Neurimminfl2018018622 1..3

Volume 6, Number 2, March 2019 Neurology.org/NN

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CLINICAL/SCIENTIFIC NOTE Balint syndrome in anti-NMDA receptor encephalitis e532

ARTICLE Real-world validation of the 2017 McDonald criteria for pediatric MS e528

ARTICLE HIV is associated with endothelial activation despite ART, in a sub-Saharan African sett ing e531

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Articles

e538 Anti-MOG encephalitis mimicking small vessel CNS vasculitis K. Patterson, E. Iglesias, M. Nasrallah, V. Gonz´alez-Alvarez,´ M. Suñol, J. Anton, A. Saiz, E. Lancaster, and T. Armangue Open Access Editorial, p. e544

e528 Real-world validation of the 2017 McDonald criteria for pediatric MS Y.Y.M. Wong, C.L. de Mol, R.M. van der Vuurst de Vries, E.D. van Pelt, I.A. Ketelslegers, C.E. Catsman-Berrevoets, R.F. Neuteboom, and R.Q. Hintzen, On behalf of the Dutch pediatric MS and ADEM study group Open Access Class of Evidence

e529 Mouse model of anti-NMDA receptor post–herpes simplex encephalitis J. Linnoila, B. Pulli, T. Armangu´e, J. Planagum`a, R. Narsimhan, S. Schob, M.W.G. Zeller, J. Dalmau, and J. Chen Open Access

e530 MRI phenotypes in MS: Longitudinal changes and miRNA signatures C.C. Hemond, B.C. Healy, S. Tauhid, M.A. Mazzola, F.J. Quintana, R. Gandhi, H.L. Weiner, and R. Bakshi Open Access

e531 HIV is associated with endothelial activation despite ART, in a sub-Saharan African setting J. Kamtchum-Tatuene, H. Mwandumba, Z. Al-Bayati, J. Flatley, M. Griffiths, T. Solomon, and L. Benjamin Open Access

e535 Inﬂammatory myopathy with myasthenia gravis: Thymoma association and polymyositis pathology N. Uchio, K. Taira, C. Ikenaga, M. Kadoya, A. Unuma, K. Yoshida, S. Nakatani-Enomoto, Y. Hatanaka, Y. Sakurai, Y. Shiio, K. Kaida, A. Kubota, T. Toda, and J. Shimizu Editor’s Corner Open Access e543 Cranial nerve involvement in patients with MOG e540 Rectifying neurologic diagnosis through – autoantibodies antibody associated disease A. Cobo-Calvo, X. Ayrignac, P. Kerschen, P. Horellou, F. Cotton, J. Dalmau P. Labauge, S. Vukusic, K. Deiva, C. Serguera, and R. Marignier Open Access Open Access

Editorial Clinical/Scientific Notes e544 Diagnostic challenges and pitfalls of myelin e533 pRNFL as a marker of disability worsening in the – oligodendrocyte glycoprotein antibody associated medium/long term in patients with MS demyelination: Lessons from neuropathology C. Cordano, B. Nourbakhsh, M. Devereux, V. Damotte, D. Bennett, R. Hoftberger¨ and E. Gelpi S.L. Hauser, B.A.C. Cree, J.M. Gelfand, and A.J. Green Open Access Companion article, p. e538 Open Access

Continued TABLE OF CONTENTS Volume 6, Number 2, March 2019 Neurology.org/NN

e536 Episodic ataxia in CASPR2 autoimmunity A.S. Lopez´ Chiriboga and S. Pittock Open Access Video e537 Anti-NMDAR encephalitis with concomitant varicella zoster virus detection and nonteratomatous malignancy P.A. Prakash, J. Jin, K. Matharu, T. Garg, W.C. Tsai, P. Patel, J.E. Weatherhead, and J.S. Kass Open Access Cover image e539 A fatal case of daclizumab-induced liver failure in a Pathologic ﬁndings included inﬁltrates of T and B lymphocytes in patient with MS association with edema, perivascular demyelination, and reactive gliosis M. Stettner, C.C. Gross, A.K. Mausberg, R. Pul, A. Junker, H.A. Baba, (luxol fast blue staining). Stylized by Andrea Rahkola, Neurology A. Schulte-Mecklenbeck, H. Wiendl, C. Kleinschnitz, and S.G. Meuth Production Editor. See “Anti-MOG encephalitis mimicking small vessel Open Access CNS vasculitis.” See page e538 e542 Rituximab during pregnancy in neuromyelitis optica: A case report J. Miranda-Acuña, E. Rivas-Rodr´ıguez, M. Levy, M. Ansari, R. Stone, V. Patel, and L. Amezcua Open Access ’ Volume 6, Number 2, March 2019 Editor sCorner Neurology.org/NN

Josep Dalmau, MD, PhD, Editor, Neurology® Neuroimmunology & Neuroinﬂammation Rectifying neurologic diagnosis through autoantibodies

Neurol Neuroimmunol Neuroinﬂamm March 2019 vol. 6 no. 2 e540. doi:10.1212/NXI.0000000000000540

Tools that improve our ability to provide patients with a speedy and accurate diagnosis can come in many forms, and I have chosen several articles in this issue of N2 to highlight this point.

The 2017 McDonald diagnostic criteria for MS include modifications of the 2010 criteria to MORE ONLINE facilitate an earlier diagnosis of MS while preserving the accuracy of the previous criteria. Up to Editor Summary 10% of patients with MS have their first symptoms before the age of 18 years. However, MS NPub.org/N2/edsum diagnosis based on the baseline MRI obtained during the first attack of an acquired demyelinating syndrome (ADS) in children younger than 12 years or in those presenting with acute disseminated encephalomyelitis (ADEM) should be considered with caution because of the lower specificity and sensitivity of the McDonald 2010 criteria in these 2 groups. In the current issue of N2, Wong et al.1 compared the diagnostic accuracy of the 2017 and 2010 McDonald criteria in children with ADS at first presentation paying particular attention to patients younger than 12 or presenting with ADEM. Among 164 patients, 110 (67%) presented without encephalopathy and 54 (33%) with encephalopathy or ADEM. Of the 110 ADS patients, 47% were diagnosed with clinically definite MS within a median follow-up of 4.5 years. The sensitivity was higher for the 2017 criteria than for the 2010 criteria (83% vs 49%), but the specificity was lower (73% vs 87%). At baseline, 48 patients fulfilled the 2017 criteria and 27 patients fulfilled the 2010 criteria. For ADEM cases, 10% fulfilled the 2017 criteria and 8% fulfilled the 2010 criteria. The authors conclude that the 2017 criteria are more sensitive than the 2010 criteria for predicting definite MS at baseline; these criteria can be applied in children younger than 12 years without encephalopathy, but not in children with ADEM. As the authors indicate, this study has several limitations related to the choice of brain and spinal MRI, inclusion of contrast or not, and the decision to include CSF testing for oligoclonal bands, which were left to the discretion of the treating physicians. However, the data suggest that the McDonald 2017 criteria perform well in children.

In another study, Patterson et al.2 describe 2 patients diagnosed with small-vessel primary CNS vasculitis who, in fact, had myelin oligodendrocyte glycoprotein (MOG) antibody-associated encephalitis. In both cases, the initial diagnosis was influenced by biopsy findings showing interstitial and perivascular lymphocytic infiltrates affecting but not confined to the vessel wall. As indicated by the authors, and the accompanying editorial comment, the presence of inflammatory cells in the vessel wall is sufficient for the diagnosis of medium- or large-sized vessel vasculitis; however, for small vessel vasculitis, the diagnostic criteria require evi- denceofvesseldamagesuchasfibrin deposition or necrosis, which were absent in both patients. In these patients, an earlier diagnosis of MOG-associated encephalitis would have prevented the brain biopsy. Additionally, important implications were illustrated by one of the patients, who despite treatment with cyclophosphamide (aimed at the diagnosis of vasculitis) was having clinical relapses; these resolved after the diagnosis of MOG antibody-associated encephalitis was made and a B-cell depletion therapy was implemented. The authors indicate that there are previous reports of patients with CNS small vessel

Funding information and disclosures are provided at the end of the article. Full disclosure form information provided by the authors is available with the full text of this article at Neurology.org/NN. 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 © 2019 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1 vasculitis who later developed symptoms compatible with (e.g., NMO, anti-NMDA receptor encephalitis) is strongly MOG-antibody-associated syndromes (myelitis or optic encouraged. neuritis), suggesting that this diagnostic confusion may not be unusual. In addition to these studies, the March issue of N2 contains an infographic summary of the study “Characterization of MS Autoimmune episodic ataxia (EA) has been reported in phenotypes using MRI and miRNA profiles” by Hemond patients with Caspr2 antibody-associated encephalitis.3 In et al.,8 as well as other interesting articles that I hope will catch the current issue, Drs. Lopez Chiriboga and Pittock4 report your attention. a 64-year-old man who developed cognitive dysfunction and seizures, followed by episodes of transient speech and gait Study funding disturbances lasting several minutes and occurring multiple No targeted funding. times per day. The brain MRI was normal, and CSF analysis showed only elevated protein concentration. The diagnosis Disclosure of Caspr2 antibody-associated encephalitis led to treatment J. Dalmau is the editor of Neurology: Neuroimmunology & with immunotherapy and dramatic improvement of symp- Neuroinflammation, is on the editorial board for Neurology toms. Overall, considering this and a previous report of 6 UpToDate; holds patents for and receives royalties from patients, EA should be added to the list of paroxysmal neu- Ma2 autoantibody test, NMDA receptor autoantibody test, rologic symptoms caused by autoantibodies, such as facio- GABA(B) receptor autoantibody test, GABA(A) receptor brachial dystonic seizures and anti-LGI1 antibodies, and autoantibody test, DPPX autoantibody test, and IgLON5 orthostatic myoclonus and anti-Caspr2 antibodies. In con- autoantibody test; and receives research support from Euro- trast to faciobrachial dystonic seizures that usually precede immun, NIH, Fundació CELLEX, Instituto Carlos III limbic encephalitis, Caspr2-antibody associated EA more (CIBERER, and Fondo de Investigaciones Sanitarias). Full often develops after, or concomitantly with, limbic enceph- disclosure form information provided by the authors is avail- alitis. The similarities between autoimmune and genetic EA able with the full text of this article at Neurology.org/NN. type I, caused by mutations of KCNA1 that codes for the voltage-gated potassium channel Kv1.1 and interacts with Publication history Caspr2, have been previously noted. One suspects that if Received by Neurology: Neuroimmunology & Neuroinflammation January “ ” a forme fruste of Caspr2-antibody associated encephalitis 4, 2019. Accepted in final form January 4, 2019. manifests as isolated or predominant EA, the diagnosis may be missed. References 1. Wong YYM, Louk de Mol C, van der Vuurst de Vries RM, et al. Real world validation 5 of the 2017 McDonald criteria for pediatric multiple sclerosis. Neurol Neuroimmunol Miranda-Acuña et al. report the favorable outcome of Neuroinflamm 2019;6:e528. doi: 10.1212/NXI.0000000000000528. a patient with neuromyelitis optica (NMO) treated during 2. Patterson K, Iglesias E, Nasrallah M, et al. Anti-MOG encephalitis mimicking small vessel CNS vasculitis. Neurol Neuroimmunol Neuroinflamm 2019;6:e538. doi: pregnancy with rituximab. The patient conceived 3 months 10.1212/NXI.0000000000000538. after the last dose of rituximab and received another in- 3. Joubert B, Gobert F, Thomas L, et al. Autoimmune episodic ataxia in patients with anti-CASPR2 antibody-associated encephalitis. Neurol Neuroimmunol Neuro- fusion of 1,000 mg at 24 weeks of pregnancy, maintaining inflamm 2017;4:e371. doi: 10.1212/NXI.0000000000000371. the CD19+ count <1%. The newborn APGAR score was 4. Lopez-Chiriboga AS, Pittock S. Episodic ataxia in CASPR-2 autoimmunity. Neurol + Neuroimmunol Neuroinflamm 2019;6:e536. doi: 10.1212/NXI.0000000000000536. normal, and the CD19 cell count was 1% at birth; 2 months 5. Miranda-Acuña J, Rivas-Rodriguez E, Levy M, et al. Rituximab during pregnancy in later, the infant’s CD19+ count was 23%. Previous reports have neuromyelitis optica: A case report. Neurol Neuroimmunol Neuroinflamm 2019;6:e542. doi: 10.1212/NXI.0000000000000542. explored the potential complications of rituximab during 6. Das G, Damotte V, Gelfand JM, et al. Rituximab before and during pregnancy: 6,7 pregnancy ; however, systematic reviews are often con- a systematic review, and a case series in MS and NMOSD. Neurol Neuroimmunol Neuroinflamm 2018;5:e453. doi: 10.1212/NXI.0000000000000453. founded by limited available information or concomitant 7. Chakravarty EF, Murray ER, Kelman A, Farmer P. Pregnancy outcomes after maternal use of drugs with potential effects on pregnancy. Therefore, exposure to rituximab. Blood 2011;117:1499–1506. 8. Hemond CC, Healy BC, Tauhid S, et al. MRI phenotypes in MS: Longitudinal reporting detailed information of pregnant patients af- changes and miRNA signatures. Neurol Neuroimmunol Neuroinflamm 2019;6:e530. fected with severe diseases that may benefitfromrituximab doi: 10.1212/NXI.0000000000000530.

2 Neurology: Neuroimmunology & Neuroinflammation | Volume 6, Number 2 | March 2019 Neurology.org/NN EDITORIAL OPEN ACCESS Diagnostic challenges and pitfalls of myelin oligodendrocyte glycoprotein antibody–associated demyelination Lessons from neuropathology

Romana Hoftberger,¨ MD, and Ellen Gelpi, MD, EFN, PhD Correspondence Dr. Hoftberger¨ Neurol Neuroimmunol Neuroinﬂamm 2019;6:e544. doi:10.1212/NXI.0000000000000544 romana.hoeftberger@ meduniwien.ac.at

Antibodies against myelin oligodendrocyte glycoprotein (MOG) are associated with a wide RELATED ARTICLE spectrum of demyelinating CNS diseases such as clinically isolated syndrome (CIS), neuromyelitis optica spectrum disorder (NMOSD), optic neuritis (ON), myelitis, acute demyelinating Article encephalomyelitis (ADEM), or multiphasic demyelinating encephalomyelitis (MDEM).1 Several Anti-MOG encephalitis brain biopsy–based cases of MOG antibody–associated demyelination have been described so far mimicking small vessel and have reported perivascular (ADEM) or confluent (CIS, MS, NMOSD) demyelination with CNS vasculitis relative preservation of axons, numerous macrophages, and well-preserved astrocytes. The in- Page e538 flammatory infiltrates have been mostly described as perivascular and parenchymal T cells along with some perivascular B cells. Moreover, actively demyelinating lesions may also show terminal complement complex (C9neo) deposition and can display relatively well-preserved, partly MOG- – negative preoligodendrocytes.2 4

The article by Patterson et al. published in this issue of Neurology® Neuroimmunology & Neuroinflammation addresses the challenges and potential pitfalls in the differential diagnostic work-up of brain biopsies in patients with MOG-spectrum disorders. The authors described 2 patients, 1 child and 1 adult, who clinically presented with meningoencephalitis, and brain biopsy revealed predominantly perivascular lymphocytic infiltrates involving the vessel wall, which led to the (mis)diagnosis of small-vessel vasculitis. Both patients were retrospectively tested positive for MOG antibodies and the diagnosis was changed into anti-MOG encephalitis.5 The treatment of the pediatric patient was subsequently changed into a B-cell depletion with good clinical and serologic response. In the second patient, the finding of MOG antibodies did not change the treatment strategy as she was already clinically stable; however, an earlier detection of MOG antibodies could have prevented the brain biopsy.5

The histopathologic diagnosis in small brain biopsies in the context of an inflammatory disease can be a major challenge, and an algorithmic approach for the neuropathologic work-up has been recommended to establish a correct diagnosis.6 Lymphocytic infiltrates within the brain parenchyma involving gray and/or white matter and around vessels may raise several differential diagnoses, including different inflammatory demyelinating diseases (e.g., ADEM, acute hemorrhagic leukoencephalitis, and X-linked adrenoleukodystrophy), autoimmune or para- neoplastic disorders, other inflammatory or infectious diseases (e.g., vasculitis, viral infections, and sarcoidosis), sentinel lesions preceding lymphomas, tumors (e.g., lymphomas and astro- cytomas), and infarcts, among other rare conditions. The neuropathologic hallmark of CNS vasculitis comprises inflammatory infiltration of the vessel wall, fibrinoid necrosis, and inflammatory destruction of the vessels resulting in luminal thrombosis, ultimately causing ischemic brain lesions or hemorrhages. The vasculitis may be accompanied by mild-to-severe

From the Institute of Neurology, Medical University of Vienna, Austria.

Copyright © 2019 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1 inflammatory infiltrates of the CNS parenchyma, but in specimens are frequently small and often represent only part of contrast to ADEM or MS, demyelination is typically absent.6 the entire pathologic process. Small CNS biopsies with inflammation should be thoroughly analyzed for demyelination, In the 2 brain biopsies presented by Patterson et al.,5 the ret- extent, and localization of inflammation (perivascular vs paren- rospective analysis of the histopathology could not confirm the chymal), and axonal damage. Additional immunohistochemical initial diagnosis of small-vessel CNS vasculitis. In both patients, stainings for T and B cells, plasma cells, astrocytes, and macro- the inflammatory infiltrates did not affect the vessel walls, and phages may narrow the differential diagnosis. Neuropathology fibrinoid necrosis, hemorrhage, or ischemia was absent. In the requires a multidisciplinary approach, and the findings should first (pediatric) case, the inflammation was accompanied by be assessed together with neurologic and neuroradiologic data. a small rim of perivascular demyelination, fulfilling the neuro- The current study emphasizes that in some cases, testing for pathologic criteria of ADEM. At the time of biopsy, the brain autoantibodies in serum and CSF provides useful additional MRI showed T2 abnormalities in the basal ganglia, cerebellar information for the proper clinicopathologic classification of peduncles, and supratentorial white matter. Several months after adisease. disease onset, the child developed an episode of ON and 2 years later, another relapse with extensive white matter abnormalities. Study funding Children with ADEM and MOG antibodies are at risk of de- No targeted funding reported. veloping clinical relapses, often with ON, subsequently leading to a diagnosis of ADEM associated with recurrent ON (ADEM- Disclosure 1 ON), MDEM, or NMOSD. The MRI of the second (adult) The Medical University of Vienna receives payments for anti- patient showed unilateral hyperintense gyriform fluid-attenuated body assays (MOG, AQP4, and other autoantibodies). inversion recovery (FLAIR) signal abnormalities over the left Dr. Höftberger received speakers’ honoraria from Euroimmun temporal, parietal, and occipital lobes. The brain biopsy revealed and research support from the Jubiläumsfonds der perivascular inflammatory infiltrates, but no clear evidence of Osterreichischer¨ Nationalbank, Project 16919, and the Austrian 5 demyelination. Unilateral cortical lesions depicted in FLAIR Science Fund (FWF):I3334-B27; E. Gelpi served on the scien- images were previously described in adult patients with steroid- tific advisory board of Institut du Cerveau et de la Moelle epi- 7 responsive encephalitis associated with MOG antibodies. Sub- niere, served as an editorial board member of Neuropathology and sequently, this syndrome has been included in the spectrum of Applied Neurobiology, served as an associate editor of Clinical MOG antibody–associated diseases; however, neuropathologic Neuropathology, received research support from Fundacion descriptions were lacking so far. The current case may add Tatiana Perez de Guzman el Bueno and Fundacio la Marato de this information to the existing literature. The absence of de- TV3 Convocatoria Malalties Neurogeneratives. Full disclosure myelination described by Patterson et al. may result from form information provided by the authors is available with the a sampling error of the small brain biopsy; alternatively, it may full text of this article at Neurology.org/NN. reflect the far end of a spectrum of MOG antibody–associated diseases with only minor or even absent demyelination. From Publication history experimental autoimmune encephalitis models, we learned that Received by Neurology: Neuroimmunology & Neuroinflammation January the extent of demyelination in MOG-induced autoimmunity 4, 2019. Accepted in final form January 9, 2019. seems to depend on the balance between the levels of encephalitogenic T cells and demyelinating MOG antibodies. Although References 1. Reindl M, Waters P. Myelin oligodendrocyte glycoprotein antibodies in neurological an excess of T cells occurred with inflammatory infiltrates in disease. Nat Rev Neurol Epub 2018 Dec 17. 2. Höftberger R, Lassmann H. Inflammatory demyelinating diseases of the central meninges and perivascular spaces, an excess of MOG antibodies – 8 nervous system. Handb Clin Neurol 2017;145:263 283. was found associated with confluent demyelinating lesions. 3. Lang J, Biebl A, Gruber A, et al. Teaching case 5-2018: integrated morphological and Accordingly, intensive perivascular inflammation but sparse or immunological work-up of neurosurgical specimen allows accurate diagnosis of neuroinflammatory lesions: an example of acute disseminated encephalomyelitis even absent demyelination in human MOG-spectrum diseases (ADEM) associated with anti-MOG antibodies. Clin Neuropathol 2018;37:206–208. may, among other factors (e.g., genetic, infectious), reflect 4. Spadaro M, Gerdes LA, Mayer MC, et al. Histopathology and clinical course of MOG- antibody-associated encephalomyelitis. Ann Clin Transl Neurol 2015;2:295–301. a scenario, where an encephalitogenic T-cell response dominates 5. Patterson K, Iglesias E, Nasrallah M, et al. Anti-MOG encephalitis mimicking small over a demyelinating antibody response (with variable antibody vessel CNS vasculitis. Neurol Neuroimmunol Neuroinflamm 2019;XX:XXX. ffi 6. Kuhlmann T, Lassmann H, Brück W. Diagnosis of inflammatory demyelination in titer, a nity) against conformational MOG epitopes. biopsy specimens: a practical approach. Acta Neuropathol 2008;115:275–287. 7. Ogawa R, Nakashima I, Takahashi T, et al. MOG antibody-positive, benign, unilateral, cerebral cortical encephalitis with epilepsy. Neurol Neuroimmunol Neuroinflamm The 2 patients described by Patterson et al., and other reports of 2017;4:e322. doi: 10.1212/NXI.0000000000000322. patients diagnosed with CNS small-vessel vasculitis by brain 8. Lassmann H, Brunner C, Bradl M, Linington C. Experimental allergic encephalo- 7 myelitis: the balance between encephalitogenic T lymphocytes and demyelinating biopsy, who later developed ON or myelitis raise the possibility antibodies determines size and structure of demyelinated lesions. Acta Neuropathol that misdirecting diagnoses may not be unusual because biopsy 1988;75:566–576.

