The Netherlands) using the Hu- patients with paranodal antibodies compared with patients man Magnetic Luminex Assay (LXSAHM; R&D Systems, without antibodies (p < 0.01) and lowest in patients with Minneapolis, MN) according to the manufacturer’s instruc- CNTN1 antibodies. All 6 samples that were below the LLOQ tions. Samples were randomized and analyzed in duplicate in a were present in this subgroup (figure). Compared with healthy 6-fold dilution, blinded for the presence of paranodal anti- controls, median sCNTN1 levels were lower in patients with bodies. Assay validation is described elsewhere.4 The intra‐ CIDP, both in patients without (p:0.04) and in patients with assay CV was 3.2%, and measurements with an intra-assay CV paranodal antibodies (p < 0.01). There was no correlation 2 Neurology: Neuroimmunology & Neuroinflammation | Volume 8, Number 5 | September 2021 Neurology.org/NN for the presence of paranodal antibodies in patients with CIDP. Table Clinical Data of Patients With CIDP Serum measurements of CNTN1 may therefore aid in di- Patients agnosing paranodal CIDP mediated by IgG4 antibodies. Testing without Patients with for CIDP antibodies is not yet widely available, which can delay paranodal paranodal antibodies antibodies diagnosis, whereas sCNTN1 level testing is simple, cheap, and (N = 146) (N = 41) fast to perform. The high specificity of sCNTN1 in combination Age (y), mean (±SD)a 60 (±15) 55 (±16) with the low overall prevalence of paranodal antibodies (esti- mated at 10%) indicates that normal sCNTN1 values may have a Male, n (%)b 101 (69%) 32 (80%) high negative predictive value and may therefore by used as a Clinical phenotypec screening assay preceding focused assays testing for the presence of specific paranodal antibodies in patients with CIDP with a Classic, n (%) 121 (84%) 26 (74%) clinical picture suggesting the presence of paranodal antibodies. Acute onset classic, n (%) 14 (10%) 10 (29%) Also, low or undetectable sCNTN1 may guide early treatment Asymmetric, n (%) 15 (10%) 0 choices as traditional first-line treatments are frequently in- effective in paranodal CIDP mediated by IgG4 antibodies.8 Sensory predominant, n (%) 3 (2%) 3 (9%) Motor predominant, n (%) 4 (3%) 2 (6%) Other studies in demyelinating disorders of the CNS also found decreased sCNTN1 levels in serum, although not as low as seen Distal predominant, n (%) 2 (1%) 4 (11%) in patients with CIDP with paranodal antibodies.4,5 Although Presence of paranodal antibodies we found a significant difference on group level between pa- NF-155 +, n (%) 18 (44%) tients with CIDP without paranodal antibodies and healthy controls, the difference was small with considerable overlap in CNTN1 +, n (%) 13 (32%) sCNTN1 levels between patients with CIDP without para- Caspr1 +, n (%) 6 (14%) nodal antibodies and healthy controls. It is therefore unlikely NF-186 +, n (%) 4 (10%) that sCNTN1 can be used to confirm the diagnosis of CIDP in general. Longitudinal studies during various disease stages are Treatment status at moment needed to further study the potential role of sCNTN1 as of samplingd biomarker of disease activity in patients with CIDP with and Untreated 53 (37%) 5 (13%) without paranodal antibodies. Receiving treatment 92 (63%) 33 (87%) In this study, there was uneven recruitment of patients across Abbreviations: CNTN1 = contactin-1; Caspr1 = contactin-1 associated protein different states of disease activity reflected that could partly 1; NF-155 = neurofascin-155; NF-186 = neurofascin-186. a Missing for 6 patients. contribute to the results. The lack of patients with acute neu- b Missing for 1 patient. c Missing for 7 patients. ropathies such as the Guillain-Barre syndrome can also be seen d Missing for 4 patients. as limitation of this study. Also, in the subgroup of patients with paranodal antibodies, there is a risk of selection bias due to nonconsecutive recruitment in some of the cohorts. We cannot between sCNTN1 levels and antibody titers at the time of rule out interference with our assay by CNTN1 antibodies measurement, including in those patients with CNTN1 anti- binding to the antigen at the same epitope as the assay anti- bodies (data not shown). For discriminating between the bodies as an explanation for the low and unmeasurable presence and absence of paranodal antibodies, the AUC of the sCNTN1 levels seen in this subgroup. We presume that assay ROC curve was 0.84 (95% CI: 0.76–0.93). The associated interference is not a major factor influencing our results be- optimal cutoff value was 5,810 pg/mL, indicating that lower cause sCNTN1 levels were also distinctly reduced in patients values had a sensitivity 71% (95% CI: 56%–85%) and a