2 Neurology: Neuroimmunology & Neuroinflammation | Volume 6, Number 2 | March 2019 Neurology.org/NN ARTICLE OPEN ACCESS Anti-MOG encephalitis mimicking small vessel CNS vasculitis

Kristina Patterson, MD, PhD, Estibaliz Iglesias, MD, PhD, Maclean Nasrallah, MD, PhD, Veronica´ Gonz´alez- Correspondence Alvarez,´ MD, Mariona Suñol, MD, Jordi Anton, MD, PhD, Albert Saiz, MD, PhD, Eric Lancaster, MD, PhD, Dr. Armangue [email protected] and Tha´ıs Armangue, MD, PhD

Neurol Neuroimmunol Neuroinﬂamm 2019;6:e538. doi:10.1212/NXI.0000000000000538

Abstract RELATED ARTICLE Objective Editorial To report 2 patients with anti–myelin oligodendrocyte glycoprotein (MOG)-associated en- Diagnostic challenges and cephalitis who were initially misdiagnosed with small vessel primary CNS vasculitis. pitfalls of myelin oligodendrocyte Methods glycoprotein – Review of symptoms, MRI and neuropathologic features, and response to treatment. MOG antibody associated antibodies were determined in serum and CSF using a cell-based assay. demyelination: Lessons from neuropathology Results Page e544 Symptoms included fever, headache, and progressive mental status changes and focal neurologic deficits. CSF studies revealed lymphocytic pleocytosis, and both patients had abnormal brain MRIs. Brain biopsy samples showed prominent lymphocytic infiltration of the wall of small vessels; these findings initially suggested small vessel CNS vasculitis, and both patients were treated accordingly. Although 1 patient had a relapsing-remitting course not responsive to cyclophosphamide, the other one (also treated with cyclophosphamide) did not relapse. Retrospective assessment of serum and CSF demonstrated MOG antibodies in both cases, and review of biopsy specimens showed absence of fibrinoid necrosis (a pathologic requirement for small vessel CNS vasculitis).

Conclusions Anti–MOG-associated encephalitis can be mistaken for small vessel CNS vasculitis. This is important because the diagnosis of anti–MOG-associated encephalitis does not require brain biopsy and can be established with a serologic test.

From the Neurology Department (K.P., E.L.), University of Pennsylvania, Philadelphia; Rheumatology Department, Sant Joan de Deu Children’s Hospital (E.I., J.A.), University of Barcelona, Spain; Department of Pathology (M.N.), University of Pennsylvania, Philadelphia; Pediatric Neuroimmunology Unit (V.G.-A.,´ T.A.), Sant Joan de D´eu Children’s Hospital, University of Barcelona, Spain; Department of Pathology (M.S.), Sant Joan de D´eu Children’s Hospital, University of Barcelona, Spain; and Neuroimmunology Program (A.S., T.A.), IDIBAPS-Hospital Clinic, University of Barcelona, Spain.

The Article Processing Charge was funded by the Clinic Foundation. 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 © 2019 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1 Glossary MMF = mycophenolate mofetil; MOG = myelin oligodendrocyte glycoprotein.

The diagnosis of small vessel primary CNS vasculitis is the brain biopsy showed that the inflammatory infiltrates challenging because conventional and brain MRI angiog- were not confined to the vessel wall and also involved the raphy are negative, and brain biopsy remains as the only white and gray matter. With these findings, the patient was definite diagnostic test.1 However, brain biopsy is invasive diagnosed with anti-MOG encephalitis, and treatment with and may be uninformative because of sampling error. Here, rituximab, azathioprine, and low-dose prednisone was initi- we describe 2 patients with myelin oligodendrocyte gly- ated. No more relapses were observed; at the last follow-up, coprotein (MOG) antibody–associated encephalitis2 who 3 years later, he remained clinically and radiologically stable were initially misdiagnosed with small vessel CNS vascu- on azathioprine and low-dose prednisone (eventually dis- litis based on biopsy findings. Physicians should be aware of continued), and the serum titer of MOG immunoglobulin this potential misdiagnosis because it has important clinical G (IgG) antibodies had decreased (1:80) below the con- implications. sensus limit of positivity (≥1:160).2,3

Case 1 Case 2 A 5-year-old boy presented with 2 weeks of frontal headache A 39-year-old woman with a history of ulcerative colitis and fever. His physical examination showed decreased treated with mesalamine and budesonide developed severe, alertness and bilateral papilledema (table). Brain CT and progressive left temporo-parietal headache associated with MRI (figure 1A) were normal, and the CSF showed pleo- nausea and photophobia. Shortly after neurologic symptom cytosis. Meningoencephalitis was suspected, and he was onset, she developed intermittent fever. Peripheral leukocy- started on steroids and acyclovir. During the following days, tosis with neutrophilic predominance and elevated C-reactive fi he developed visual hallucinations. There was gradual clin- protein were identi ed (table). Brain MRI showed hyperin- fl ical improvement until complete recovery, and the patient tense gyriform uid-attenuated inversion recovery signal over fi – was discharged on steroid taper 1 month later. In the en- the left temporal, parietal, and occipital lobes ( gure 1G H). suing 4 months, he was readmitted 3 times for relapsing CSF analysis showed pleocytosis and elevated protein con- symptoms while weaning from steroids. Repeat brain MRI centration, and the patient was started on empiric antibiotics showed T2 abnormalities in the basal ganglia, cerebellar and acyclovir. Six days later, she developed expressive aphasia, peduncles, and supratentorial white matter (figure 1B-D), confusion, and agitation, and bilateral VI cranial nerve palsies. and CSF pleocytosis was identified in all episodes (table). Repeat brain MRI showed no changes, and CT angiography All relapses substantially improved after treatment with demonstrated no clear evidence of vasculitis. A biopsy of the steroids. At the last relapse, a conventional brain angiogra- left parietal lobe and dura showed interstitial and perivascular fi phy was inconclusive. Brain biopsy showed infiltrates lymphocytic in ltrates with marked involvement of the vessel fi – of lymphocytes involving the wall of small vessels and per- wall ( gure 2E H). There was no clear evidence of de- fi ivascular areas accompanied by perivascular demyelination myelination (data not shown). These ndings led to suspect (figure 2A–D). The patient was diagnosed with primary CNS vasculitis, and high-dose IV steroids and oral cyclophos- CNS vasculitis, and he was started on monthly pulses of phamide were started. Four months later, cyclophosphamide cyclophosphamide. After the 5th pulse, he developed acute was discontinued because of elevated transaminases. The dose right optic neuritis that was treated with steroids, resulting in of prednisone was tapered over the course of 2 years. During little improvement. Extensive blood testing identified an the follow-up, the brain MRI normalized at 4 months and the elevation of lipoprotein A (also present in his asymptomatic neurologic examination at 9 months; her only complaints were father), and oral aspirin was added, together with myco- mild aphasia when tired and chronic daily headache. Retro- phenolate mofetil (MMF) and prednisone. He remained spective analysis of stored CSF obtained by the time of clinically and radiologically stable (figure 1E), with a right symptom onset showed MOG-IgG antibodies (1:8). eye visual deficitfor2years;atthistime,immunosuppres- sion was weaned, and shortly after stopping the steroids Discussion (while on MMF and aspirin), he developed confusion and decreased level of consciousness. MRI showed extensive We describe 2 patients initially diagnosed with small vessel white matter abnormalities (figure 1F) and high serum titer CNS vasculitis but who in fact had anti–MOG-associated of MOG antibodies (1:640). Retrospective assessment of encephalitis. The first patient received standard treatment for stored serum and CSF obtained at onset of the disease were CNS vasculitis, including cyclophosphamide and steroid- also positive for MOG antibodies (serum titer 1:20,480 and sparing drugs. Although this treatment led to initial stabili- CSF 1:320, table). Review of the paraffin block containing zation of symptoms, the patient developed new relapses until

Table Clinical and laboratory data of 2 patients with anti-MOG encephalitis initially misdiagnosed with small CNS vessel vasculitis

Patient 1 Patient 2

Age, sex 5 years, male 39 years, female

Previous history None Ulcerative colitis

Attack 1234 56 1

Time from disease onset Onset +4 wk +6 wk +4 mo +8 mo +30 mo Onset

Symptoms Headache, LGF, Headache and LGF Headache, blurry LGF, right leg paresis, Pain, severe loss of Confusion and DLC Headache, nausea, papilledema, bilateral vision, unsteady gait, hallucinations, and DLC vision on the right eye, photophobia, fatigue, visual loss, confusion, and DLC and papilledema diarrhea, LGF, and hallucinations aphasia, and irritability. Later: bilateral VI cranial nerve palsy

Systemic inflammatory Normal CRP and ESR Normal CRP, ESR, and Normal CRP and ESR Normal CRP, ESR, n.d. Normal CRP, ESR, and vWF Mildly elevated CRP markers ferritin elevation of lipoprotein A,a (3.62 mg/dL) and and elevated vWF (150%)b normal ferritin

CSF studies 1st d; 4th d 1st d; 7th d; 8th d

WBC (cell/mm3) 50; 5 40 10 30 n.d. 5 64; 146; 112

Protein (mg/dL) 31; 26 54 50 68 n.d. 29 49; 65; 98

Neopterin (nmol/L) 115 528 n.d. 146 n.d. 36 (NR 9–55)

OCB Positive Negative

Anti-MOG Ab serum n.d.; positive (1: 320) Positive (1:20480); n.d. n.d.; n.d. n.d.; n.d. Positive (1:2,560); n.d. Positive (1:640); n.d. n.d.; positive (1:8) titer; CSF titerc

Brain MRI Normal (figure 1A) Basal ganglia (figure Cerebral peduncle Asymmetric large hazy No new lesions; New large hazy white T2/FLAIR cortical 1B) (figure 1C) white matter and basal residual extensive matter lesions (figure 1F) hypersignal, edema, ganglia lesions (figure 1D) white matter lesions meningeal (figure 1E) enhancement. Solitary enhancing T2 hyperintense white matter lesions (figure 1, G–H)

Treatment Acyclovir, IV Acyclovir, IV IVIg, oral steroids, and IV methylprednisolone, IV IV methylprednisolone, Vancomycin, dexamethasone, oral methylprednisolone, nifedipine oral steroids, and pulses methylprednisolone, AZA, RTX,d oral steroids, ceftriaxone, steroids, and and oral steroids of CYC oral steroids, MM, and and aspirin doxycycline acyclovir, levomepromazine aspirin and IVIg. After brain biopsy: IV steroids, CYC, MM, and oral steroids

Continued 3 he was transitioned to a B-cell depletion therapy with good clinical and serologic response. In the second patient, the ﬁnding of MOG antibodies did not change the treatment strategy because she was already clinically stable; however, an earlier detection of these antibodies could have prevented the brain biopsy. Patient 2 9 mo: mild aphasia when tired and chronic daily headache Nearly complete; normalization of brain MRI

In retrospect, none of these patients fulfilled the criteria for small vessel CNS vasculitis. Although the presence of inflammatory cells in the vessel walls is sufficient to diagnose medium or large vessel vasculitis, signs of vessel damage such as fibrin deposition and/or necrosis (absent in our patients) must also be present to meet the criteria for small vessel Nearly complete; residual MRI lesions. (continued) vasculitis. Diapedesis of leukocytes takes place in small vessels, and the presence of inflammatory cells in the walls does not necessarily imply a pathologic process. Moreover, the pathology of CNS vasculitis is usually focal or segmental,

one; NR = normal range; OCB = oligoclonal band; RTX = rituximab, S = and the brain biopsy specimen can be negative or not meet 4 throcyte sedimentation rate ; FLAIR = fluid-attenuated inversion recovery; the full diagnostic criteria. As our patients reveal, the presence of lymphoid cells in vessel walls must be interpreted

Right visual deficit, with optic atrophy with caution, particularly when the diﬀerential diagnosis includes encephalitis with lymphocytic inﬁltrates. In this context,thepresenceoflymphocytesinthebrainparen- chyma away from the vessel walls favors secondary involvement of the vessels rather than a primary vasculitic process. Whether other cases of anti-MOG encephalitis may have been misdiagnosed as CNS vasculitis is unknown. There are reports of patients diagnosed with CNS small lesions vessel vasculitis by brain biopsy who later developed myelitis or optic neuritis5 (symptoms suggestive of MOG encephalitis),2,3 making us to postulate that this misdiagnosis may not be unusual.

Acknowledgment The authors thank Professor Josep Dalmau for his valuable critical review of the manuscript.

Study funding This study was supported in part by Fundaci´o Marat´o de TV3 (20141830). 2,3 Disclosure 1:160).

≥ K. Patterson received research support from the NIH/NINDS. doses) due to CD19 counts <2% of lymphocytes since then.

2 E. Iglesias, M. Narallah, V. Gonzalez-Alverez, and M. Sunol ﬁ

d report no disclosures. J. Anton served on the scienti cad- visory boards of Novartis, Sobi, and Gebro; received travel funding and/or speaker honoraria from AbbVie, Roche, 1:2), and serum ( (1:80); current ≥ fi ’ c P zer, Sobi, and Gebro; and served on the speakers bureau of AbbVie, Pfizer, Roche, and Gebro. A. Saiz received con- treatment: AZA and low-dose aspirin Patient 1 Complete Complete Complete Complete; MRI residual 6 y, left eye visualand loss mild deficit of attention; extensive MRI white matter residual lesions. Serum anti-MOG titers sulting compensation and speaker honoraria from Bayer- Schering, Merck Serono, Biogen Idec, Sanofi-Aventis, Teva, Novartis, and Roche. E. Lancaster served on the advisory boards of Grifols, Amgen, and Janssen; received speaker honoraria and travel funding from Grifols; consulted for 120%. – Clinical and laboratory data of 2 patients with anti-MOG encephalitis initially misdiagnosed with small CNS vessel vasculitis Medimmune, Merck, and Novartis; received fees from the Federal Vaccine Injury Compensation Program; received NR: 60% Only 1 cycle of RTX was given (4 weekly 375 mg/m Also present in his asymptomatic father. Threshold for positive results, CSF ( Table a b c d Clinical response to immunotherapy Abbreviations: Ab = antibody; AZA = azathioprine;IV CRP = = intravenous; C-reactive IVIg protein; = CYC IV = cyclophosphamide; immunoglobulin; DLC LGF = = decreased low-grade level fever; of consciousness; MM ESR = = mycophenolate ery mofetil; MOG = myelin oligodendrocyte glycoprotein; n.d. = not d serum; vWF = von Willebrand factor; WBC = white blood cell. Last follow-up research support from Grifols and the NINDS; and provided

4 Neurology: Neuroimmunology & Neuroinflammation | Volume 6, Number 2 | March 2019 Neurology.org/NN Figure 1 MRI of 2 patients with anti-MOG encephalitis initially misdiagnosed with small vessel CNS vasculitis

Patient 1: (A) Axial T2 MRI sequence showing no abnormalities at disease onset; (B) bilateral involvement of the basal ganglia 4 weeks after disease onset while steroids were being decreased; (C) left cerebral peduncle abnormality at 6-week follow-up; (D) asymmetric large hazy white matter and basal ganglia lesions at 4 months; (E) residual white matter lesions and enlargement of ventricles due to brain atrophy; and (F) new asymmetric large hazy white matter lesions 30 months after disease onset when steroids were discontinued. Patient 2: (G and H) Axial FLAIR sequences showing gyriform hyperintensities with edema similar to abnormalities previously reported in cases of anti–MOG-associated cortical encephalitis.6 medical legal consultation for Rogers Towers PA, Orlando Madrileña Foundation. Full disclosure form information pro- Health, and Wilson, Elser, Moskowitz, Edelman & Dicker vided by the authors is available with the full text of this article LLP. T. Armangue received research support from the Mutua at Neurology.org/NN.

Figure 2 Brain biopsy of 2 patients with anti-MOG encephalitis initially misdiagnosed with small vessel CNS vasculitis

In patient 1, biopsy of the right temporal lobe showed small vessel perivascular lymphocytic infiltration (A, hematoxylin-eosin staining; B, magnification of the vessel shown in panel A). Inflammatory infiltrates included T and B lymphocytes (not shown) in association with edema, perivascular demyelination, and reactive gliosis (C and D, luxol fast blue staining). In patient 2, biopsy of the left temporal lobe showed marked perivascular lymphocytic infiltrates involving the vessel wall (E, hematoxylin-eosin staining). The infiltrates were also composed of T lymphocytes (F, CD3 immunostaining), B lymphocytes (G, CD20 immunostaining), and macrophages (H, anti-CD68 immunostaining). Myelin staining did not show clear evidence of demyelination (not shown). No necrosis or fibrin deposition was identified (not shown). Scale bar 200 μm in A and C, 500 μm in E, and 100 μm in B, D, and F–H.

Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 6, Number 2 | March 2019 5 Publication history Received by Neurology: Neuroimmunology & Neuroinflammation August References 16, 2018. Accepted in final form October 19, 2018. 1. Benseler SM, deVeber G, Hawkins C, et al. Angiography-negative primary central nervous system vasculitis in children: a newly recognized inflammatory central nervous system disease. Arthritis Rheum 2005;52:2159–2167. 2. Jarius S, Paul F, Aktas O, et al. MOG encephalomyelitis: international recom- fl Appendix 1 Author contributions mendations on diagnosis and antibody testing. J Neuroin ammation 2018;15: 134. 3. Höftberger R, Sepulveda M, Armangue T, et al. Antibodies to MOG and AQP4 in Name Location Role Contribution adults with neuromyelitis optica and suspected limited forms of the disease. Mult Scler 2015;21:866–874. Kristina University of Author Analyzed the data and 4. Giannini C, Salvarani C, Hunder G, Brown RD. Primary central nervous Patterson, Pennsylvania, drafted the manuscript system vasculitis: pathology and mechanisms. Acta Neuropathol 2012;123: MD, PhD Philadelphia 759–772. 5. Benseler S, Pohl D. Childhood central nervous system vasculitis. Handb Clin Neurol Estibaliz University of Author Analyzed the data and 2013;112:1065–1078. Iglesias, Barcelona, critically reviewed the 6. Ogawa R, Nakashima I, Takahashi T, et al. MOG antibody-positive, benign, unilateral, MD, PhD Barcelona manuscript cerebral cortical encephalitis with epilepsy. Neurol Neuroimmunol Neuroinflamm 2017;4:e322. doi: 10.1212/NXI.0000000000000322. Maclean University of Author Analyzed the data, critically Nasrallah, Pennsylvania, reviewed the manuscript, MD, PhD Philadelphia and developed figure 2

Veronica´ University of Author Analyzed the data and Gonzalez-´ Barcelona, critically reviewed the Alvarez,´ MD Barcelona manuscript

Mariona University of Author Analyzed the data, critically Suñol, MD Barcelona, reviewed the manuscript, Barcelona and developed figure 2

Jordi Anton, University of Author Analyzed the data and MD, PhD Barcelona, critically reviewed the Barcelona manuscript

Albert Saiz, University of Author Analyzed the data and MD, PhD Barcelona, critically reviewed the Barcelona manuscript

Eric University of Author Analyzed the data and Lancaster, Pennsylvania, critically reviewed the MD, PhD Philadelphia manuscript

Tha´ıs University of Author Designed and Armangue, Barcelona, conceptualized the study; MD, PhD Barcelona analyzed the data; drafted the manuscript; and developed the figures

6 Neurology: Neuroimmunology & Neuroinflammation | Volume 6, Number 2 | March 2019 Neurology.org/NN ARTICLE OPEN ACCESS CLASS OF EVIDENCE Real-world validation of the 2017 McDonald criteria for pediatric MS

Yu Yi M. Wong, MD, C. Louk de Mol, BSc, Roos M. van der Vuurst de Vries, MD, E. Dani¨elle van Pelt, MD, PhD, Correspondence Immy A. Ketelslegers, MD, PhD, Coriene E. Catsman-Berrevoets, MD, PhD, Rinze F. Neuteboom, MD, PhD,* Dr. Hintzen [email protected] and Rogier Q. Hintzen, MD, PhD*, On behalf of the Dutch pediatric MS and ADEM study group

Neurol Neuroimmunol Neuroinﬂamm 2019;6:e528. doi:10.1212/NXI.0000000000000528

Abstract MORE ONLINE

Objective Class of Evidence To compare the diagnostic accuracy of the McDonald 2017 vs the McDonald 2010 criteria to Criteria for rating predict a second attack of MS (clinically definite MS [CDMS]) at the first attack of acquired therapeutic and diagnostic demyelinating syndromes (ADS). studies NPub.org/coe Methods One hundred sixty-four children (aged <18 years) with an incident attack of ADS were included in a prospective multicenter study between June 2006 and December 2016. Brain (and spinal if available) MRI was performed ≤3 months after symptom onset. Sensitivity, specificity, positive predictive value, negative predictive value, and accuracy were compared at baseline between the 2010 and 2017 criteria.

Results Among the 164 patients, 110 patients (67%) presented without encephalopathy (ADS–, female 63%; median age 14.8 years, IQR 11.3–16.1years) and 54 (33%) with encephalopathy (acute disseminated encephalomyelitis [ADEM], female 52%; median age 4.0 years, IQR 2.6–6.1 years). Of the 110 ADS– patients, 52 (47%) were diagnosed with CDMS within a median follow-up of 4.5 years (IQR 2.6–6.7 years). The sensitivity was higher for the 2017 criteria than for the 2010 criteria (83%; 95% CI 67–92, vs 49%; 95% CI 33–65; p < 0.001), but the specificity was lower (73%; 95% CI 59–84 vs 87%; 95% CI 74–94, p = 0.02). At baseline, 48 patients fulfilled the 2017 criteria compared with 27 patients when using the 2010 criteria. The results for children aged <12 years without encephalopathy were similar. In patients with ADEM, 8% fulfilled the 2010 criteria and 10% the 2017 criteria at baseline but no patient fulfilled the criteria for CDMS.

Conclusions The McDonald 2017 criteria are more sensitive than the McDonald 2010 criteria for predicting CDMS at baseline. These criteria can also be applied in children aged <12 years without encephalopathy but not in children with ADEM.

Classification of evidence This study provides Class II evidence that in children with ADS, the 2017 McDonald criteria are more sensitive but less speciﬁc than the 2010 McDonald criteria for predicting CDMS.

*Joint senior authors.

From the Department of Neurology (Y.Y.M.W., C.L.d.M., R.M.v.d.V.d.V., E.D.v.P., I.A.K., R.Q.H.), MS Centre ErasMS, Erasmus MC, Rotterdam, The Netherlands; and Department of Pediatric Neurology (C.E.C.-B., R.F.N.), Erasmus MC, Rotterdam, The Netherlands.

The Article Processing Charge was funded by the 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 © 2018 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1 Glossary ADEM = acute disseminated encephalomyelitis; ADS = acquired demyelinating syndrome; CDMS = clinically deﬁnite MS; DIS = dissemination in space; DIT = dissemination in time; DMT = disease modifying treatment; FLAIR = ﬂuid-attenuated inversion recovery; FU = follow-up; HR = hazard ratio; IPMSSG = International Pediatric MS Study Group; NPV = negative predictive value; OCB = oligoclonal band; PPV = positive predictive value.

MS is a chronic demyelinating disorder of the CNS.1 Up to multicenter, and prospective study for children with ADS 10% of all patients with MS have their first symptoms before between June 2006 and December 2016 (PROUD-kids age 18 years.2,3 Recently, the international panel on diagnosis study).16,17 Patients were assessed at baseline and were proof MS proposed the McDonald 2017 criteria by reviewing and spectively followed up. MRI was performed within 3 months revising the previous 2010 McDonald criteria.4,5 These re- after symptom onset as part of a routine diagnostic process to vised criteria include modifications to facilitate earlier MS rule out alternative diagnoses. Patients with alternative non- diagnosis while attempting to preserve the diagnostic accu- demyelinating disorders were excluded from our study. All racy of the criteria.5 Important modifications included rein- patients had a follow-up (FU) duration of at least 1 year troducing CSF oligoclonal bands (OCBs) into the criteria as because the interval between the first and second attacks in – a substitute for dissemination in time (DIT) and allowing pediatric MS is typically less than 1 year.18 21 symptomatic lesions to contribute to dissemination in space (DIS) and DIT. Furthermore, cortical lesions have been Patients were included for analysis when presenting with combined with the juxtacortical lesion component to dem- ADS without (ADS–) and with encephalopathy (ADEM).13 onstrate DIS. Patients with neuromyelitis optica spectrum disorders or with relapsing disease other than MS were excluded from analyses The applicability of the adult McDonald 2010 criteria in (e.g., patients presenting with relapsing anti–MOG antibody – children was supported by several studies.6 12 These criteria related disorders) because there is emerging evidence that were implemented in the revised 2012 diagnostic criteria for these patients have a distinct clinical phenotype.22,23 children with acquired demyelinating syndromes (ADS) proposed by the International Pediatric MS Study Group Standard protocol approvals and (IPMSSG).13 ADS encompass the first attack of demyelin- patient consents ationintheCNS.13,14 According to the IPMSSG criteria, MS The PROUD-kids study protocol was approved by the Ethics diagnosis based on the first baseline MRI in children with Committee Erasmus MC, Rotterdam, and by the other par- a first attack should be performed with caution for children ticipating centers in the Netherlands. Written informed con- aged <12 years and patients presenting with acute dissemi- sent was obtained from all patients and/or their families. nated encephalomyelitis (ADEM) because of a lower specificity and sensitivity of the McDonald 2010 criteria in these Definitions fi groups.13 ADS in children encompass the rst attack of demyelination in the CNS, including patients presenting with (ADS+) and without encephalopathy (ADS–). ADEM was defined as A Canadian study reported that the revised McDonald 2017 13 criteria apply well in children with a first attack of ADS across a polyfocal onset with encephalopathy (ADS+). Clinically fi fi the age span.15 Validation of these criteria in different study de nite MS (CDMS) was de ned as a second attack of MS, fi populations is imperative because of the long-term adminis- with 2 nonencephalopathic con rmed attacks with clinical 13 – tration of disease-modifying treatment (DMT) after MS di- evidence of 2 separate lesions. ADS patients who remained fi – 13 agnosis. Overtreatment in patients must be prevented, as well as monophasic were de ned as monophasic ADS . Patients undertreatment, because of delayed diagnosis. Early and accu- were reassessed annually. The patients were instructed to rate identification of patients with MS is therefore essential. contact the outpatient clinic if new symptoms occurred to be clinically assessed. A relapse was defined as new neurologic fi We aimed at evaluating the diagnostic accuracy of the revised de cits or subacute worsening of existing symptoms after 30 fi days of improvement or stable disease, without evidence of an 2017 MS criteria in children with ADS at the time of the rst 24 presentation. Extra attention is paid to children younger than alternative diagnosis. age of 12 years and to patients presenting with ADEM. Procedures Brain and spine MRI scans were performed at 1.5 Tesla scanners. Available T1-, axial T2-, axial and/or sagittal fluid- Methods attenuated inversion recovery (FLAIR)-, and T1-weighted Patients and design images with gadolinium administration were evaluated cen- Children younger than 18 years with a first attack of de- trally. The MRI scan closest to the date of symptom onset was myelination were consecutively included in the nationwide, evaluated as the baseline scan.

2 Neurology: Neuroimmunology & Neuroinflammation | Volume 6, Number 2 | March 2019 Neurology.org/NN For DIS, all baseline MRI scans were scored using the Statistical analyses McDonald 2010 criteria and the modified components as For statistical analyses, we used SPSS software, version 24.0 described in the revised McDonald 2017 criteria for the first (SPSS Inc.) and GraphPad Prism5. CDMS diagnosis was MRI scan (table 1). MRI techniques, such as double in- used as an endpoint for all following analyses. For group version recovery, that are required to reliably demonstrate comparisons, the χ2 test and Fisher exact test were used for cortical lesions were not part of the routine MRI protocol. categorical data. The Mann-Whitney U test was used for Therefore, the cortical lesion component was not taken into continuous data. account in our analyses. If a spinal MRI was performed within 30 days after or before brain MRI, this scan was taken Diagnostic performance and accuracy into account in scoring the DIS components. For DIT, all Patients fulfilling the diagnostic criteria at the time of the first MRI scans with post-gadolinium T1 images were used; or attack with a subsequent diagnosis of CDMS during FU were scans that did not have gadolinium administered, but did considered true positives. False positives did fulfill the di- not show any FLAIR/T2 hyperintense lesions either. CSF agnostic criteria for MS at baseline MRI but were not di- analyses for OCBs were performed in local laboratories agnosed with CDMS during FU. Patients who did not fulfill using isoelectric focusing.25 OCB status was considered the diagnostic criteria at baseline MRI and who were not positive if there were ≥2 unique bands in CSF compared diagnosed with CDMS during FU were considered true with serum. negatives. False negatives were patients who did not fulfill the diagnostic criteria on baseline MRI but were diagnosed Rationale with CDMS during FU. We set out to analyze our data in a manner that is representative for clinical practice. As acknowledged by the International Sensitivity, specificity, positive predictive value (PPV), nega- Panel on Diagnosis of MS, spinal cord MRI and/or lumbar tive predictive value (NPV), and accuracy were calculated, puncture was not performed in every case depending on clinical including a 95% CI. presentation and was left to the decision of the local treating physician.5 We did not exclude patients without a spinal cord Comparison between the sensitivity and specificity for the MRI or lumbar puncture because this would probably in- 2010 and 2017 criteria was made using the McNemar test. troduce selection bias in our study. Kaplan-Meier analysis was used to analyze the time to CDMS diagnosis using the 2010 and 2017 criteria (log-rank test). DIS was based on 3 parameters (periventricular, juxtacortical, Patients who did not meet the criteria for CDMS diagnosis and infratentorial) or 4 including spinal localization if a spinal during FU were considered censored observations. Cox haz- MRI was performed. We allowed OCB status to contribute to ard regression analyses were performed to calculate hazard the fulfillment of DIT 2017 in patients with no gadolinium ratios (HRs) for time to MS diagnosis based on the McDo- enhancement. nald 2010 criteria (applied at baseline), McDonald 2017

Table 1 Baseline MRI criteria for MS diagnosis derived from the McDonald 2010 and revised 2017 criteria

McDonald 2010 criteria for baseline MRI Revised McDonald 2017 criteria for baseline MRI

DIS DIS

At least 2 out of 4 of: At least 2 out of 4 of:

≥1 periventricular lesion ≥1 periventricular lesion

≥1 juxtacortical lesion ≥1 juxtacortical or cortical lesion

≥1 infratentorial lesion ≥1 infratentorial lesion

≥1 spinal cord lesion (symptomatic brainstem ≥1 spinal cord lesion (asymptomatic and symptomatic brainstem syndromes or spinal syndromes or spinal cord lesions are excluded) cord lesions are included)

McDonald 2010 criteria for baseline MRI Revised McDonald 2017 criteria for baseline MRI

DIT DIT

Simultaneous presence of asymptomatic At least 1: gadolinium-enhancing lesions

Simultaneous presence of asymptomatic or symptomatic gadolinium-enhancing lesions

Presence of unique CSF oligoclonal bands compared with serum as a substitute for DIT

Abbreviations: DIS = dissemination in space; DIT = dissemination in time.

Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 6, Number 2 | March 2019 3 criteria (applied at baseline), and CDMS diagnosis. p Values < A subanalysis evaluating the fulfillment of DIS in patients who 0.05 were considered statistically significant. had T1 images with gadolinium administration (n = 93) yielded no difference in the results. Data availability statement The raw data used in this article could be shared in an ano- Of the 110 ADS– patients, 61 patients (55%) had spinal cord nymized format by request from a qualified investigator. imaging, and spinal lesions were detected in 45/61 scans (74%). In a subgroup analysis, we included only patients who underwent a spinal scan. The test characteristics of DIS were Results comparable to the results mentioned earlier. Patient characteristics The inclusion and exclusion process of eligible ADS children Dissemination in time – is displayed in figure 1. Among the 164 included patients To evaluate the DIT for both criteria, we selected ADS with a first demyelinating event, 54 patients (33%) pre- patients who had T1 images with gadolinium administration sented with encephalopathy (ADS+) and were diagnosed (n = 93). Of these patients, 41 were diagnosed with CDMS fi with ADEM. The other 110 (67%) were ADS– at the time of during FU (44%). The 2010 DIT criteria were ful lled in inclusion. Of these, 52/110 (47%) were diagnosed with 35 patients (38%). Of these, 23 were diagnosed with CDMS ff CDMS during FU (median FU time, 4.5 years, interquartile (66%). Regarding the di erence between the symptomatic and range (IQR) 2.6–6.6 years). The median time to CDMS asymptomatic lesions for the DIT component, we observed diagnosis was 10.2 months (IQR 3.8–20.7 months). None of that the 2017 DIT criteria (excluding OCBs) yielded 3 more fi the patients with ADEM had a second attack within a median patients ful lling the DIT criteria (n = 38, 41%), of whom 26/38 FU of 5.1 years (IQR 2.7–7.8). Patient characteristics and were diagnosed with CDMS (68%). A major increase in fi statistical comparisons between monophasic ADS– and patients ful lling DIT with the 2017 criteria is caused by CDMS are displayed in table 2. allowing OCBs to contribute to DIT when no gadolinium enhancement was present: an additional 20 patients fulfilled DIS and DIT performance on baseline MRI 2017 DIT criteria at baseline (n = 58 patients, 62%). Thirty- All test characteristics of analyses with and without children seven (64%) of these 58 patients were diagnosed with CDMS. aged <12 years are presented in table 3. The test characteristics for 2010 and 2017 DIT criteria, ex- Dissemination in space cluding OCB status, were similar. When adding OCB status, – Hundred and ten ADS patients were included for this the DIT criteria yield 27% in sensitivity (p < 0.001) but lost analysis, of whom 52 were diagnosed with CDMS during FU 17% in specificity (p = 0.004) (table 3). (47%). Spinal MRI was included when available. The 2010 fi DIS criteria were ful lled in 54 patients (49%). Of these, 41 McDonald 2010 vs McDonald 2017 criteria were diagnosed with CDMS (76%). Additional 16 patients To evaluate the McDonald 2010 and 2017 criteria, we se- fi ful lled the 2017 DIS criteria (n = 70, 64%), and 48 of these lected ADS– patients who had T1 images with gadolinium 70 patients were diagnosed with CDMS (69%). The 2017 administration (n = 93). The McDonald 2010 DIS + DIT DIS criteria yield an increase in sensitivity of 13% compared criteria were fulfilled in 27/93 patients at baseline (29%), of fi with the 2010 criteria (p =0.1)andalossinspeci city of whom 20 were diagnosed with CDMS (74%) after a median 16% (p = 0.008). FU of 4.5 years (IQR 2.6–7.1 years).

The McDonald 2017 criteria identified 21 additional patients Figure 1 Flow chart for patient inclusion who fulfilled the criteria at baseline (n = 48, 52%) compared with the 2010 criteria, and 34/48 patients (71%) were diagnosed with CDMS. The sensitivity was higher in the McDonalds 2017 criteria (83% vs 49%; p < 0.001), and the specificity was lower (73% vs 87%; p = 0.02) (table 3).

The 7 patients who caused the loss in specificity were identified (fulfilling the 2017 criteria and not the 2010 criteria at baseline, but not having a second attack during an FU of median 2.7 years [IQR: 1.2–6.5 years]). New lesions on subsequent MRI were observed in 4 of these 7 patients during FU. The other 3 patients did not undergo a second MRI.

Of the 41 patients diagnosed with CDMS, the McDonald 2010 criteria led to the identiﬁcation of 20 patients (49%) at baseline. A second attack occurred in 17/20 patients (85%)

4 Neurology: Neuroimmunology & Neuroinflammation | Volume 6, Number 2 | March 2019 Neurology.org/NN Table 2 Patient characteristics

Monophasic CDMS ADS+ ADS–(n = 110) ADS– (n = 58) (n = 52) (n = 54) All (n = 164) p Valuea

Sex, female, n (%) 69 (63) 32 (55) 37 (71) 28 (52) 97 (59) 0.11

Age at onset, median (IQR), y 14.8 (11.3–16.1) 13.3 (9.1–16.0) 15.4 (13.7–16.2) 4.0 (2.6–6.1) 12.1 (5.1–15.8) <0.004

Age <12 years, n (%) 30 (27) 23 (40) 7 (14) 51 (94) 81 (49) 0.002

Presenting phenotype, n (%) 0.001

Optic neuritis 36 (33) 23 (40) 13 (25) 0 36 (22) —

Transverse myelitis 18 (16) 15 (26) 3 (6) 0 18 (11) —

Other monofocal ADS– 25 (23) 11 (19) 14 (27) 0 25 (15) —

Polyfocal ADS– 31 (28) 9 (16) 22 (42) 0 31 (19) —

Polyfocal ADS with encephalopathy (ADEM) 0 0 0 54 (100) 54 (33) —

Spinal MRI, n (%) 61 (56) 34 (59) 27 (52) 14 (26) 75 (46) 0.57

Spinal cord lesions present, n (%) 45/61 (74) 23/34 (68) 22/27 (82) 11/14 (79) 56/75 (75) 0.27

Symptomatic spinal cord lesion, n (%) 29/45 (64) 18/23 (78) 11/22 (50%) 7/11 (64%) 36/56 (64) 0.07

Gadolinium administration, n (%) 89 (81) 48 (83) 41 (79) 49 (91) 138 (84) 0.63

Gadolinium enhancement, n (%) 38/89 (43) 12/48 (25) 26/41 (63) 8/49 (16) 46/138 (33) <0.001

OCBs tested, n (%) 86/110 (78) 44/58 (76) 42/52 (81) 33/54 (61) 119 (73) 0.65

OCBs present, n (%) 54/86 (49) 17/44 (39) 37/42 (88) 1/33 (3) 55 (34) <0.001

Time to baseline MRI, median (IQR), wk 1.6 (0.6–3.4) 1.4 (0.4–3.1) 2.3 (0.8–4.7) 1.4 (0.7–2.4) 1.4 (0.6–3.1) 0.02

Time to lumbar puncture, median (IQR), wk 2.5 (0.7–9.3) 1.8 (0.4–8.4) 3.3 (1.0–10.1) 1.1 (0.4–2.4) 2.0 (0.7–6.5) 0.11

Time to CDMS, median (IQR), mo 10.2 (3.8–20.7) NA 10.2 (3.8–20.7) NA NA NA

Follow-up time, median (IQR), y 4.5 (2.6–6.7) 3.4 (2.1–5.2) 6.0 (4.2–7.8) 5.1 (2.7–7.8) 4.6 (2.6–7.1) <0.001

DMT use, n (%) 63/110 (57) 15/58 (26) 48/52 (92) 1/54 (2) 64 (39) <0.001

DMT use before CDMS diagnosis, n (%) 14/63 (22) NA 14/48 (30) NA 14 (9) NA

Presence of MOG antibodies, n (%) 5/69 (7) 5/34 (15) 0/35 (0) 16/35 (46) 21/104 (20) 0.03

Presence of AQP4 antibodies, n (%) 0/61 (0) 0/40 (0) 0/21 (0) 0/27 (0) 0/88 (0) NA

Abbreviations: ADEM = acute disseminated encephalomyelitis; ADS = acquired demyelinating syndrome; AQP4 = anti-aquaporin 4; CDMS = clinically definite MS; DMT = disease-modifying treatment; MOG = anti–myelin oligodendrocyte glycoprotein; NA = not applicable; OCB = oligoclonal band. Patient characteristics for patients with acquired demyelinating syndromes without encephalopathy (ADS–), CDMS, and ADEM (ADS+). In the Dutch pediatric setting, DMTs are prescribed when the patient fulfills the criteria for MS, either clinically or radiologically. a Comparison between monophasic ADS– and CDMS. Statistical significance p < 0.05 within 3 years of FU and in 19/20 (95%) within 5 years. earlier in patients with ADS using the 2017 than the 2010 With the McDonald 2017 criteria, 34/41 (83%) patients criteria. For both criteria, the HRs for the DIS, DIT, and full with CDMS were identified at baseline. At 3 and 5 years of criteria at baseline are displayed in table 4. FU, 31/34 (91%) and 33/34 (97%) had a second attack within3and5years. Contribution of symptomatic enhancing lesions, OCBs, and spinal cord imaging Only 1 patient who fulfilled the 2010 and 2017 diagnostic As described earlier, the McDonald 2017 criteria identified 21 criteria at baseline did not have a second attack (CDMS) additional patients (n = 48) compared with the 2010 criteria within 5 years of FU, yet this patient showed new MRI lesions (n = 27). on FU scans. These 21 patients were identified with the contribution of 2 The survival curves for CDMS diagnosis, the McDonald 2010 major changes in the criteria. First, symptomatic lesions can criteria, and the revised McDonald 2017 criteria on baseline be included to demonstrate DIS and DIT for the McDonald MRI are presented in figure 2. MS diagnosis could be made 2017 criteria. This led to 7 more MS diagnoses (n = 34) at