spec- with other paranodal antibodies and because we did not find a ificity of 97% (95% CI: 83%–100%) to discriminate between correlation between sCNTN1 and antibody titers. CIDP with or without paranodal antibodies. Classification of Evidence This study provides class II evidence that serum contactin-1 Conclusion levels can discriminate between patients with CIDP with or These findings indicate that serum contactin-1 level is a prom- without paranodal antibodies with a sensitivity of 71% (95% ising new diagnostic biomarker of paranodal injury in CIDP. CI: 56%–85%) and a specificity of 97% (95% CI: 83%–100%). Study Funding Discussion This study was supported by an Amsterdam UMC Neuro- We found that sCNTN1 levels were distinctly lower in patients science Alliance grant. The sponsor was not involved in de- with CIDP with paranodal antibodies and were highly specific sign, analysis, interpretation, or writing of the manuscript. Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 8, Number 5 | September 2021 3 Figure Serum Contactin-1 Levels in Patients With CIDP With or Without Paranodal Antibodies Serum contactin-1 (sCNTN1) levels in healthy controls (gray) and patients with CIDP (black) categorized according to the presence or absence of the dif- ferent nodal and paranodal antibodies. The black line indicates the median per group. CNTN1 = contactin-1; Caspr1 = contactin-1–associated protein 1; NF- 155 = neurofascin-155; NF186 = neurofascin186. Disclosure Laboratory Immunology of Oxford University Hospitals. He is L. Wieske received research grants from Grifols and the GBS/ UK Chief Investigator for the ADHERE trial and has received CIDP Foundation for the study of disease activity biomarkers consultancy payments from Argenx for the preparation and in CIDP. L. Martı́n-Aguilar has received speaking honoraria delivery of presentations regrading this trial. C.E. Teunissen’s from Roche. J. Fehmi is funded by the GBS/CIDP Founda- research is supported by the European Commission (Marie tion International Benson Fellowship (Grant 1709HM001/ Curie International Training Network, JPND), Health Hol- SB17) and previously received funding from the Guarantors land, the Dutch Research Council (ZonMW), Alzheimer Drug of Brain entry fellowship award. C. Lleixà, M.J.A. Koel- Discovery Foundation, The Selfridges Group Foundation, Simmelink, M. Chatterjee, and Z. van Lierop report no dis- Alzheimer Netherlands, and Alzheimer Association. She has a closures relevant to the manuscript. J. Killestein has con- collaboration contract with ADx Neurosciences and Quanterix, ducted contracted MS research for F. Hoffmann-La Roche performed contract research, or received grants from Axon Ltd, Biogen, Teva, Merck, Novartis, and Sanofi/Genzyme. C. Neuroscience, Brainstorm Therapeutics, Celgene, EIP Pharma, Verhamme is a member of a clinical advisory board (CAB) of iEsai, Roche, and Toyama. F. Eftimov reports grants from Inflectis France; payment was done to his organization for ZonMw (Dutch Governmental Agency) and Prinses Beatrix attending a CAB meeting, outside the submitted work. L. Spierfonds (Dutch Charity Organization) and grants from CSL Querol supported by Fondo de Investigaciones Sanitarias Behring, Kedrion, Terumo BCT, and Takeda Pharmaceutical (FIS), Instituto de Salud Carlos III, Spain, and FEDER under Company, outside the submitted work. Grants were paid to grant FIS19/01407 has provided expert testimony for Grifols, institution and are used for investigator-initiated randomized Sanofi-Genzyme, Novartis, UCB, Roche, and CSL Behring controlled trials and studies within INCbase, an international and received research funds from Novartis Spain, Sanofi- CIDP registry. In addition, he received consultancy fee from Genzyme, and Grifols. S. Rinaldi has received speaker’s UCB Pharma, paid to institution, outside the submitted work. honoraria from Fresenius, Alnylam and Excemed and pay- Go to Neurology.org/NN for full disclosures. ments to provide expert medicolegal advice on inflammatory neuropathies and their treatment. He has received compli- Publication History mentary registration and prize money from the Peripheral Received by Neurology: Neuroimmunology & Neuroinflammation Nerve Society and a travel bursary from the European School December 17, 2020. Accepted in final form May 20, 2021. of Neuroimmunology. He has received research funding from the Pathological Society of Great Britain and Ireland, the Wellcome Trust, the Academy of Medical Sciences, the Appendix Authors Medical Research Council (United Kingdom), the University of Oxford (John Fell Fund, COVID-19 Internal Research Name Location Contribution Fund), and the British Medical Association. He