Table 3 Test characteristics of the McDonald 2010 and McDonald 2017 criteria

Patients with ADS– and CDMS, DIS 2010 DIS 2017 DIT 2010 DIT 2017 (OCBs DIT 2017 (OCBs McDonald (DIS + McDonald (DIS + DIT) 2017 McDonald (DIS + DIT) 2017 excluding ADEM (n = 110) (n = 110) (n = 93) excluded) (n = 93) included) (n = 93) DIT) 2010 (n = 93) (OCBs excluded) (n = 93) (OCBs included) (n = 93)

Sensitivity % (95%CI) 79 (65–89) 92 (81–98) 56 63 (47–77) 90 (76–97) 49 (33–65) 61 (45–75) 83 (67–92) (40–71)

Specificity % (95% CI) 78 (64–87) 62 (48–74) 77 77 (63–87) 60 (45–73) 87 (74–94) 83 (69–91) 73 (59–84) (63–87)

PPV % (95% CI) 76 (62–86) 69 (56–79) 66 68 (51–82) 64 (50–76) 74 (53–88) 74 (55–87) 71 (56–83) (48–80)

NPV % (95% CI) 80 (67–89) 90 (75–97) 69 73 (59–84) 89 (72–96) 68 (55–79) 73 (60–83) 84 (70–93) (55–80)

Accuracy % (95% CI) 78 (71–86) 76 (69–84) 68 71 (62–80) 73 (64–82) 70 (61–79) 73 (64–82) 77 (69–86) (58–77)

Patients with ADS– and CDMS <12 n = 30 n = 30 n = 28 n = 28 n = 28 n = 28 n = 28 n = 28 years (excluding ADEM)

Sensitivity % (95% CI) 86 (42–99) 100 71 71 (30–95) 100 (56–100) 57 (20–88) 71 (30–95) 100 (56–100) (56–100) (30–95)

Specificity % (95% CI) 87 (65–97) 78 (56–92) 90 90 (68–98) 81 (57–94) 95 (74–100) 95 (74–100) 91 (68–98) (68–98)

PPV % (95% CI) 67 (31–91) 58 (29–84) 71 71 (30–95) 64 (32–88) 80 (30–99) 83 (37–99) 78 (40–96) (30–95)

NPV % (95% CI) 95 100 90 90 (68–98) 100 (77–100) 87 (65–97) 91 (69–98) 100 (79–100) (74–100) (78–100) (68–98)

Accuracy % (95% CI) 87 (75–99) 83 (70–96) 86 86 (73–99) 86 (73–99) 86 (73–99) 89 (78–100) 93 (83–100) (73–99)

Abbreviations: ADEM = acute disseminated encephalomyelitis; ADS = acquired demyelinating syndrome; DIS = dissemination in space; DIT = dissemination in time; CDMS = clinically definite MS. Test characteristics of DIS criteria, DIT criteria, full McDonald 2010, and full McDonald 2017 criteria (DIS + DIT) for CDMS diagnosis in patients without encephalopathy (ADS–). DIT and DIS + DIT: patients were included for analysis if gadolinium was administered or when no T2 lesions were present at baseline MRI. Spinal MRI was used in the evaluation of DIS and DIS + DIT if available. Subanalyses are presented of patients including ADEM and patients younger than 12 years (excluding ADEM). before CDMS diagnosis (exclusion n = 12). In the included Figure 2 Time from incident ADS event to MS diagnosis patients (n = 81), test characteristics for the full criteria (DIS + DIT) remained comparable to the total group (including DMT use before CDMS) for both the 2010 (sensitivity 41% vs 49%; speciﬁcity 87% for both selections) and 2017 criteria (sensitivity 83% and speciﬁcity 73% for both selections).

Analysis in ADS– patients aged <12 years The IPMSSG recommended that the McDonald 2010 criteria at baseline should be used with caution in patients aged <12 years. We applied the novel 2017 criteria to the group of patients younger than 12 years only after exclusion of ADEM cases.

Seven of 28 ADS– patients (25%) were diagnosed with CDMS. Five patients (18%) fulfilled the baseline criteria for 2010, and 4 of them (80%) fulfilled the criteria for CDMS. fi Survival curves for time from incident ADS event to MS diagnosis according Nine patients ful lled the 2017 criteria (32%), and 7 (78%) to the McDonald 2010 criteria (at baseline), McDonald 2017 criteria (at were diagnosed with CDMS. baseline), and CDMS. ADS = acquired demyelinating syndrome; CDMS = clinically definite multiple sclerosis. The test characteristics were even better in children aged <12 years than in the total group for both McDonald 2010 criteria baseline compared with the 2010 criteria (n = 27). Of these 34 (sensitivity 57% vs 49%; specificity 95% vs 87%) and 2017 patients, 25 were diagnosed with CDMS (74%). criteria (sensitivity 100% vs 83%; specificity 91% vs 73%).

Second, OCBs are allowed to be taken into account when Analysis in the ADEM subgroup assessing DIT. This led to the other 14 patients who fulfilled Only patients with ADEM are included in this analysis (n = the McDonald 2017 criteria at baseline (total n = 48) and 54). DIS was fulfilled for the 2010 criteria and 2017 criteria in caused the major increase in the sensitivity of the criteria. 24 (44%) vs 28 (52%) patients with ADEM, respectively. Among these 48 patients, 34 (71%) were diagnosed with CDMS. Regarding DIT, 7/49 (14%) patients with ADEM fulfilled the 2010 DIT criteria and 9/49 (18%) fulfilled the 2017 DIT No additional patients fulfilled the McDonald 2010 criteria criteria, including OCB status. Of note, 4/49 (8%) patients depending on spinal imaging. In contrast, the presence of would have fulfilled the McDonald 2010 criteria and 5/49 a spinal cord lesion contributed to fulfilling the McDonald (10%) the McDonald 2017 criteria (gain of 1 patient due to 2017 criteria at baseline in 6 patients. Three of these patients OCBs). None of the patients fulfilled the criteria for CDMS were eventually diagnosed with CDMS. Thus, 3/41 (7%) during FU. patients with future CDMS fulfilled the DIS component at baseline by performing spinal cord imaging. Discussion Relevant subgroup analyses We investigated the application of the novel McDonald 2017 criteria for pediatric MS in clinical practice. We show that the DMT use before CDMS McDonald 2017 criteria have a higher sensitivity than the DMTs have the potency to postpone a second attack and could previous 2010 criteria for CDMS diagnosis for pediatric therefore influence CDMS diagnosis. We performed a sub- patients (83% vs 49%, p < 0.001). However, the specificity was group analysis after excluding patients who received DMT lower (73% vs 87%, p = 0.02). Overall, the diagnostic accuracy

Table 4 Hazard ratios for CDMS diagnosis after applying DIS, DIT, and full criteria (DIS + DIT) for McDonald 2010 and 2017 on baseline MRI

McDonald 2010 p Value McDonald 2017 p Value

DIS (n=110) HR 5.8 (95% CI 3.0–11.4) p < 0.001 HR 11.3 (95% CI 4.0–31.6) p < 0.001

DIT (n=93) HR 2.9 (95% CI 1.5–5.4) p = 0.001 HR 8.3 (95% CI 3.0–23.4) p < 0.001

DIS + DIT (n=93) HR 3.1 (95% CI 1.7–5.8) p < 0.001 HR 8.5 (95% CI 3.5–20.4) p < 0.001

Abbreviations: CDMS = clinically definite MS; DIS = dissemination in space; DIT = dissemination in time; HR = hazard ratio.

Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 6, Number 2 | March 2019 7 of the 2017 criteria was higher than that for the 2010 criteria Regarding age, ADS– patients aged <12 years seemed to have (77% vs 70%). The revised criteria are easier to apply than the better accuracy for the McDonald 2017 criteria than the total McDonald 2010 criteria, mainly because of the major change group (sensitivity 100% vs 90%, specificity 81% vs 60%, PPV of accepting all lesions to contribute to DIS and DIT, without both 64%, NPV 100% vs 89%), despite the small sample size taking the clinical symptoms into account. Moreover, we for this analysis. This implies that both the McDonald 2010 show that the 2017 McDonald criteria lead to more MS di- criteria and McDonald 2017 criteria can be used across the age agnosis at baseline; therefore, MS diagnosis can be made span, including children aged <12 years with ADS, excluding earlier using the 2017 criteria. patients with ADEM, in keeping with the results from previous studies.12,15,27 A high sensitivity is important to start DMT as soon as possible,26 which might lead to overtreatment in the group of Our study has several limitations. The choice of brain and/or patients who have a less active clinical disease course. Our main spinal MRI, inclusion of contrast or not, and the decision to findings are supported by a recent extensive study in a cohort of include testing for CSF OSBs were left to the discretion of the patients with ADS that also included evaluation of the appli- treating physician. For example, spinal imaging is not always cability of the McDonald 2017 criteria.15 We validate their justified (sedation may be needed), CSF is not always tested finding that the sensitivity is increased and specificity is de- in isolated optic neuritis, and a paired serum sample is not creased mainly by including OCB status into the 2017 criteria. always available with CSF (e.g., in the case of exclusion of However, the specificity of the McDonald 2017 criteria in our suspected infection). Therefore, similar to other studies on study is somewhat lower. This is probably due to our study the diagnostic criteria for pediatric MS, there was not com- design by taking CDMS, a more clinical primary endpoint plete coverage of all potentially relevant parameters. How- instead of new T2 lesions on a second MRI. We identified ever, our main goal was to evaluate the revised 2017 criteria in a minor subgroup of 7 patients who were responsible for the real-world data; therefore, we did not exclude patients from loss in specificity and who had an FU duration of 2.3 years. New the analyses. Instead, a few subanalyses have been performed. lesions on subsequent MRI were observed in 4 of these Despite our considerable FU duration of median 4.6 years, it patients. The other 3 patients did not undergo a second MRI. is possible that some patients may develop a second attack in However, given the presence of typical MS lesions at baseline the future. DMT could have postponed CDMS diagnosis; and OCB positivity in these 3 patients, it is quite likely that however, our subanalysis excluding these patients showed no these patients would also have developed new MRI lesions after differences in test characteristics. Second, the PROUD-kids a longer FU. Taken together, we believe that initiation of DMT study did not have a standardized MRI FU; therefore, we did based on the novel criteria is warranted. Yet, clinicians should not take FU MRIs into account for the analysis of the 2010 be aware that with the McDonald 2017 criteria, more patients and 2017 criteria. However, the scope of this article was to will be identified at baseline and that a proportion of these analyze the diagnostic accuracy for CDMS at the first attack patients will clinically have a less active disease course. of ADS.

Performing spinal MRI led to fulfillment of the McDonald Our data suggest that the McDonald 2017 criteria perform 2017 criteria at baseline in 3/41 (7%) patients with CDMS, well in children. MS diagnosis can be made earlier and leads to but did not have additional value in the McDonald 2010 a higher number of patients with MS at baseline. Both the criteria, in line with another study that indicated limited value McDonald 2010 and 2017 criteria show similar results for the of spinal cord imaging in the McDonald 2010 criteria in patients aged <12 years presenting with ADS and can there- children.6 Fadda et al.15 argued whether spinal cord acquisi- fore be applied in this population as well. As proposed by the tion would meaningfully add to the performance of the IPMSSG for the McDonald 2010 criteria, application of both McDonald 2017 criteria because only a real small proportion 2010 and 2017 criteria in patients with ADEM should be of their patients fulfilled the criteria based on spinal cord MRI. avoided. However, in our cohort, 7% of the patients with CDMS could have been identified at baseline, which could significantly Acknowledgment reduce the time to diagnosis in these patients. The exact place The authors thank all the children and their families who of spinal cord imaging as part of the MS diagnostic procedure participated in the Dutch PROUD-kids study for acquired deserves further investigation. demyelinating syndromes in children.

Our data show that approximately 10% of the patients with Study funding ADEM fulﬁll the McDonald 2010 and 2017 criteria at base- The study was supported by the Dutch MS Research Foun- line. However, no patient with ADEM was diagnosed with dation. This study was not industry sponsored. CDMS in our cohort. The IPMSSG 2012 criteria explicitly mention not to apply the McDonald 2010 criteria to patients Disclosure with ADEM. Our data support this view for the McDonald Y.Y.M.Wong,C.L.deMol,R.M.vanderVuurstdeVries,E.D. 2017 criteria to prevent the incorrect initiation of treatment in van Pelt, and I.A. Ketelslegers report no disclosures. C.E. these monophasic patients. Catsman-Berrevoets received travel funding from the European

8 Neurology: Neuroimmunology & Neuroinflammation | Volume 6, Number 2 | March 2019 Neurology.org/NN Paediatric Neurology Society. R.F. Neuteboom served on the multiple sclerosis and immune-mediated central nervous system demyelination dis- fi orders. J Neurol Neurosurg Psychiatry 2014;85:790–794. scienti c advisory board of LAREB and data safety monitory 13. Krupp LB, Tardieu M, Amato MP, et al. International Pediatric Multiple Sclerosis board of the EXCEL study. R.Q. Hintzen participated in trials Study group criteria for pediatric multiple sclerosis and immune-mediated central nervous system demyelinating disorders: revisions to the 2007 definitions. Mult Scler with Biogen Idec, Merck Serono, Roche, and Novartis and is on 2013;19:1261–1267. the editorial board of Multiple Sclerosis and Related Disorders. 14. Hintzen RQ, Dale RC, Neuteboom RF, Mar S, Banwell B. Pediatric acquired CNS demyelinating syndromes: features associated with multiple sclerosis. 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Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 6, Number 2 | March 2019 9 Appendix 1 Author contributions

Name Degrees Location Role Contribution

Yu Yi M. Wong MD Department of Neurology, MS Centre First author Study design, acquisition of data, statistical analysis, ErasMS, Erasmus MC, Rotterdam, The interpretation of data, and drafting of the Netherlands manuscript

C. Louk de Mol BSc Department of Neurology, MS Centre Coauthor Study design, acquisition of data, statistical analysis, ErasMS, Erasmus MC, Rotterdam, The interpretation of data, and drafting of manuscript Netherlands

Roos M. van der Vuurst MD Department of Neurology, MS Centre Coauthor Interpretation of data and revision of the manuscript de Vries ErasMS, Erasmus MC, Rotterdam, The for content Netherlands

E. Dani¨elle van Pelt MD, PhD Department of Neurology, MS Centre Co-author Acquisition of data and revision of the manuscript ErasMS, Erasmus MC, Rotterdam, The for content Netherlands

Immy A. Ketelslegers MD, PhD Department of Neurology, MS Centre Coauthor Acquisition of data and revision of the manuscript ErasMS, Erasmus MC, Rotterdam, The for content Netherlands

Coriene E. Catsman- MD, PhD Department of Pediatric Neurology, Coauthor Acquisition of data, revision of manuscript for Berrevoets Erasmus MC, Rotterdam, The content Netherlands

Rinze F. Neuteboom MD, PhD Department of Pediatric Neurology, Last author Study design, study supervision, acquisition of data, Erasmus MC, Rotterdam, The interpretation of data, and revision of the Netherlands manuscript for content

Rogier Q. Hintzen MD, PhD Department of Neurology, MS Centre Last author, Study design, study supervision, interpretation of ErasMS, Erasmus MC, Rotterdam, The corresponding data, and revision of the manuscript for content Netherlands author

10 Neurology: Neuroimmunology & Neuroinflammation | Volume 6, Number 2 | March 2019 Neurology.org/NN Appendix 2 Coinvestigators

Name Degrees Location Role Contribution

D.P. Bakker MD, PhD Departments of Pediatric Neurology, VU Medical Centre, Coinvestigator Collection of data Amsterdam

M. Boon MD, PhD Department of Pediatric Neurology, UMCG, Groningen Coinvestigator Collection of data

K.P.J. Braun MD, PhD Department of Pediatric Neurology, University Medical Coinvestigator Collection of data Centre Utrecht, Utrecht

K.G.J. van Dijk MD, PhD Department of Pediatrics, Rijnstate Hospital, Arnhem Coinvestigator Collection of data

M.J. Eikelenboom MD, PhD Department of Neurology, Westfriesgasthuis, Hoorn Coinvestigator Collection of data

M. Engelen MD, PhD Department of Pediatric Neurology, Academic Medical Coinvestigator Collection of data Centre Amsterdam, Amsterdam

K. Geleijns MD, PhD Department of Pediatric Neurology, University Medical Coinvestigator Collection of data Centre Utrecht, Utrecht

C.A. Haaxma MD, PhD Department of Pediatric Neurology, Radboud UMC, Coinvestigator Collection of data Nijmegen

J.M.F. Niermeijer MD, PhD Department of Neurology, Elisabeth-Tweesteden Coinvestigator Collection of data Hospital, Tilburg

E.H. Niks MD, PhD Department of Neurology, Leiden University Medical Coinvestigator Collection of data Centre, Leiden

C.M.P.C.D. Peeters-Scholte MD, PhD Department of Neurology, Leiden University Medical Coinvestigator Collection of data centre, Leiden

E.A.J. Peeters MD, PhD Department of Neuropediatrics, Haga Hospital, the Coinvestigator Collection of data Hague

B.T. Poll MD, PhD The Department of Pediatric Neurology, Emma Coinvestigator Collection of data Children’s Hospital/AMC, Amsterdam

R.P. Portier MD Department of Neurology, Medisch Spectrum Twente, Coinvestigator Collection of data Enschede

J.F. de Rijk-van Andel MD, PhD Department of Neurology, Amphia Hospital, Breda Coinvestigator Collection of data

J.P.A. Samijn MD, PhD Department of Neurology, Maasstad Hospital, Coinvestigator Collection of data Rotterdam

H.M. Schippers MD Department of Neurology, St. Antonius Hospital, Coinvestigator Collection of data Nieuwegein

I.N. Snoeck MD Department of Neuropediatrics, Haga Hospital, the Coinvestigator Collection of data Hague

H. Stroink MD, PhD Department of Neurology, Canisius-Wilhelmina Hospital, Coinvestigator Collection of data Nijmegen

R.J. Vermeulen MD, PhD Department of Pediatric Neurology, Maastricht UMC, Coinvestigator Collection of data Maastricht

A. Verrips MD, PhD Department of Neurology, Canisius-Wilhelmina Hospital, Coinvestigator Collection of data Nijmegen

F. Visscher MD Department of Pediatric Neurology, Admiraal de Ruyter Coinvestigator Collection of data Hospital, Goes

J.H.S. Vles MD, PhD Department of Pediatric Neurology, Maastricht UMC, Coinvestigator Collection of data Maastricht

M.A.A.P. Willemsen MD, PhD Department of Pediatric Neurology, Radboud UMC, Coinvestigator Collection of data Nijmegen

Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 6, Number 2 | March 2019 11 ARTICLE OPEN ACCESS Mouse model of anti-NMDA receptor post–herpes simplex encephalitis

Jenny Linnoila, MD, PhD,* Benjamin Pulli, MD,* Tha´ıs Armangu´e, MD, PhD, Jesus´ Planagum`a, PhD, Correspondence Radha Narsimhan, PhD, Stefan Schob, MD, Matthias W.G. Zeller, MD, Josep Dalmau, MD, PhD,* Dr. Linnoila [email protected] and John Chen, MD, PhD*

Neurol Neuroimmunol Neuroinﬂamm 2019;6:e529. doi:10.1212/NXI.0000000000000529 Abstract Objective To develop an endogenous rodent model of postinfectious anti-NMDA receptor (NMDAR) encephalitis.

Methods Six mice were inoculated intranasally with herpes simplex virus (HSV) 1 and subsequently treated with acyclovir for 2 weeks. Serum was collected at 3, 6, and 8 weeks postinoculation and tested for NMDAR antibodies through a cell-based assay. Eight weeks postinoculation, mice were killed and their brains were sectioned and immunostained with antibodies to postsynaptic density (PSD)-95 and NMDARs. Colocalization of hippocampal PSD-95 and NMDAR clusters, representing postsynaptic membrane NMDARs, was quantiﬁed via confocal imaging. Hippocampi were additionally analyzed for NMDAR and PSD-95 protein using Western blot analysis.

Results Four of 6 mice (67%) developed serum antibodies to NMDARs: 1 at 3 weeks, 1 at 6 weeks, and 2 at 8 weeks postinoculation. As compared to inoculated mice that did not develop NMDAR antibodies, immunoﬂuorescence staining revealed decreased hippocampal postsynaptic membrane NMDARs in mice with serum antibodies at 8 weeks postinoculation. Western blot analysis showed that mice that had NMDAR antibodies at 8 weeks had decreased total NMDAR but not PSD-95 protein in hippocampal extracts (p < 0.05).

Conclusions Mice inoculated intranasally with HSV-1 developed serum NMDAR antibodies. These antibodies were associated with reduced hippocampal NMDARs, as has been shown in previous models where antibodies from patients with anti-NMDAR encephalitis were infused into mice, paving the way for future studies into the pathophysiology of autoimmune encephalitides.

*These authors contributed equally to the manuscript.

From the Center for Systems Biology (CSB), Department of Neurology (J.L., R.N.), Massachusetts General Hospital (MGH); CSB, Department of Radiology (B.P., S.S., M.Z.), MGH, Boston; Neuroimmunology Program (T.A.), Institut d’Investigacions Biom`ediques August Pi i Sunyer (IDIBAPS), Hospital Cl´ınic, Universitat de Barcelona; Pediatric Neuroimmunology Unit (T.A.), Neurology Department, Sant Joan de D´eu Children’s Hospital, University of Barcelona; Neuroimmunology Program (J.P., J.D.), IDIBAPS, Barcelona, Spain; Department of Neurology (J.D.), University of Pennsylvania, PA; Catalan Institute for Research and Advanced Studies (ICREA) (J.D.), Barcelona, Spain; and Institute for Innovation in Imaging (J.C.), Department of Radiology, MGH, Boston.

The Article Processing Charge was funded by the NIH/NINDS. 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 © 2018 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1 Glossary ACV = acyclovir; HSE = HSV encephalitis; HSV = herpes simplex virus; IgG = immunoglobulin G; NMDAR = NMDA receptor; PSD = postsynaptic density.

Herpes simplex virus (HSV)-1 is neurotropic1 and highly de- Immunofluorescence and confocal structive when it infects the brain. Despite treatment, HSV en- microscopy cephalitis (HSE) has a mortality of 10%–30%. Many patients At 8 weeks, mice were killed, brains were harvested, and 7-μm- with HSE are left with devastating sequelae, including short-term thick sections were stained for NMDARs and postsynaptic memory deficits, behavioral changes, and seizures. It was long density (PSD)-95, similarly to previously described.8 Spot thought that HSE recurs in a subset of patients despite treatment detection and three-dimensional colocalization of NMDARs and known that some children develop post-HSE chor- and PSD-95 were performed to quantify postsynaptic mem- eoathetosis.2 However, repeat CSF analysis in these patients was brane NMDAR clusters. negative for HSV. Surprisingly, retrospective analyses3,4 detected NMDA receptor (NMDAR) antibodies in patients’ sera and/or Immunoblot analyses CSF, suggesting that the new neurologic symptoms actually Hippocampi were dissected and homogenized from thawed represented autoimmune encephalitis. A subsequent prospective brain halves, nuclei were removed, and 10 μg protein (con- study has shown that 27% of patients with HSE develop auto- firmed by bicinchoninic acid assay) of each sample was immune encephalitis, often associated with NMDAR antibodies.5 immunoblotted for NMDARs, PSD-95, and actin, similarly to previously described.8 Based on these observations, the current study was un- dertaken to develop an endogenous rodent model of post- Statistics HSE NMDAR encephalitis to allow for detailed studies into Results are expressed as mean with standard error of the mean the pathogenesis of autoimmune encephalitides. In the model error bars. NMDAR and PSD-95 colocalized spots, as well as described below, mice were infected with HSV-1 and sub- hippocampal NMDAR and PSD-95 protein normalized to actin sequently treated with acyclovir (ACV), mirroring the clinical (loading control), were compared with one-way analysis of – course of patients with HSE. variance (ANOVA). Further pooled (NMDAR antibody negative vs positive samples) were also compared with a Mann- Whitney U test. p < 0.05 was considered statistically significant. Methods Data availability Please refer to the online supplemental methods (e-methods, Data will be shared upon request from any qualified links.lww.com/NXI/A92) for detailed descriptions of pro- investigator. cedural details. Standard protocol approvals Results All animal procedures were approved by our Institutional Animal Care and Use Committee. Collection and banking of Mice inoculated intranasally with HSV-1 CSF from patients with autoimmune encephalitis was ap- develop antibodies to NMDARs proved by our institutional review board. Four of 6 mice (67%) inoculated intranasally with HSV-1 developed NMDAR antibodies in their sera, as demon- Inoculation with HSV-1 and treatment strated by a GluN1-transfected HEK293 cell–based assay with ACV (figure 1, A, B): HSE mouse 4 transiently at 3 weeks, HSE Six female BALB/c mice were intranasally inoculated with 0.5 mouse 3 transiently at 6 weeks, and HSE mice 5 and 6 at 8 (mouse 2) or 1 × 106 (mice 1 and 3–6) plaque-forming units of weeks postinoculation. No mice had NMDAR antibodies HSV-1 (strain 17 syn+)6 and treated with ACV (AuroMedics, preinoculation, and serum antibodies were not detected in HSE 20 mg/kg/dose twice daily) via intraperitoneal injections for mice 1 and 2 at any time point. HSE mouse 6 had immuno- 2 weeks. Two age- and sex-matched control mice were in- globulin G (IgG) to an unknown antigen at 6 weeks (figure 1, oculated with vehicle solution. B, arrows) and developed additional IgG to NMDARs at 8 weeks (figure 1, A, B). Serum collection and immunofluorescent cell-based assay for NMDAR Ab detection Mice with antibodies to NMDARs have Retro-orbital bleeds were performed preinoculation and at 3, decreased hippocampal postsynaptic 6, and 8 weeks post-HSV inoculation, and the presence of membrane NMDARs NMDAR antibodies in mouse sera was determined using Immunostaining for NMDARs (figure 2A) and colocalization a GluN1-NMDAR–transfected HEK293 cell–based assay as with PSD-95 (figure 2C) revealed a decrease in hippocampal previously reported.7 postsynaptic membrane NMDAR clusters in HSE mice 4–6

2 Neurology: Neuroimmunology & Neuroinflammation | Volume 6, Number 2 | March 2019 Neurology.org/NN Figure 1 Mice inoculated intranasally with HSV-1 develop antibodies to NMDARs

(A) NMDAR (GluN1 subunit)-expressing HEK293 cells incubated with mouse serum and probed with anti-mouse immunoglobulin G to evaluate for serum NMDAR antibodies (Abs) in HSE mice preinoculation and at 3, 6, and 8 weeks postinoculation with HSV-1. +: NMDAR Abs in serum; –: no NMDAR Abs in serum; #: antibody to unknown antigen in serum. (B) Representative images of GluN1-NMDAR–transfected HEK293 cell–based assay incubated with 8-week sample from HSE mouse 6 serum (a–c) or control serum (d–f) probed with anti-mouse immunoglobulin G (IgG; a, d) or commercial anti-NMDAR (GluN1) probed with anti- rabbit IgG (b and e). Merged images (c and f) show that whereas HSE mouse 6 developed serum antibodies that bind to NMDARs (as evident by colocalization with the commercial NMDAR Ab, c, yellow cell surface labeling), no NMDAR Abs are seen in control serum (f). Note the additional IgG Ab toward an unknown target (a, c; green dots, two of which are highlighted by arrows). Scale bar (a) = 10 μm. compared with control mice and HSE mice 1–3. A significant Discussion difference was found between HSE mice 1 and 2 and HSE mice 5 and 6 (231 ± 23 vs 117 ± 12, respectively, p = 0.03) In this mouse model of anti-NMDAR post-HSE encephalitis, (figure 2B.a), as well as a trend toward a decrease between we showed that two-thirds of mice inoculated intranasally with control mice and HSE mice 5 and 6 (204 ± 4 vs 117 ± 12, HSV-1 and treated with ACV developed serum NMDAR respectively, p = 0.07). When data were pooled based on antibodies using the same cell-based assay that is used to detect serum NMDAR antibody status (control mice and HSE mice NMDAR antibodies in human samples. One mouse (no. 6) 1 and 2 vs HSE mice 3–6, figure 2B.b), a trend toward de- developed antibodies to an unknown target in addition to creased hippocampal postsynaptic membrane NMDAR NMDAR antibodies, further mirroring what has been reported 5,9 clusters was found (217 ± 12 vs 155 ± 25, p = 0.20). in some patients after HSE. Although NMDAR antibodies were not directly demonstrated to be binding to the mouse Mice with antibodies to NMDARs have hippocampi in these studies, a decreased number of hippo- decreased hippocampal NMDAR protein campal postsynaptic NMDAR clusters and decreased hippo- Immunoblot analysis (figure 3A) revealed that HSE mice 5 campal NMDAR protein were found in mice that had serum and 6 had a significant decrease in total extranuclear hip- NMDAR antibodies at the time of sacrifice. The decrease in pocampal NMDAR but not PSD-95 protein compared with hippocampal NMDARs suggests that these mouse antibodies control mice and HSE mice 1 and 2 (figure 3B) (63.2 ± 9.3 vs are similar to patient-derived NMDAR antibodies.8 Effects on 100.0 ± 8.3, p = 0.0498, and 101.3 ± 2.7, p = 0.045, re- hippocampal NMDARs were less pronounced for mice that spectively, normalized to mean of control as 100%). When transiently developed NMDAR antibodies, suggesting that data were pooled based on serum NMDAR antibody status their effects may have been short lived. (control mice and HSE mice 1 and 2 vs HSE mice 3–6), a similar decrease was found for hippocampal NMDAR The previously published animal model of NMDAR (100.6 ± 3.6 vs 68.9 ± 5.0, p = 0.029) but not PSD-95 protein encephalitis8,10 relies on adoptive transfer, infusing human levels (figure 3C). NMDAR antibodies into mouse brains. Mice in that model have

Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 6, Number 2 | March 2019 3 Figure 2 Mice with antibodies to NMDARs have decreased hippocampal postsynaptic membrane clusters of NMDARs

(A) Epifluorescence composites of hippocampi from mice inoculated with vehicle solution (control mice) 1 and 2 (b and a) and mice inoculated with HSV-1(HSE)1–6 (c–h) immunostained to assess the levels of membrane clusters of NMDARs. HSE mice 1 and 2 (c and d) did not develop serum antibodies (Abs) to NMDARs, HSE mice 3 and 4 (e and f) transiently made Abs at 6 and 3 weeks, and HSE mice 5 and 6 (g and h) had serum NMDAR Abs at the time of sacrifice (8 weeks). Boxes in b demonstrate sites that were further imaged under a confocal microscope for all mice. Scale bar = 500 μm. (B) Quantification of colocalized NMDAR and PSD95 clusters (= postsynaptic membrane NMDAR clusters) averaged from 3-μm stacks across the 15 hippocampal regions outlined in A.b for all groups (a) and then further grouped by serum NMDAR antibody status (b). Data are presented as mean ± standard error of the mean (SEM). Significance of NMDAR Ab presence on postsynaptic NMDAR cluster number was assessed by one-way ANOVA for all groups and with Mann-Whitney U test for pooled analysis; *p < 0.05. (C) Epifluorescence composites of NMDAR staining (a) and PSD95 staining (b) for control mouse 1 (scale bar = 500 μm). Square represents an area that was further imaged on a confocal microscope (c–j). Representative confocal 2D images of NMDAR clusters (c and d) and PSD95 clusters (e and f) for control mouse 1 and HSE mouse 5 from the region outlined in a and b. Note the decrease in NMDAR cluster numbers, with relatively preserved PSD95 cluster numbers for HSE mouse 5 compared with control mouse 1 (scale bar = 10 μm). Representative NMDAR (green) and PSD95 (red) clusters for 3D Z-stacks from the region outlined in a and b for control mouse 1 (g) and HSE mouse 5 (h). Note the decrease in NMDAR (green) clusters in stack from HSE mouse 5 compared with control mouse 1. (i and j) Colocalized NMDAR and PSD95 clusters (= postsynaptic membrane NMDAR clusters) in the Z-stacks from g and h for control mouse 1 and HSE mouse 5. Note the decrease in postsynaptic membrane NMDAR clusters in this region in HSE mouse 5 vs control mouse 1 (scale bar = 3 μm).

memory and behavioral deficits, their brains are bound by patient had transient antibody synthesis without overt clinical mani- IgG, and they show a decreased density of postsynaptic mem- festations, and patients often developed additional antibodies brane NMDARs, NMDAR-mediatedcurrents,andsynaptic against yet unknown protein antigens. Transient antibody plasticity. In the model described here, mice produce their own synthesis and an IgG to an unknown target were similarly NMDAR antibodies after HSE, mirroring what has been ob- noted in the current animal model, the significance of which is served in patients with autoimmune encephalitis post-HSE. left for future studies to clarify. Of interest, the outcome of Behavioral studies were not a focus of this proof of principle patients with autoimmune encephalitis post-HSE was sub- study. They are planned for future investigations. stantially worse than that of patients with classic (not HSE triggered) autoimmune encephalitis. A recent prospective study of patients with HSE showed that 27% developed autoimmune encephalitis within 3 months The recent findings in the human disease5 raise important after the viral infection.5 In most cases, the disorder associated questions related to where and how the immune response is with novel synthesis of NMDAR antibodies, but a few patients triggered and potential measures to prevent the subsequent

4 Neurology: Neuroimmunology & Neuroinflammation | Volume 6, Number 2 | March 2019 Neurology.org/NN Figure 3 Mice with antibodies to NMDARs have decreased hippocampal total protein levels of NMDARs

(A) Representative immunoblots of extranuclear proteins extracted from hippocampi of control mice and HSE mice, which were probed for NMDAR (GluN1 subunit), PSD-95, and actin (loading control). Muscle lysates were included as a control. Protein size is expressed as kilodaltons (kD). Note that there is less visible NMDAR protein in the hippocampi of mice that made antibodies (Abs) to NMDARs (mice 3–6), whereas the PSD-95 and actin protein amounts are similar in all the mice. Blots were run in duplicates. a NMDAR Abs transiently present. b NMDAR Abs present at the time of sacrifice. (B) Quantification of hippocampal NMDAR and PSD-95 in control mice, HSE mice that never made NMDAR Abs, HSE mice that transiently made NMDAR Abs, and HSE mice that made NMDAR Abs at the time of sacrifice (8 weeks). (C) Quantification of hippocampal NMDAR and PSD-95 based on serum NMDAR antibody status. Results in B and C were normalized to actin (loading control), and mean band density of control mice was defined as 100%. Data are presented as mean ± SEM. Significance of serum NMDAR Ab presence on hippocampal NMDAR protein amount was assessed by one-way ANOVA (B) and Mann-Whitney U test (C), respectively; *p < 0.05. autoimmune disorder. To answer these questions, we need an (T.A.), and the Radiologic Society of North America (Prince animal model, similar to that reported here, which will allow for Research Grant; B.P.). The Microscopy Core facility of the investigations into the pathogenesis of this disorder, with the Massachusetts General Hospital Program in Membrane Biology potential to provide insights that are generalizable to non- receives support from Boston Area Diabetes and Endocrinology parainfectious autoimmune encephalitides, as they likely share Research Center grant DK57521 and Center for the Study of common mechanisms and pathways. The task is now to de- Inflammatory Bowel Disease grant DK43351. The Zeiss LSM termine in our model the behavioral impact of the NMDAR 800 Airyscan confocal microscope was purchased using NIH antibodies, what other antibodies are produced in this model, Shared Instrumentation Grant 1S10OD021577-01. where and how the immune system is primed to produce neuronal antibodies in response to HSE, and to develop Disclosure a treatment strategy, adjuvant to ACV, that prevents neuronal J. Linnoila is an expert respondent for the National Vaccine antibody synthesis. Injury Compensation Program and received research support from the NIH/NINDS and Harvard Medical School/ Acknowledgment Massachusetts General Hospital. B. Pulli received research The authors thank Esther Aguilar, Dr. Ilona Linnoila, Dr. support from the Radiologic Society of North America. T. AnilkumarNair,Dr.DanielWilton,andDr.AnastasiaZekeridou Armangue received research support from the Mutua for their technical assistance. Research reported in this Madrilena Foundation. J. Planaguama, R. Narsimhan, and S. publication was supported by the National Institute of Schob report no disclosures. M. Zeller owns stock in Bayer. Neurological Disorders and Stroke of the National Institutes J. Dalmau is editor of Neurology: Neuroimmunology & Neu- of Health under the award numbers listed below. The content is roinflammation; is on the editorial board of Neurology UpTo- solely the responsibility of the authors and does not necessarily Date; receives royalties from Ma2 autoantibody test, NMDAR represent the official views of the National Institutes of Health. autoantibody test, GABA(B) receptor autoantibody test, GABA(A) receptor autoantibody test, DPPX autoantibody Study funding test, and IgLON5 autoantibody test; and received research This work was supported by the NIH/NINDS (R25 NS065743 support from the NIH, Instituto Carlos III/FEDER, Agència [J.L.], K08 NS101084 [J.L.], R01 NS077851 [J.D.], R01 de Gestiód’Ajuts Universitaris i de Recerca, and CERCA NS103998 [J.C.], and R01 NS070835 [J.C.]), Instituto Carlos Programme Generalitat de Catalunya–Fundació Cellex. J. III/FEDER 17/00234, CIBERER 15/00010, FundacióCEL- Chen receives publishing royalties from Elsevier and research LEX (J.D.), Agència de Gestiód’Ajuts Universitaris i de Recerca support from the NIH and National MS Society. Full dis- (AGAUR; J.D.), CERCA Programme Generalitat de Catalunya closure form information provided by the authors is available (J.D.), Mutua Madrileña Foundation Award AP162572016 with the full text of this article at Neurology.org/NN.

Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 6, Number 2 | March 2019 5 Appendix 1 Author contribution

Name Location Role Contribution

Jenny Linnoila, MD, PhD Massachusetts General Author Study concept and design, acquisition of data, analysis and interpretation Hospital (MGH), Boston, MA of data, and writing of the manuscript

Benjamin Pulli, MD MGH, Boston Author Study concept and design, implementation of experiments, analysis and interpretation of data, and critical revision of the manuscript for intellectual content

Tha´ıs Armangu´e, MD, PhD University of Barcelona, Author Acquisition of data and critical review of the manuscript for Barcelona, Spain intellectual content

Jesus´ Planagum`a, PhD University of Barcelona, Author Implementation of experiments and critical review of Barcelona, Spain the manuscript

Radha Narsimhan, PhD MGH, Boston Author Acquisition of data and critical review of the manuscript

Stefan Schob, MD University Hospital, Leipzig, Author Study concept and design and critical review of the manuscript Germany

Matthias Zeller, MD Kepler University Hospital, Author Implementation of experiments and critical review of the manuscript Linz, Austria

Josep Dalmau, MD, PhD University of Barcelona, Author Study concept and design, analysis and interpretation of data, critical Barcelona, Spain review of the manuscript for intellectual content, and study supervision

John Chen, MD, PhD MGH, Boston Author Study concept and design, analysis and interpretation of data, critical review of the manuscript for intellectual content, and study supervision

a prospective observational study and retrospective analysis. Lancet Neurol Publication history – fl 2018;17:760 772. Received by Neurology: Neuroimmunology & Neuroin ammation 6. Brown SM, Ritchie DA, Subak-Sharpe JH. Genetic studies with herpes simplex virus September 4, 2018. Accepted in final form October 16, 2018. type 1. The isolation of temperature-sensitive mutants, their arrangement into complementation groups and recombination analysis leading to a linkage map. J Gen Virol 1973;18:329–346. References 7. Dalmau J, Gleichman AJ, Hughes EG, et al. Anti-NMDA-receptor encephalitis: 1. Braun E, Zimmerman T, Hur TB, et al. Neurotropism of herpes simplex virus type 1 in case series and analysis of the effects of antibodies. Lancet Neurol 2008;7: brain organ cultures. J Gen Virol 2006;87(pt 10):2827–2837. 1091–1098. 2. De Tiège X, Rozenberg F, Des Portes V, et al. Herpes simplex encephalitis relapses in 8. Planagumà J, Leypoldt F, Mannara F, et al. Human N-methyl D-aspartate receptor children: differentiation of two neurologic entities. Neurology 2003;61:241–243. antibodies alter memory and behaviour in mice. Brain 2015;138:94–109. 3. Prüss H, Finke C, Höltje M, et al. N-methyl-D-aspartate receptor antibodies in herpes 9. Linnoila JJ, Binnicker MJ, Majed M, Klein CJ, McKeon A. CSF herpes virus and simplex encephalitis. Ann Neurol 2012;72:902–911. autoantibody profiles in the evaluation of encephalitis. Neurol Neuroimmunol 4. Armangue T, Leypoldt F, Málaga I, et al. Herpes simplex virus encephalitis is a trigger Neuroinflamm 2016;3:e245. doi: 10.1212/NXI.0000000000000245. of brain autoimmunity. Ann Neurol 2014;75:317–323. 10. Planagumà J, Haselmann H, Mannara F, et al. Ephrin-B2 prevents N-methyl-D- 5. Armangue T, Spatola M, Vlagea A, et al. Frequency, symptoms, risk factors, aspartate receptor antibody effects on memory and neuroplasticity. Ann Neurol 2016; and outcomes of autoimmune encephalitis after herpes simplex encephalitis: 80:388–400.

6 Neurology: Neuroimmunology & Neuroinflammation | Volume 6, Number 2 | March 2019 Neurology.org/NN ARTICLE OPEN ACCESS MRI phenotypes in MS Longitudinal changes and miRNA signatures

Christopher C. Hemond, MD, Brian C. Healy, PhD, Shahamat Tauhid, MD, Maria A. Mazzola, MD, Correspondence Francisco J. Quintana, PhD, Roopali Gandhi, PhD, Howard L. Weiner, MD, and Rohit Bakshi, MD, MA Dr. Bakshi [email protected] Neurol Neuroimmunol Neuroinﬂamm 2019;6:e530. doi:10.1212/NXI.0000000000000530 Abstract Objective To classify and immunologically characterize persons with MS based on brain lesions and atrophy and their associated microRNA proﬁles.