is a member of Luuk Wieske, Amsterdam UMC, location Designed and the GBS and Associated Inflammatory Neuropathies (GAIN) MD, PhD AMC, Amsterdam, the conceptualized the study; Netherlands major role in the patient charity medical advisory board. He runs a not-for- acquisition of data; profit nodal/paranodal antibody testing service at the Nuffield analyzed the data; and drafted the manuscript for Department of Clinical Neurosciences, John Radcliffe Hos- intellectual content pital, Oxford, United Kingdom, in partnership with Clinical 4 Neurology: Neuroimmunology & Neuroinflammation | Volume 8, Number 5 | September 2021 Neurology.org/NN Appendix (continued) Appendix (continued) Name Location Contribution Name Location Contribution Lorena Hospital de la Santa Creu i Major role in the Luis Querol, Hospital de la Santa Creu i Sant Interpreted the data and Martı́n- Sant Pau, Universitat acquisition of data; MD, PhD Pau, Universitat Autònoma de revised the manuscript for Aguilar, MD Autònoma de Barcelona, interpreted the data; and Barcelona, Spain intellectual content Spain revised the manuscript for intellectual content Simon John Radcliffe Hospital, Interpreted the data and Rinaldi, MD, Oxford, United Kingdom revised the manuscript for Janev Fehmi, John Radcliffe Hospital, Major role in the PhD intellectual content MD Oxford, United Kingdom acquisition of data; interpreted the data; and Charlotte E. Amsterdam UMC, location Designed and revised the manuscript for Teunissen, Vrije Universiteit, Amsterdam, conceptualized the study; intellectual content PhD the Netherlands analyzed the data; and drafted the manuscript for Cinta Lleixà, Hospital de la Santa Creu i Major role in the intellectual content MD Sant Pau, Universitat acquisition of data; Autònoma de Barcelona, interpreted the data; and Filip Eftimov, Amsterdam UMC, location Designed and Spain revised the manuscript for MD, PhD AMC, Amsterdam, the conceptualized the study; intellectual content Netherlands analyzed the data; and drafted the manuscript for Marleen J.A. Amsterdam UMC, location Major role in the intellectual content Koel- Vrije Universiteit, Amsterdam, acquisition of data; Simmelink, the Netherlands interpreted the data; and PhD revised the manuscript for References intellectual content 1. Faivre-Sarrailh C, Devaux JJ. Neuro-glial interactions at the nodes of Ranvier: im- plication in health and diseases. Front Cell Neurosci. 2013;7:196. Madhurima Amsterdam UMC, location Major role in the 2. Bunschoten C, Jacobs BC, Van den Bergh PYK, Cornblath DR, van Doorn PA. Chatterjee, Vrije Universiteit, Amsterdam, acquisition of data; Progress in diagnosis and treatment of chronic inflammatory demyelinating poly- PhD the Netherlands interpreted the data; and radiculoneuropathy. Lancet Neurol. 2019;18(8):784-794. revised the manuscript for 3. Durbec P, Gennarini G, Goridis C, Rougon G. A soluble form of the F3 neuronal cell intellectual content adhesion molecule promotes neurite outgrowth. J Cell Biol. 1992;117(4):877-887. 4. van Lierop ZY, Wieske L, Koel-Simmelink MJ, et al. Serum contactin-1 as a biomarker Zoë van Amsterdam UMC, location Interpreted the data and of long-term disease progression in natalizumab-treated multiple sclerosis. Mult Scler Lierop, MD Vrije Universiteit, Amsterdam, revised the manuscript for J. 2021:135245852110100. the Netherlands intellectual content 5. Ai N, Liu H, Zhou H, et al. Cytokines and chemokines expression in serum of patients with neuromyelitis optica. Neuropsychiatr Dis Treat. 2019;15:303-310. Joep Amsterdam UMC, location Designed and 6. Joint Task Force of the EFNS and the PNS, Van den Bergh PYK, Hadden RDM, Illa AHI, Killestein, Vrije Universiteit, Amsterdam, conceptualized the study et al. European Federation of Neurological Societies/Peripheral Nerve Society guideline MD, PhD the Netherlands and revised the on management of chronic inflammatory demyelinating polyradiculoneuropathy: report manuscript for intellectual of a joint task force of the European Federation of Neurological Societies and the Peripher. content Eur J Neurol. 2010;17(4):356-363. 7. Martı́n-Aguilar L, Pascual-Goñi E, Lleixà C, et al. Antibodies against nodo-paranodal Camiel Amsterdam UMC, location Interpreted the data and proteins are not present in genetic neuropathies. Neurology. 2020;95(4):e427-e433. Verhamme, AMC, Amsterdam, the revised the manuscript for 8. Querol L, Rojas-Garcı́a R, Diaz-Manera J, et al. Rituximab in treatment-resistant CIDP MD, PhD Netherlands intellectual content with antibodies against paranodal proteins. Neurol Neuroimmunol Neuroinflammation. 2015;2(5):e149. Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 8, Number 5 | September 2021 5