Methods Cerebral T2-hyperintense lesion volume (T2LV) and brain parenchymal fraction (BPF) were quantiﬁed and used to deﬁne MRI phenotypes as follows: type I: low T2LV, low atrophy; type II: high T2LV, low atrophy; type III: low T2LV, high atrophy; type IV: high T2LV, high atrophy, in a large cross-sectional cohort (n = 1,088) and a subset with 5-year lngitudinal follow- up (n = 153). Serum miRNAs were assessed on a third MS cohort with 2-year MRI phenotype stability (n = 98).

Results One-third of the patients had lesion-atrophy dissociation (types II or III) in both the cross- sectional and longitudinal cohorts. At 5 years, all phenotypes had progressive atrophy (p < 0.001), disproportionally in type II (BPF −2.28%). Only type IV worsened in physical disability. Types I and II showed a 5-year MRI phenotype conversion rate of 33% and 46%, whereas III and IV had >90% stability. Type II switched primarily to IV (91%); type I switched primarily to II (47%) or III (37%). Baseline higher age (p = 0.006) and lower BPF (p < 0.001) predicted 5-year phenotype conversion. Each MRI phenotype demonstrated an miRNA signature whose underlying biology implicates blood-brain barrier pathology: hsa.miR.22.3p, hsa.miR.361.5p, and hsa.miR.345.5p were the most valid diﬀerentiators of MRI phenotypes.

Conclusions MRI-deﬁned MS phenotypes show high conversion rates characterized by the continuation of either predominant neurodegeneration or inﬂammation and support the partial independence of these 2 measures. MicroRNA signatures of these phenotypes suggest a role for blood-brain barrier integrity.

From the Departments of Neurology (C.C.H., B.C.H., S.T., M.A.M., F.J.Q., R.G., H.L.W, R.B.) and Department of Radiology (R.B.); Brigham and Women’s Hospital (C.C.H., B.C.H., S.T., M.A.M., F.J.Q., R.G., H.L.W, R.B.); Laboratory for Neuroimaging Research (C.C.H., S.T., R.H.); Partners Multiple Sclerosis Center (C.C.H., B.C.H., S.T., M.A.M., F.J.Q., R.G., H.L.W, R.B.); Ann Romney Center for Neurologic Diseases (C.C.H., B.C.H., S.T., M.A.M., F.J.Q., R.G., H.L.W, R.B.); and Harvard Medical School (C.C.H., B.C.H., S.T., M.A.M., F.J.Q., R.G., H.L.W., R.B.), Boston, MA.

Copyright © 2019 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1 Glossary BPF = brain parenchymal fraction; Cq = cycle quantiﬁcation; EDSS = Expanded Disability Status Scale; miRNA = microRNA; SP = secondary progressive; T2LV = T2-hyperintense lesion volume.

MS is an autoimmune disease of the CNS, characterized by to characterize MRI-defined phenotypes in a large cross- recurrent episodes of inflammatory demyelination and acceler- sectional cohort and examine their (5-year) longitudinal sta- ated global CNS tissue loss. MRI is the most useful clinical bility and second, to identify differences in miRNA signatures modality to diagnose and monitor MS-related pathology and is among the MRI-phenotype groups. We additionally assessed sensitive to both inflammatory and destructive pathologic pro- the demographic, clinical, and MRI features associated with cesses as measured by T2-hyperintense lesion volume (T2LV) stability vs switching of the phenotypes over 5 years. and whole-brain atrophy, respectively. There is increasing evidence that these 2 processes are at least partially independent.1 Methods We have recently described an MRI-based, 4-phenotype classification that is based on the relationship between (1) brain Cohort identification and volume loss and (2) brain T2LV.2 This MRI-based classification patient characteristics scheme showed that approximately one-fourth of the patients This was a single-center, retrospective longitudinal study of have a disassociation between atrophy and lesions (e.g., high- patients who met baseline inclusion criteria as follows: (1) atrophy and low T2LV or low-atrophy/high T2LV). This dis- diagnosis of MS on the relapsing-remitting (RR) spectrum, association is consistent with previous work, indicating that brain including clinically isolated syndrome, RR MS, or secondary – atrophy progresses somewhat independent of lesions.3 6 progressive (SP) MS19; (2) age 18–60 years; (3) availability of Moreover, recent work has also demonstrated serum immuno- brain MRI acquired at 1.5T by a consistent acquisition pro- logic biomarkers, which may uniquely be linked to destructive or tocol; and (4) neurologic examination, including assessment of lesional pathology, such as serum microRNAs (miRNAs),7 lipid Expanded Disability Status Scale (EDSS) score20 within 6 antibodies,8 free hemoglobin,9 retinol binding protein,10 mito- months of neuroimaging. Patients with primary progressive MS chondrial metabolites,11 and neurofilament light chains.12 CSF were excluded. This initial group composed the cross-sectional biomarkers have also been linked to MRI atrophy or T2 lesion cohort. Among these patients, additional cohorts were identi- volume including CXCL13,13 tau levels,14 oligoclonal bands,15 fied. A longitudinal cohort was also identified as those with increased B-cell activation,16 glial fibrillic acidic protein,17 and the 5-year follow-up clinical and MRI data (henceforth referred to CSF-serum albumin quotient.18 as the 5-year longitudinal cohort). To identify relationships between MRI phenotypes and serum miRNAs, we defined With the goal of investigating the contributions of in- a third cohort, which included only patients with MRI phe- flammation and neurodegeneration to MS pathophysiology, notype congruence/stability between baseline and 2-year we established 2 major objectives for the present study: first, follow-up who had baseline serum blood samples available. A

2 Neurology: Neuroimmunology & Neuroinflammation | Volume 6, Number 2 | March 2019 Neurology.org/NN Table 1 Patient baseline characteristics

Cross-sectional cohort 5-year longitudinal cohort microRNA cohort Historic reference cohorta n 1,088 153 98 175

Women n (%) 818 (75%) 119 (78%) 76 (78%) 124 (71%)

Age (y) 41.1 ± 9.3 42.5 ± 8.6 44.6 ± 7.3 42.7 ± 9.1

Disease duration (y) 8.3 ± 7.7 9.7 ± 7.7 14.5 ± 7.9 9.6 ± 8.6

Smoking history n (%) 456 (48%) 61 (43%) n/a n/a

MRI follow-up (y) n/a 5.0 ± 0.3 2.0 ± 0.1 n/a

Clinical category n (%)

CIS 97 (9%) 3 (2%) 0 18 (10%)

RRMS 913 (84%) 140 (92%) 91 (93%) 115 (66%)

SPMS 78 (7%) 10 (7%) 7 (7%) 42 (24%)

Gd-enhancing lesion n (%) n/a 32 (21%) n/a n/a

Receiving DMT n (%) 792 (73%) 112 (73%) 66 (67%) n/a

EDSS score 1.7 ± 1.7 1.7 ± 1.4 1.9 ± 1.5 2.5 ± 2.3

Brain T2LV (mL) 6.7 ± 8.5 7.2 ± 8.1 8.0 ± 7.5 8.7 ± 10.2

Brain parenchymal fraction 0.857 ± 0.046 0.849 ± 0.046 0.84 ± 0.05 0.84 ± 0.05

Abbreviations: CIS = clinically isolated syndrome; DMT = disease-modifying therapy; EDSS = Expanded Disability Status Scale; RRMS = relapsing-remitting MS; SPMS = secondary progressive MS; T2LV = cerebral T2-hyperintense lesion volume. Data are mean ± SD unless otherwise listed. a Used to define median split values: Tauhid et al.2 cohort of 98 patients who met these criteria was identified, Classification of MRI-defined phenotypes hereon referred to as the 2-year cohort. Patients’ clinical Patients were assigned MRI phenotypes based on a previously and demographic characteristics are summarized in table 1, defined median split values of T2LV (4.21 mL) and BPF and the cohort selection process is depicted in figure e-1, (0.8502) as described in our index cases, selected based on links.lww.com/NXI/A93. After MRI quality control analysis, 3 similarity to a broad cohort of patients with MS2: patients in the 5-year cohort were excluded because of poor segmentation and/or motion artifact. 1. Type I = mild atrophy (BPF > 0.8502) and low lesion burden (T2LV < 4.21 mL) Standard protocol approvals, registrations, 2. Type II = mild atrophy (BPF > 0.8502) and high lesion and patient consents burden (T2LV > 4.21 mL) This study was approved by our institutional local ethics 3. Type III = moderate/severe atrophy (BPF < 0.8502) and committee on human experimentation. All patients provided low lesion burden (T2LV < 4.21 mL) written informed consent. 4. Type IV = moderate/severe atrophy (BPF < 0.8502) and high lesion burden (T2LV > 4.21 mL) MRI acquisition and segmentation fl All brain MRI scans were obtained on a eet of 1.5T Signa Thus, types I and IV had concordance between lesions and ’ GE scanners at the Brigham and Women s Hospital using atrophy and types II and III a discordance. similar acquisition parameters to produce axial dual echo images as follows: TR = 2,800–3,000 msec, TE1/TE2 = Serum samples 30/80 msec, slice thickness = 3 mm (gapless), pixel size = Serum samples were collected in red-top Vacutainer tubes 0.93 × 0.93 mm, producing 54 slices. Automated template- without additives, centrifuged at 2000 rpm for 10 minutes to driven segmentation was performed to derive cerebral separate the serum, and stored at −70°C until RNA extraction. T2LV and brain parenchymal fraction (BPF),21 followed by Isolation of RNA was then performed (miRcury kit; Exiqon) expert manual correction. The number of gadolinium and converted to complementary DNA using a synthesis kit lesions was determined by an expert observer on axial T1- (Exiqon). Prepared complementary DNA samples were weighted spin-echo images, 5–7 minutes after an IV in- stored at −20°C. We selected 186 candidate miRNAs based fusion of gadolinium contrast agent at a concentration of upon their relevance in our previous studies. Expression of 0.1-mmoL/kg. these miRNAs was tested in prepared complementary DNA

Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 6, Number 2 | March 2019 3 Table 2 Full cohort: baseline characteristics comparison by MRI phenotype

Type I Type II Type III Type IV p Value

N 466 (43%) 181 (17%) 168 (15%) 273 (25%)

Clinical type

CIS 65 (14%) 11 (6%) 15 (9%) 6 (2%)

RRMS 392 (84%) 161 (89%) 142 (85%) 218 (80%)

SPMS 9 (2%) 9 (5%) 11 (6%) 49 (18%)

No. of women n (%) 374 (80%) 143 (79%) 118 (70%) 183 (67%) <0.001

Receiving DMT n (%) 323 (69%) 129 (71%) 133 (79%) 207 (76%) 0.051

Age (y) 38.6 ± 8.8 38.1 ± 9.7 45.8 ± 7.8 44.4 ± 8.5 <0.001

EDSS score 1.2 ± 1.3 1.5 ± 1.5 1.7 ± 1.6 2.7 ± 2.0 <0.001

Disease duration (y) 5.5 ± 5.5 8.0 ± 7.4 8.9 ± 7.2 12.8 ± 9.2 <0.001

T2LV (mL) 2.1 ± 0.9 8.2 ± 5.1 2.4 ± 1.0 16.1 ± 11.4 <0.001

BPF 0.890 ± 0.023 0.879 ± 0.022 0.826 ± 0.023 0.804 ± 0.036 <0.001

Smoking historya n (%) 166 (41%) 68 (44%) 75 (49%) 147 (59%) <0.001

Abbreviations: BPF = brain parenchymal fraction; CIS = clinically isolated syndrome; DMT = disease-modifying therapy; EDSS = Expanded Disability Status Scale; RRMS = relapsing-remitting MS; SPMS = secondary progressive MS; T2LV = brain T2-hyperintense lesion volume. Values are mean ± SD unless otherwise noted. p-values derived from group mean differences (age, EDSS, disease duration, T2LV, and BPF) using an ANOVA statistic and frequency variables (sex, DMT use, and smoking history) using a Pearson χ2 test. a n = 958.

samples using a locked nucleic acid, SYBR green-based interpreted using miRPath v.3 with default settings and Tar- quantitative real time polymerase chain reaction method Base v7.0, accessed online between October 11, 2017 and (LNA-SYBR-qRT-PCR). Normalization was performed us- October 13, 2017 at snf-515788.vm.okeanos.grnet.gr/.22 ing the mean expression of 4 miRNAs, with the best stability “Biologic directionality” of miRNAs was determined by index determined by NormFinder software. Normalized cycle comparing mean expression values between phenotypes: quantification (Cq) was calculated as mean Cq–assay Cq. higher expression in a numerically higher phenotype was presumed to represent a “pathologic” association, and con- Statistical analysis and data interpretation versely, lower expression levels were presumed “protective.” Mean values of group-level characteristics with continuous Comparisons of types II and III were labeled “unknown” variables (EDSS score, T2LV, BPF, and disease duration) were because of the lesion/atrophy dissociation. “Biologic MRI compared using paired t-tests (baseline vs follow-up) and one- associations” were assigned based on the defined neuro- way analysis of variance (ANOVA) (baseline MRI phenotype imaging feature(s) to separate MRI phenotypes as follows: comparison); ordinal and dichotomous variables (age, sex, type I vs type II, “lesions”; type I vs type III, “atrophy”; type I smoking history, presence of gadolinium-enhancing lesion(s), vs type IV, “both”; type II vs type III, “unknown”; type II vs and disease category) were compared using the Pearson chi- “ ” “ ” square test. A linear mixed effect model was used to compare type IV, atrophy ; and type III vs type IV, lesions. MRI phenotype group differences over time; predictors of phenotype conversion were estimated using a linear/logistic All statistics were performed using the R (R-project.org) or STATA v14.0 (stata.com) software. regression or odds ratios. Data availability MicroRNA expression was compared among the 4 groups Anonymized data will be shared on request from any qualified using a proportional odds logistic regression model, with investigator. phenotype as the predictor and miRNA expression as the outcome. A proportional odds logistic regression model was used so that patients who had missing expression could be Results given a value lower than the smallest detected expression. If Baseline clinical, MRI, and demographic characteristics are a significant difference between the groups was observed for summarized in table 1, with the inclusion of a comparison his- the global test, pairwise group comparisons were completed. torical reference cohort initially used to define the median Differentially expressed miRNA pathways (at p < 0.05) were split values based on a broad representation of MS disease

4 Neurology: Neuroimmunology & Neuroinflammation | Volume 6, Number 2 | March 2019 Neurology.org/NN phenotypes similar to that of the New York State Multiple sectional cohort): sex, age, EDSS score, disease duration, T2LV, Sclerosis Consortium.2 Our median split criteria defined 4 MRI- BPF, and smoking history (table 2). As shown in table e-1, links. based phenotypes (see Methods); data for the full cross-sectional lww.com/NXI/A93, notable baseline pairwise phenotype dif- cohort are summarized in table 2, with visual depiction of ar- ferences included (1) types III and IV both being older and with chetypal phenotypes in figure 1. In both the cross-sectional co- a higher proportion of men compared with types I and II (all p < hort (n = 1,088), as well as the 5-year longitudinal cohort (n = 0.001); (2) disease duration, EDSS, and SPMS prevalence, all 153), ;one-third of all patients (32% and 33%, respectively) increased linearly from type I (mean disease duration 5.5 years, were categorized as having dissociation between T2LV and at- EDSS 1.2, SPMS 2%) to type IV (mean disease duration 12.8 rophy (types II or III); the proportion of types II and III was very years, EDSS 2.7, SPMS 18%, all p < 0.01) with the exception of similar (33%) when using median split values obtained from the pairwise comparison of types II and III, which were not signifi- cross-sectional cohort instead of the historic cohort (data not cant for any of these comparisons (p > 0.05); (3) smoking shown). The overall unadjusted correlation between T2LV and history was significantly overrepresented in type IV compared BPF was −0.476 (p < 0.0001) in the longitudinal cohort and with others (all p ≤ 0.05); (4) BPF decreased linearly from type I −0.538 in the cross-sectional cohort (p < 0.0001). Both of these to type IV and differed significantly among all phenotypes (all associations remained significant after adjusting for age (p < p < 0.001); (5) T2LV differed significantly among all pheno- 0.0001). We additionally analyzed the associations between BPF types (p < 0.001) except between types I and III. and T2LV using quartile splits of BPF; T2LV decreased as BPF increased: BPF quartile 1, T2LV 13.5 ± 12.4 mL; quartile 2, Longitudinal comparisons: Table 3 and figure 1 depict T2LV 6.1 ± 6.7 mL; quartile 3, T2LV 4.0 ± 4.1 mL; and quartile within-phenotype changes over 5 years, and table e-2, links. 4, T2LV 3.0 ± 2.5 mL; p < 0.001. In both cohorts, the following lww.com/NXI/A93 shows between-phenotype comparisons. significant baseline group differences between phenotypes were All phenotypes demonstrated significant decreases in BPF (all observed by one-way ANOVA (p values all <0.001 for the cross- p < 0.001); the 5-year rate of brain atrophy was highest in type II (vs type I: p <0.001,vstypeIII:p = 0.056, and vs type IV: p = 0.013, figure e-2, links.lww.com/NXI/A93). T2LV significantly increased over 5 years only in type I (p <0.01)andwasnot Figure 1 MRI phenotype 5-year conversion rates significantly different in between-group comparisons. EDSS score increased in types II and IV over 5 years, but the change was only significantly different when comparing group IV with group I (p =0.048,figure e-3, links.lww.com/NXI/A93). In regard to 5-year phenotype conversion, type I was 67% stable, with 33% converting to type II (16%), type III (12%), or type IV (5%); type II was 54% stable, with 42% converting to type IV, none to type III, and 4% to type I. types III and IV were >90% stable and type III converted exclusively to type IV (4%). Predictors of 5-year conversion of MRI phenotype (types I and II combined, table e-3, links.lww.com/NXI/A93) included baseline lower BPF (p < 0.001) and older age (p =0.008).

In summary, clinical and demographic data generally differentiated all 4 MRI phenotypes from one another with the exception of types II and III, which were similar on most pairwise comparisons despite stark MRI differences. Higher phenotypes (e.g., III and IV) were characterized by more men, a history of smoking, higher age, and longer disease durations. Over the 5-year follow-up period, type I converted to either an inflammatory-predominant (type II) or degenerative- predominant (type III) phenotype. Types III and IV were terminal phenotypes that rarely interconvert. T2 FLAIR images showing changes in MS-MRI phenotypes over a 5-year follow-up period. Type 1 patients (33%) converted: 16% to type II, 12% to type III, and 5% to type IV; 46% of type II patients converted: 42% to type IV miRNA study: 2-year longitudinal cohort with and 4% to type I. Types III and IV were predominantly stable (<10% MRI-phenotype stability interconversion). Higher age, higher T2LV, and lower BPF were predictive of phenotype conversion for types I and II. We observe conversion pathways, The 2-year cohort was composed of 98 patients with MS (91 depicted by arrows: adaptive immunity/inflammatory-predominant RR, 7 SP). Baseline characteristics are summarized in table 1. changes characterize conversion from type I to type II, or type III to type IV, whereas degenerative-predominant changes are characterized by type I to The MRI-defined phenotype breakdown was as follows: type type III or type II to type IV. Conversion of type I to type IV involves both I: n = 24; type II: n = 19, type III: n = 21, and type IV: n = 34. pathways. BPF = brain parenchymal fraction; FLAIR = fluid-attenuated inversion-recovery; T2LV = T2-hyperintense lesion volume. Forty-one percent (41%) of all patients were categorized as having dissociation between T2LV and atrophy (types II or

Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 6, Number 2 | March 2019 5 Table 3 MRI phenotype changes at 5-year follow-up

Type I Type II Type III Type IV

N 57 24 26 46

5-year phenotype 19 (33%) 11 (46%) 1 (4%) 4 (9%) conversion n (%)

Receiving DMT +17.5%* +12.5% −3.8% −8.7%

BPF (difference, % −0.011; 95% CI: (−0.014, −0.023; 95% CI: (−0.031, −0.015; 95% CI: (−0.019, −0.013; 95% CI: (−0.019, change) −0.008); p < 0.001 −0.014); p < 0.001 −0.01); p < 0.001 −0.008); p < 0.001

(% change: −1.2%) (% change: −2.6%) (% change: −1.8%) (% change: −1.6%)

T2LV (mL) 0.45; 95% CI: (0.18, 0.72); 1.19; 95% CI: (−0.60, 2.98); 0.22; 95% CI: (−0.17, 0.60); 0.34; 95% CI: (−1.07, 1.75); p = 0.002 p = 0.182 p = 0.256 p = 0.628

EDSS score 0.15; 95% CI: (−0.19, 0.48); 0.04; 95% CI: (−0.29, 0.38); 0.1; 95% CI: (−0.27, 0.46); 0.66; 95% CI: (0.16, 1.16); p = 0.376 p = 0.799 p = 0.589 p = 0.011

Abbreviations: BPF = brain parenchymal fraction; DMT = disease-modifying therapy; EDSS = Expanded Disability Status Scale; T2LV = brain T2-hyperintense lesion volume. All values and statistical results are differences in mean values between baseline and 5-year follow-up using paired t-tests (BPF, T2LV, and EDSS) or χ2 comparison of proportions. *p < 0.05.

III). Serum miRNA analysis (see Methods) of this cohort using serum miRNA analysis (n = 98). The central findings of revealed 16 miRNAs (tables e-4 and e5, table 4, links.lww. our work include (1) evidence for a dissociation between brain com/NXI/A93) that significantly differed in their expression lesions and brain atrophy, as demonstrated by large numbers of among the 4 MRI phenotypes at p < 0.05 after adjustment for patients classified as type II or type III; (2) longitudinal as- age and sex; none survived corrections for multiple compar- sessment showing dynamic changes in phenotypes and the rate isons (table e-4). Among these 16 miRNAs, 15 overlapped and predictors of change of these MRI classifications; and (3) with significant findings from our previous work examining preliminary serum miRNAs that differentiate the MRI pheno- correlations between MRI involvement and miRNAs in sep- types and implicate potential targetable biological pathways arate sets of patients with MS.7 Table e-4 shows pairwise involved in disease heterogeneity. group comparisons of the level of miRNA expression of these 16 miRNAs. Three of the miRNAs showed pairwise differ- We first aimed at extending and validating previous findings ences between phenotypes and were both internally consis- from the development of an MRI classification of MS based tent with phenotype definitions (see Methods), as well as on the relationship between brain lesions and atrophy.2 Using externally consistent with our previous work7: hsa.miR.22.3p, a large sample size, we show a high rate of patients with MS hsa.miR.345.5p, and hsa.miR.361.5p. Both hsa.miR.22.3p and (32%), with a dissociation between whole-brain atrophy and hsa.miR.345.5p are significantly and pathogenically upregu- the total volume of cerebral T2-hyperintense lesions (types II lated in types II and IV compared with types I and III, sug- or III). A similar proportion was observed in our longitudinal gesting an association with inflammation/lesions. cohort (33%, n = 153) and our miRNA cohort (41%, n = 98). Hsa.miR.361.5p was upregulated in type I compared with Taken together, these observations provide evidence that types II and III, suggesting a protective association against these 2 pathologic processes are at least partially independent both lesions and atrophy. DIANA miRPath analysis revealed among a subset of persons with MS. biological associations of these miRNAs (table 4, figure e-4, links.lww.com/NXI/A93); of the 16 significant miRNAs, 7 Over a 5-year follow-up period, the patients at the highest risk of are involved in adherens junction and 4 in extracellular matrix disease progression as shown by conversion to more advanced functions; of the 3 notable miRNAs above, 2 seem to be phenotypes are types I (33% converting) and II (46% convert- involved in adherens junction function. A heatmap (figure 2) ing), with conversion risk at baseline linked to higher age and depicts miRNA expression levels differentiating the 4 MRI lower BPF (table e-3, links.lww.com/NXI/A93). These results phenotypes. are consistent with previous studies, showing that higher age is a risk factor for disease progression23,24 and that more brain atrophy at baseline predicts disability progression at 2- and 5-year Discussion 25,26 follow-up periods. Similarly, a group who tested MRI clas- In this study, we present cross-sectional (n = 1,088) and lon- sification systems like ours27 showed that 1 longitudinal model gitudinal (n = 153) data using a novel MRI-based classification (the development of whole-brain atrophy and >4 new lesions) system of MS based on lesions and atrophy and demonstrate could predict short-term disability worsening. Another group exploratory immunologic correlates of these MRI phenotypes classified 4 MRI phenotypes using gadolinium enhancement as

6 Neurology: Neuroimmunology & Neuroinflammation | Volume 6, Number 2 | March 2019 Neurology.org/NN Figure 2 MicroRNA expression heatmap stratified by MRI phenotype

MRI phenotypes are noted by color in the leftmost column (Type I = purple, Type II = green, Type III = red, Type IV = blue). The level of microRNA (miRNA) expression level is noted by color from blue (lowest/absent expression) to red (highest expression), and or- ganized in numerical order from left to right. Each row represents an individual with MS.

aproxyforinflammation and BPF + T1-hypointense lesion not complete. Of note, the 5-year change in T2LV was not volumes to determine neurodegeneration, finding that greater significantly different in types II or IV, suggesting that these atrophy was associated with activated B-cell activity.16 patients may have attained a stabilized T2 lesion burden plateau characteristic of later-stage MS.31 Our findings provide insights into the relative contributions of inflammation and neurodegeneration in MS.1,6,28 Our data The 5-year brain atrophy rate was highest in patients who were support partial independence of these processes, as reflected by type II at study entry, a finding we interpret as the result of the individual patient phenotypes at baseline and the pathways cascade of events linking the appearance of lesions to later leading to progression/conversions. Among the type I indi- (secondary) atrophy, which may take years to unfold.32,33 The viduals at baseline who converted during the 5-year observation patients may also have had “pseudo-expansion” of brain volume period (33%), roughly equal numbers proceed to type II, at the time of new or recent lesion appearance/expansion in the characterized by increased T2LV without significant global at- early stages of the disease, which would mask any atrophy rophy, or type III, characterized by high whole-brain tissue loss present until the equilibration of fluid shifts.34 Type I patients are without much lesion progression. In contrast, type II converted equally likely to proceed through an adaptive inflammation- nearly exclusively to type IV. This type I to type II to type IV dominant path (type II) as they are to an atrophy-dominant path “inflammation-then-degeneration” pathway (figure 1) supports (type III); this observation supports innate immunity or neu- arguments linking early inflammation to later neuro- rodegeneration as a predominant process in some patients, degeneration, mechanisms believed to be predominantly me- separate from inflammatory white matter lesions.1 This obser- diated by adaptive autoimmunity (inflammation), followed by vation also raises the possibility that this type of individual may innate (degeneration related) immune changes,29,30 and dem- be biologically distinct from others; we speculate that type III onstrates that a portion of type II phenotypes are in a temporal may represent greater involvement of gray matter de- transition phase with age and lower BPF being risk factors for myelination, cortical lesions, and tissue destruction related to subsequent conversion (table e-3, links.lww.com/NXI/A93). leptomeningeal ectopic deposits or soluble inflammatory However, 54% of type II patients remained stable at follow-up, mediators not visible on conventional 1.5T MRI.35 Type III demonstrating that this temporal dispersion of dissociation is patients may also represent greater resistance to inflammatory

Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 6, Number 2 | March 2019 7 Table 4 Interpretation of differentially expressed miRNAs among the 4 MRI phenotypes

miRNA Relevant pathwaysa Relevant findings from previous publications

hsa.miR.10b.5p None relevant Pathogenic association with T2LVb, downregulated in MS vs HCb

hsa.miR.1271.5p None relevant Pathogenic association with BPF, GMF, protective association with T1:T2b, and risk factor for anxietyd

hsa.miR.139.5p Neurotrophin signaling, AJ, TNF signaling, NF- Pathogenic association with T2LVb and EDSSc kappa B signaling, and estrogen signaling

hsa.miR.145.5p ECM-receptor interaction, focal adhesion, AJ, Pathogenic association with T2LV, spinal cord T2LV, and UCCAb and TGF-β signaling

hsa.miR.193a.5p ECM-receptor Pathogenic association with BPV and cortical GMV,b downregulated in PTSD with increased IL-12Bf

hsa.miR.195.5p AJ, EBV infection, and TGF-β signaling Pathogenic association with T2LV, GMF, and EDSS,b,c upregulated in MS vs HCb

hsa.miR.1972 None relevant Pathogenic association with UCCAb and EDSSc

hsa.miR.22.3p* TGF-β signaling, AJ, EBV infection, and focal Downregulated in MS vs HCc; pathogenic association with T2LVa and risk factor for adhesion anxietyd

hsa.miR.28.5p AJ and ECM-receptor interaction Protective association with T1:T2b and EDSS,c upregulated in MS vs other autoimmune disease, SP&PP vs other neurologic disease, SPMS vs RRMS, and downregulated in MS vs HCc

hsa.miR.32.3p AJ Protective association with T2LV and BPFb

hsa.miR.330.5p None relevant Protective association with T2LVb, dysregulated in bipolar disordere

hsa.miR.345.5p* None relevant Pathogenic association with T2LVb, upregulated in MS vs HCc and dysregulated in both major depression and bipolar disordere

hsa.miR.361.5p* AJ and ECM-receptor interaction Pathogenic association with GMF, protective association with T1:T2b, abnormally expressed in Alzheimer disease, and major depressiong

hsa.miR.503.5p None relevant Pathogenic association BPFb

hsa.miR.651.5p None relevant None relevant

hsa.miR.660.5p None relevant Protective association with BPF,b upregulated in SP/PP vs other neurologic disease, RR vs SP, and protective association with EDSSc

Abbreviations: AJ = adherens junction; BPF = brain parenchymal fraction; BPV = brain parenchymal volume; EBV = Epstein-Barr virus; ECM = extracellular matrix; EDSS = Expanded Disability Status Scale score; GMF = (cortical) gray matter fraction; GMV = brain gray matter volume; HC = healthy control; IL = interleukin; miRNA = microRNA; PP = primary progressive; PTSD = post-traumatic stress disorder; SP = secondary progressive; T2LV = brain T2 hyperintense lesion volume; TGF-β = transforming growth factor beta; UCCA = upper cervical cord area. MicroRNAs are arranged in numerical order. a Derived from DIANA miRPath v3.0 KEGG analysis using TarBase v7.0 at p < 0.05.22 b Regev et al.7 c Regev et al.43. d Hommers et al.48 e Maffioletti et al.44 f Bam et al.49 g Mendes-Silva et al.50

processes, a greater responsiveness to their disease modifying Of note, our data also showed a strong overrepresentation of therapy (DMT), or an enhanced reparative/remyelinating smoking history, specifically in the type IV phenotype at ability, reducing the load of T2-hyperintense lesions.36 Overall, baseline, a finding which corroborates previous studies our study lends insights into the inflammation-neurodegeneration demonstrating smoking as a risk factor for accelerated ac- debate by highlighting the dissociation between cerebral lesions cumulation of T2LV, disability, atrophy, and conversion to – and atrophy; we argue that although two-thirds of the patients SPMS.37 40 have congruent measures of lesions and atrophy, the one-third that shows incongruence demonstrates that for at least a subset We characterized MRI phenotypes based on serum circu- of patients, these processes are somewhat independent. Early lating miRNA profiles, using a 2-year cohort selected for age stratification of MS into specific pathobiological pathways may and sex balance, as well as stable MRI phenotype. Each MRI offer novel translational insights into individually targeted phenotype demonstrated a unique miRNA signature, and we therapeutics, especially for persons characterized by a more found 16 miRNAs that differentiated the 4 phenotypes. degenerative (type III) phenotype that may derive reduced Three of these miRNAs were both internally consistent (e.g., benefit from traditional anti-inflammatory DMTs. no contradictory expression-level data in pairwise phenotype

8 Neurology: Neuroimmunology & Neuroinflammation | Volume 6, Number 2 | March 2019 Neurology.org/NN comparisons) and externally valid as compared to the liter- Acknowledgment ature (table e-4, links.lww.com/NXI/A93): hsa.miR.22.3p, The authors thank Svetlana Egorova, Sheena L. Dupuy, Mark hsa.miR.345.5p, and hsa.miR.361.5p. DIANA miRPath Anderson, and Mariann Polgar-Turcsanyi for technical assistance. analysis22 of these specific miRNAs revealed that hsa.miR.22.3p and hsa.miR.361.5p are both involved in blood-brain barrier Study funding function including adherens junctions (sealing the endothelial This work was supported by a Clinical Research Training barrier), focal adhesion (of lymphocytes to the endothelium), Fellowship in Multiple Sclerosis to Dr. Hemond from the and extracellular matrix integrity (endothelial-lymphocyte American Academy of Neurology Institute and Sanofi Gen- communication; table 4, figure e-4, links.lww.com/NXI/ zyme. This study was also supported by the Ann Romney 41 A93), a theme in microRNA regulation. Hsa.miR.22.3p is Center for Neurologic Diseases. additionally associated with TGF-beta signaling, involved in the maturation of Th-17 cells believed to be important in Disclosure 42 the development of MS. Hsa.miR.345.5p demonstrated C.C. Hemond received research support from American 7 a pathologic association with T2LV in previous work, is Academy of Neurology Institute and Sanofi Genzyme; 43 upregulated in MS vs HC, andisdysregulatedinboth B.C. Healy served on the editorial board of Statistical 44 major depression and bipolar disorder. Ofnote,4ofthe Methods in Medical Research and received research support miRNAs (hsa.miR.145.5p, hsa.miR.193a.5p, hsa.miR.195.5p, from Merck Serono, Genzyme, Novartis, Verily Life Sci- and hsa.miR.1972) were also consistently upregulated in type ences, NIH, and NMSS; S. Tauhid served as the managing I, suggestive of a protective role in MS pathology. However, editor for the Journal of Neuroimaging; M.A. Mazzola this protective aspect was not consistent with previous liter- reports no disclosures; F.J. Quintana served on the edi- ature. Nonetheless we suggest these 16 miRNAs, especially torial boards of Biomedicine, Immunologia (Spain), and hsa.miR.22.3p, hsa.miR.345.5p, and hsa.miR.361.5p, may be American Journal of Clinical and Experimental Immunology; considered potentially relevant for future investigation. served as an associate editor for Immunology (UK); served as an advisory board member for Seminars in Immunopa- Our study results are limited based on several factors. As thology; received research support from EMD Serono, a retrospective analysis (of prospective data), data are subject to Sanofi, Novartis, Teva, ONO Pharmaceutical, NIH, systemic and selection biases, and we did not capture any dy- NMSS, the American Cancer Society, Israel Cancer Re- ff namic treatment e ects (substitution of 1 DMT with another, search Fund, NIH-NIAID, King Abdulaziz City for Sci- IV steroid use) or comorbid medical conditions that could ences & Technology, Harvard Medical School, and contribute to miRNA or MRI results. However, we did com- BADERC; and holds stock or stock options in AnTolRx; pare our cross-sectional and longitudinal cohorts for major R. Gandhi is employed by Sanofi Genzyme; received re- fi forms of selection bias; this did not reveal any signi cant search support from Sanofi Genzyme, the NIH, and the ff baseline di erences in age, sex, disease duration, BPF, T2LV, Race to Erase MS; and holds stock or stock options in EDSS, and DMT usage (p > 0.05, data not shown). In addition, Sanofi; H.L. Weiner served on the advisory boards of The the MRI parameters used (T2LV, BPF) are unlikely to com- Guthy Jackson Charitable Foundation, Teva, Biogen, fl pletely re ect the immunopathogenic process, especially at Novartis, Sanofi-Aventis, Tilos, CBridge Capital, Gen- 1.5T. Spatial patterns of cerebral lesion topography have also entech, Genzyme, vTv Therapeutics, and MedDay; con- 45 been demonstrated to be important predictors of disability, sulted for Biodextris, Biogen, Novartis, Serono, Teva, and we did not model this process here; similarly, we did not Sanofi Genzyme, Tilos, Tiziana Life Sciences, vTv Ther- quantify spinal cord disease, which could contribute to whole- apeutics, MedDay, Genentech, and CBridge Capital; and brain atrophy through Wallerian degeneration. However, our received research support from EMD Serono, Google Life results are more generalizable because of what would be ob- Sciences, the NIH, and the NMSS; R. Bakshi received served in the routine clinical setting. The interpretation of bi- consulting fees from the advisory boards of Bayer, Biogen, ological associations of circulating (extracellular) miRNAs is an Celgene, EMD Serono, Genentech, Guerbet, Sanofi evolving process and requires further investigations; although Genzyme, and Shire; is Editor-in-Chief for the Journal of some authors present evidence that these circulating miRNAs Neuroimaging; consulted for Bayer, Biogen, Celgene, represent byproducts of cellular metabolism and death, there EMD Serono, Genentech, Guerbet, Sanofi Genzyme, and are also data supporting their role in cell-cell signaling as active Shire; and received research support from EMD Serono 46,47 paracrine or endocrine molecules. Last, the miRNA anal- and SanofiGenzyme. Dr. Bakshi’s spouse holds stock in ysis was hypothesis-generating; none of our results remained Biogen. Full disclosure form information provided by the fi signi cant after correction for multiple comparisons. Results authors is available with the full text of this article at may thus represent type I statistical errors and must be inter- Neurology.org/NN. preted with caution. Results are of a correlative nature only and cannot be extrapolated to causation. They nonetheless provide Publication history insights into potentially important biological pathways, which Received by Neurology: Neuroimmunology & Neuroinflammation July 31, may help explain MRI and clinical heterogeneity in MS. 2018. Accepted in final form November 9, 2018.

Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 6, Number 2 | March 2019 9 cortical atrophy in multiple sclerosis at clinical onset. J Neuroinflammation 2017; – Appendix 1 Author contributions 14:5 13. 14. Pietroboni AM, Schiano Di Cola F, Scarioni M, et al. CSF β-amyloid as a putative biomarker of disease progression in multiple sclerosis. Mult Scler 2017;23: Name Location Role Contribution 1085–1091. 15. Farina G, Magliozzi R, Pitteri M, et al. Increased cortical lesion load and intrathecal Christopher Brigham and Author Acquired, analyzed, and inflammation is associated with oligoclonal bands in multiple sclerosis patients: ’ C. Hemond, Women s interpreted the data and a combined CSF and MRI study. J Neuroinflammation 2017;14:1–11. MD Hospital, wrote the manuscript 16. Comabella M, Cantó E, Nurtdinov R, et al. MRI phenotypes with high neuro- Boston MA degeneration are associated with peripheral blood B-cell changes. Hum Mol Genet 2016;25:308–316. Brian C. Brigham and Author Statistical analysis, drafted 17. Kassubek R, Gorges M, Schocke M, et al. GFAP in early multiple sclerosis: a bio- ’ Healy, PhD Women s figure(s), and contributed marker for inflammation. Neurosci Lett 2017;657:166–170. Hospital, to the manuscript 18. Uher T, Horakova D, Tyblova M, et al. Increased albumin quotient (QAlb) in Boston MA patients after first clinical event suggestive of multiple sclerosis is associated with development of brain atrophy and greater disability 48 months later. Mult Scler Shahamat Brigham and Author Assisted in data acquisition 2016;22:770–781. ’ Tauhid, MD Women s and analysis 19. Polman CH, Reingold SC, Banwell B, et al. Diagnostic criteria for multiple sclerosis: Hospital, 2010 revisions to the McDonald criteria. Ann Neurol 2011;69:292–302. Boston MA 20. Kurtzke JF. Rating neurologic impairment in multiple sclerosis: an expanded disability status scale (EDSS). Neurology 1983;33:1444–1452. Maria A. Brigham and Author miRNA analysis and 21. Wei X, Warfield SK, Zou KH, et al. Quantitative analysis of MRI signal abnormalities ’ Mazzola, MD Women s interpretation of brain white matter with high reproducibility and accuracy. J Magn Reson Imaging Hospital, 2002;15:203–209. Boston MA 22. Vlachos IS, Zagganas K, Paraskevopoulou MD, et al. DIANA-miRPath v3.0: deci- phering microRNA function with experimental support. Nucleic Acids Res 2015;43: Francisco J. Brigham and Author miRNA analysis and W460–W466. ’ Quintana, Women s interpretation 23. Guillemin F, Baumann C, Epstein J, et al. Older age at multiple sclerosis onset is an PhD Hospital, independent factor of poor prognosis: a population-based cohort study. Neuro- Boston MA epidemiology 2017;48:179–187. 24. Scalfari A, Neuhaus A, Daumer M, Ebers GC, Muraro PA. Age and disability accu- Roopali Sanofi- Author miRNA analysis and mulation in multiple sclerosis. Neurology 2011;77:1246–1252. Gandhi, PhD Genzyme, interpretation 25. Jeffery DR, Di Cantogno EV, Ritter S, Meier DP, Radue EW, Camu W. The Cambridge MA relationship between the rate of brain volume loss during first 24 months and disability progression over 24 and 48 months in relapsing MS. J Neurol 2015;263: Howard L. Brigham and Author Conception and design of 299–305. ’ Weiner, MD Women s the study and reviewing the 26. Lukas C, Minneboo A, de Groot V, et al. Early central atrophy rate predicts 5 year Hospital, manuscript clinical outcome in multiple sclerosis. J Neurol Neurosurg Psychiatry 2010;81: Boston MA 1351–1356. 27. von Gumberz J, Mahmoudi M, Young K, et al. Short-term MRI measurements as Rohit Brigham and Author Conception and design of predictors of EDSS progression in relapsing-remitting multiple sclerosis: grey ’ Bakshi, MD, Women s the study, drafting, and matter atrophy but not lesions are predictive in a real-life setting. PeerJ 2016;4: MA Hospital, reviewing the manuscript e2442. Boston MA 28. Hutchinson M. Neurodegeneration in multiple sclerosis is a process separate from inflammation: no. Mult Scler 2015;21:1628–1631. 29. Weiner HL. The challenge of multiple sclerosis: how do we cure a chronic hetero- References geneous disease? Ann Neurol 2009;65:239–248. 30. Chitnis T, Weiner HL. CNS inflammation and neurodegeneration. J Clin Invest 2017; 1. Louapre C, Lubetzki C. Neurodegeneration in multiple sclerosis is a process separate 127:3577–3587. from inflammation: yes. Mult Scler 2015;21:1626–1628. 31. Li DKB, Held U, Petkau J, et al. MRI T2 lesion burden in multiple sclerosis: a pla- 2. Tauhid S, Neema M, Healy BC, Weiner HL, Bakshi R. MRI phenotypes based on teauing relationship with clinical disability. Neurology 2006;66:1384–1389. cerebral lesions and atrophy in patients with multiple sclerosis. J Neurol Sci 2014;346: 32. Chard DT, Brex PA, Ciccarelli O, et al. The longitudinal relation between brain lesion 250–254. load and atrophy in multiple sclerosis: a 14 year follow up study. J Neurol Neurosurg 3. Bermel RA, Bakshi R. The measurement and clinical relevance of brain atrophy in Psychiatry 2003;74:1551–1554. multiple sclerosis. Lancet Neurol 2006;5:158–170. 33. Rudick RA, Fisher E, Lee JC, Simon J, Jacobs L. Use of the brain parenchymal fraction 4. Bakshi R, Neema M, Healy BC, et al. Predicting clinical progression in multiple to measure whole brain atrophy in relapsing-remitting MS: Multiple Sclerosis Col- sclerosis with the magnetic resonance disease severity scale. Arch Neurol 2008;65: laborative Research Group. Neurology 1999;53:1698–1704. 1449–1453. 34. Dalton CM, Chard DT, Davies GR, et al. Early development of multiple sclerosis is 5. Kearney H, Altmann DR, Samson RS, et al. Cervical cord lesion load is associated with – associated with progressive grey matter atrophy in patients presenting with clinically disability independently from atrophy in MS. Neurology 2015;84:367 373. isolated syndromes. Brain 2004;127:1101–1107. 6. Calabrese M, Magliozzi R, Ciccarelli O, Geurts JJ, Reynolds R, Martin R. Exploring 35. Zurawski J, Lassmann H, Bakshi R. Use of magnetic resonance imaging to visualize the origins of grey matter damage in multiple sclerosis. Nat Rev Neurosci 2015;16: leptomeningeal inflammation in patients with multiple sclerosis. JAMA Neurol 2017; – 147 158. 74:100–109. 7. Regev K, Healy BC, Khalid F, et al. Association between serum MicroRNAs and 36. Stangel M, Kuhlmann T, Matthews PM, Kilpatrick TJ. Achievements and obstacles of magnetic resonance imaging measures of multiple sclerosis severity. JAMA Neurol remyelinating therapies in multiple sclerosis. Nat Rev Neurol 2017;13:742–754. – 2017;74:275 285. 37. Healy BC, Ali EN, Guttmann CR, et al. Smoking and disease progression in multiple 8. Bakshi R, Yeste A, Patel B, et al. Serum lipid antibodies are associated with cerebral sclerosis. Arch Neurol 2009;66:858–864. fl tissue damage in multiple sclerosis. Neurol Neuroimmunol Neuroin amm 2016;3: 38. Hernán MA, Jick SS, Logroscino G, Olek MJ, Ascherio A, Jick H. Cigarette smoking e200. and the progression of multiple sclerosis. Brain 2005;128:1461–1465. 9. Lewin A, Hamilton S, Witkover A, et al. Free serum haemoglobin is associated with 39. Degelman ML, Herman KM. Smoking and multiple sclerosis: a systematic review and brain atrophy in secondary progressive multiple sclerosis. Wellcome Open Res 2016; meta-analysis using the Bradford Hill criteria for causation. Mult Scler Relat Disord 1:10. 2017;17:207–216. 10. Yokote H, Kamata T, Toru S, Sanjo N, Yokota T. Serum retinol levels are associated 40. Zivadinov R, Weinstock-Guttman B, Hashmi K, et al. Smoking is associated with with brain volume loss in patients with multiple sclerosis. Mult Scler J Exp Transl Clin increased lesion volumes and brain atrophy in multiple sclerosis. Neurology 2009;73: 2017;3:2055217317729688. 504–510. 11. Lazzarino GG, Amorini AM, Petzold A, et al. Serum compounds of energy metabo- 41. Lopez-Ramirez MA, Reijerkerk A, de Vries HE, Romero IA. Regulation of brain lism impairment are related to disability, disease course and neuroimaging in multiple endothelial barrier function by microRNAs in health and neuroinflammation. FASEB sclerosis. Mol Neurobiol 2017;54:7520–7533. J 2016;30:2662–2672. 12. Kuhle J, Nourbakhsh B, Grant D, et al. Serum neurofilament is associated with 42. Okada H, Khoury SJ. Type17 T-cells in central nervous system autoimmunity and progression of brain atrophy and disability in early MS. Neurology 2017;88: tumors. J Clin Immunol 2012;32:802–808. 826–831. 43. Regev K, Paul A, Healy B, et al. Comprehensive evaluation of serum microRNAs as 13. Puthenparampil M, Federle L, Miante S, et al. BAFF Index and CXCL13 levels in biomarkers in multiple sclerosis. Neurol Neuroimmunol Neuroinflamm 2016;3: the cerebrospinal fluid associate respectively with intrathecal IgG synthesis and e267.

10 Neurology: Neuroimmunology & Neuroinflammation | Volume 6, Number 2 | March 2019 Neurology.org/NN 44. Maffioletti E, Cattaneo A, Rosso G, et al. Peripheral whole blood microRNA 48. Hommers L, Raab A, Bohl A, et al. MicroRNA hsa-miR-4717-5p regulates RGS2 and alterations in major depression and bipolar disorder. J Affect Disord 2016;200: may be a risk factor for anxiety-related traits. Am J Med Genet B Neuropsychiatr 250–258. Genet 2015;168B:296–306. 45. Bodini B, Battaglini M, De Stefano N, et al. T2 lesion location really matters: a 10 year 49. Bam M, Yang X, Zhou J, et al. Evidence for epigenetic regulation of pro-inflammatory follow-up study in primary progressive multiple sclerosis. J Neurol Neurosurg Psy- cytokines, interleukin-12 and interferon gamma, in peripheral blood mononuclear chiatry 2011;82:72–77. cells from PTSD patients. J Neuroimmune Pharmacol 2016;11:168–181. 46. Bayraktar R, Van Roosbroeck K, Calin GA. Cell-to-cell communication: microRNAs 50. Mendes-silva AP, Pereira KS, Tolentino-Araujo GT, et al. Shared biologic as hormones. Mol Oncol 2017;11:1673–1686. pathways between Alzheimer disease and major depression: a systematic 47. Turchinovich A, Tonevitsky AG, Burwinkel B. Extracellular miRNA: a collision of two review of MicroRNA expression studies. Am J Geriatr Psychiatry 2016;24: paradigms. Trends Biochem Sci 2016;41:883–892. 903–912.

Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 6, Number 2 | March 2019 11 ARTICLE OPEN ACCESS HIV is associated with endothelial activation despite ART, in a sub-Saharan African setting

Joseph Kamtchum-Tatuene, MD, MAS, MRes, FEBN, Henry Mwandumba, PhD, FRCP, Zaid Al-Bayati, PhD, Correspondence Janet Flatley, PhD, Michael Griffiths, PhD, FRCP, Tom Solomon, PhD, FRCP, and Laura Benjamin, PhD, MRCP Dr. Benjamin [email protected] Neurol Neuroimmunol Neuroinﬂamm 2019;6:e531. doi:10.1212/NXI.0000000000000531 Abstract Objective To study the relationship between endothelial dysfunction, HIV infection, and stroke in Malawians.

Methods Using a cross-sectional design, we measured plasma levels of intercellular adhesion molecule-1 (ICAM-1), plasminogen activator inhibitor-1 (PAI-1), vascular endothelial growth factor (VEGF), and soluble thrombomodulin (sTM) in stroke patients and controls, stratiﬁed by HIV status. These biomarkers were measured using ELISA. After dichotomization, each biomarker was used as the dependent variable in a multivariable logistic regression model. Primary independent variables included HIV and stroke status. Adjustment variables were age, sex, hypertension, diabetes mellitus, tobacco and alcohol consumption, personal/family history of stroke, antiretroviral therapy status, and hypercholesterolemia.

Results Sixty-one stroke cases (19 HIV+) and 168 controls (32 HIV+) were enrolled. The median age was 55 years (38.5–65.0) for controls and 52 years (38.0–73.0) for cases (p = 0.38). The median CD4+ T-cell count was 260.1 cells/mm3 (156.3–363.9) and 452 cells/mm3 (378.1–527.4) in HIV-infected cases and controls, respectively. HIV infection was independently associated with high levels of ICAM-1 (OR = 3.6, 95% CI: 1.3–10.6, p = 0.018) in controls but not in stroke cases even after excluding patients with a viral load >1,000 RNA copies/mL (OR = 4.1, 95% CI: 1.3–13.1, p = 0.017). There was no association between the clinical proﬁles of HIV-positive controls or HIV-positive stroke and high levels of PAI-1, VEGF, and sTM.

Conclusions HIV infection is associated with endothelial activation despite antiretroviral treatment. Our ﬁndings underscore the need for larger clinical cohorts to better understand the contribution of this perturbation of the endothelial function to the increasing burden of cardiovascular diseases in sub-Saharan Africa.

From the Institute of Infection and Global Health (J.K.-T., Z.A.-B., J.F., M.G., T.S., L.B.), University of Liverpool; Malawi-Liverpool-Wellcome Trust Clinical Research Programme (J.K.-T., H.M.), University of Malawi College of Medicine, Blantyre; Department of Clinical Sciences (H.M.), Liverpool School of Tropical Medicine, United Kingdom.

The Article Processing Charge was funded by the University of Liverpool on behalf of the Wellcome Trust. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (CC BY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Copyright © 2018 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1 Glossary ART = antiretroviral therapy; CV = coeﬃcient of variability; ICAM = intercellular adhesion molecule; IQR = interquartile range; PAI = plasminogen activator inhibitor; sTM = soluble thrombomodulin; VEGF = vascular endothelial growth factor.

The vascular endothelium is a critical regulator of cell adhesion, symptom onset, meeting the WHO’s clinical definition of stroke, inflammation, coagulation, and vascular remodeling. Pertur- and having a brain MRI confirming the diagnosis. Two com- bation of one or more of these functions, also known as en- munity controls were selected for each case with the goal of dothelial dysfunction, is induced by classical cardiovascular risk obtaining an age/sex frequency-matched random sample of the factors. Endothelial dysfunction is a major component of local population, with a geographical distribution in proportion to the and systemic inflammation. At the early stage of endothelial population density. The subsequent patient selection (or more activation, there is increased expression of cell adhesion mol- accurately “plasma specimen selection”) for the current study is ecules (e.g., intercellular adhesion molecule-1 [ICAM-1]) on described in figure. the surface of endothelial cells to facilitate leukocyte trans- endothelial migration. The occurrence of endothelial damage Demographic and clinical data of cases and controls included leads to the secretion of other biomarkers, notably plasminogen age, sex, hypertension, diabetes status, tobacco and alcohol activator inhibitor-1 (PAI-1) and soluble thrombomodulin consumption status, personal/family history of stroke, CD4+ (sTM) responsible for the perturbation of the coagulation T-cell count, HIV viral load, hypercholesterolemia, ART process and vascular endothelial growth factor (VEGF) re- status, recent infection, and National Institute of Health sponsible for angiogenesis.1 The interaction of these bio- Stroke Score for stroke cases only.3 The stroke type and eti- markers with other proinflammatory cytokines, as well as white ology were defined using MRI brain imaging and the trial of blood cells and smooth muscle cells, leads to vessel wall Org 10172 in acute stroke treatment criteria, respectively.3 remodeling and ultimately to various types of vasculopathy. Selection of biomarkers of Several studies conducted on HIV populations from Western endothelial dysfunction countries have reported an association between high levels of The biomarkers measured in this study were selected to assess biomarkers of endothelial dysfunction and death and/or the different stages of endothelial dysfunction as described in cardiovascular endpoints.2 However, it is unknown whether the same association is observed in a sub-Saharan African setting, where antiretroviral therapy (ART) has become more Figure Flow diagram of case and control selection widespread. This knowledge gap provides the rationale for this study conducted to evaluate the relationship between endothelial dysfunction, HIV infection, and stroke in Malawi.

Methods Standard protocol approvals, registrations, and patient consents This work was conducted as part of the study of Biomarkers of Stroke and Arterial Diseases in Malawian Adults, which was approved by the Research Ethics Committees of the Liver- pool School of Tropical Medicine and the University of Malawi College of Medicine. The study used plasma samples available in the biobank of the Malawi START study, a case- control study designed to identify risk factors of stroke in Malawi, where the prevalence of HIV infection is 10.3%.3 All patients enrolled in the Malawi START study provided a written informed consent that allowed their blood specimens to be reused for subsequent ethically cleared studies within a 5-year period from the time of collection.

Patient selection As previously described,3 stroke cases enrolled in the original Malawi START study were adult residents of Blantyre presenting at the Queen Elizabeth Central Hospital within 7 days of

2 Neurology: Neuroimmunology & Neuroinflammation | Volume 6, Number 2 | March 2019 Neurology.org/NN the introduction. The major cellular source of ICAM-1, PAI-1, plasma levels of the biomarkers as a continuous variable in and thrombomodulin is activated endothelial cells.4 VEGF, an a separate multiple linear regression analysis. essential growth factor for endothelial cells, is produced by macrophages, platelets, and keratinocytes. There is no vali- To further explore the effect of ART, the regression analyses dated normal range for the plasma concentration of bio- described above were repeated after excluding HIV-positive markers of endothelial dysfunction. Higher concentrations patients with a viral load greater than 1,000 RNA copies/mL correlate well with the magnitude of local or systemic in- (25 of 48 HIV-positive participants with viral load available). flammation and have been consistently associated with an The cutoff thresholds to distinguish between high and low increased cardiovascular risk. Because HIV-infected individ- levels of biomarkers were recalculated to adjust for the change uals have persistent systemic inflammation,5 we hypothesized in the population structure and size. The ART status was not that they would have higher levels of biomarkers of endo- included as a confounder in multivariable analyses because thelial dysfunction, explaining their greater risk of stroke. 87% of patients with a low or undetectable viral load were on ART. Measurement of biomarkers of endothelial dysfunction All the statistical tests performed were 2-tailed, and statistical The plasma concentrations of ICAM-1, PAI-1, VEGF, and significance was defined as p < 0.05. sTM were measured using ELISA kits (Abcam, Cambridge, United Kingdom). SimpleStep ELISA kits were used for Data availability ICAM-1, PAI-1, and sTM, whereas the standard kit was used The database of the Malawi START study is available for for VEGF. All samples were run in duplicates. Samples with an consultation or reuse on request to the principal investigator ffi individual coe cient of variability (CV) greater than 25% (L.B.). Sharing of all or part of this database is subject to prior were reanalyzed. authorization by the University of Malawi College of Medi- cine Research Ethics Committee. All the ELISA tests were performed according to the manufacturer’s instructions with the exception of the washing steps that were performed with an automated washer (Wellwash; Results ThermoFisher Scientific, Waltham, MA) using a mixture of ’ Tween 20 and phosphate-buffered saline (0.05% vol/vol). Participants characteristics The plates were read at 450 nm using an automated micro- The 229 participants (50.2% women) included in this study plate reader (Multiskan; ThermoFisher Scientific). The were distributed as follows: 136 HIV-negative controls, 32 standard curves were constructed using a 4-parameter logistic HIV-positive controls, 42 HIV-negative cases, and 19 HIV- regression fitting equation (online software: elisaanalysis. positive cases. The median age of the participants was 55 years – ’ com). The mean intra-assay and inter-assay CV for ICAM-1, (IQR: 38.0 65.0). Participants baseline characteristics are PAI-1, and VEGF was <10% and <15%, respectively. summarized in table 1.

Statistical analysis The etiologies of 60 of 61 ischemic strokes were as follows: 7 The statistical analyses were performed using the software (12%) atherosclerotic strokes, 5 (8%) cardio-thromboembolic STATA (version 13; StataCorp, College Station, TX). Based strokes, 16 (27%) strokes with other determined causes, and 32 on the stroke and HIV status, 4 clinical profiles were defined: (53%) strokes with an undetermined cause. HIV-negative controls, HIV-positive controls, HIV-negative stroke, and HIV-positive stroke. Demographic and clinical Factors associated with parameters were summarized as proportions for categorical endothelial dysfunction variables and median with interquartile range (IQR) for nu- In the logistic regression analysis, the adjusted OR for having merical variables. For comparisons across groups, Fisher exact high plasma levels of ICAM-1 was 3.6 (95% CI: 1.3–10.6, p = and Kruskall-Wallis tests were used for categorical and nu- 0.018) for the profile “HIV-positive controls” and 1.2 (95% merical variables, respectively. CI: 0.4–4.0, p = 0.772) for the profile “HIV-positive stroke” (table 2). The adjusted OR for having high plasma levels of The third tertile of the distribution of plasma concentration PAI-1, sTM, or VEGF was not significant for HIV-positive values was considered as the threshold for the distinction controls and HIV-positive stroke. The adjusted OR for having between high and low levels for each biomarker. To identify high plasma levels of PAI-1 was 0.4 (95% CI: 0.1–0.9, p = the predictors of endothelial dysfunction, the plasma level of 0.037) for the profile “HIV-negative stroke” (table 2). Linear each biomarker was included as a binary dependent variable regression did not modify the associations reported here. (low/high levels) in a separate multivariable logistic regression analysis. Age, sex, hypertension, diabetes mellitus, After excluding HIV-positive patients with a viral load greater tobacco and alcohol consumption, personal/family history of than 1,000 RNA copies/mL, the adjusted OR for having high stroke, ART status, and hypercholesterolemia were included plasma levels of ICAM-1 was 4.1 (95% CI: 1.3–13.1, p = in the model as potential confounders. We also explored the 0.017) for the profile “HIV-positive controls,” whereas the

Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 6, Number 2 | March 2019 3 Table 1 Demographic and clinical characteristics of study participants

Controls Ischemic stroke cases

HIV negative HIV positive HIV negative HIV positive Total (n = 136) (n = 32) (n = 42) (n = 19) (n = 229) p-Value

Women, n (%) 72 (52.9) 12 (37.5) 21 (50.0) 10 (52.6) 115 (50.2) 0.47

Age (y), median, IQR 58.0 47.5 (36.5–60.0) 63 (44.0–77.0) 42 (30.0–52.0) 55 (38.0–65.0) 0.001 (43.0–65.0)

Hypertension, n (%) 77 (56.6) 11 (34.4) 38 (90.5) 12 (63.2) 138 (60.3) <0.001

Diabetes mellitus, n (%) 2 (1.5) 1 (3.1) 5 (11.9) 0 (0.0) 8 (3.5) 0.02

Tobacco consumption, n (%) 15 (11.0) 7 (21.9) 11 (26.2) 1 (5.3) 34 (14.9) 0.04

Alcohol consumption, n (%) 26 (19.1) 9 (28.1) 8 (19.1) 2 (10.5) 45 (19.7) 0.50

Previous stroke, n (%) 4 (2.9) 0 (0.0) 2 (4.8) 0 (0.0) 6 (2.6) 0.82

Family history of stroke, n (%) 12 (8.8) 3 (9.4) 6 (14.3) 3 (15.8) 24 (10.5) 0.58

CD4 count (cell/mm3), — 452.7 (378.1–527.4) — 260.1 (156.3–363.9) 379.0 (314.3–443.6) 0.002a Mean, 95% CI

Total cholesterol (mmol/L) 3.9 (2.8–4.6) 3.6 (2.3–4.7) 3.4 (2.9–4.0) 3.3 (2.3–4.1) 3.6 (2.6–4.5) 0.63

Viral load (RNA copies/mL), — 1,836.0 — 1,883.5 1,883.5 0.80a median, IQR (1.0–68,416.0) (1.0–35,793.0) (1.0–57,596.0)

Viral load >1,000 — 15 (50.0) — 10 (55.6) 25 (52.1) 0.71a RNA copies/mL, n (%)c

On ART, n (%) — 17 (53.1) — 9 (47.4) 26 (51.0)d 0.45a

NIHSS—median, IQR —— 13.5 12.0 (9.0–14.0) 13.0 (8.0–17.0) 0.56b (8.0–18.0)

Abbreviation: IQR = interquartile range. The median age was 55 years (38.5–65.0) for controls and 52 years for cases (38.0–73.0), p = 0.38. The CD4 count was available for 29 HIV-positive controls and 18 HIV-positive cases. a p Value displayed only for comparison between HIV-positive controls and cases. b p Value displayed only for comparison between HIV-negative and HIV-positive cases. c The HIV viral load was available for 30 HIV-positive controls and 18 HIV-positive cases (3 missing values). d The denominator for the percentage displayed is 51 (total number of HIV-positive participants).

OR for having high plasma levels of PAI-1 was 0.5 (95% CI: activation and dysfunction are both risk factors for atherogenesis. 0.2–1.3, p = 0.183). For example, high levels of ICAM-1 have been associated with increasing carotid intima-media thickness, a surrogate marker of Discussion atherosclerosis and a predictor of cardiovascular events.2,8 In our cohort, more than 50% of the HIV-positive controls were on Our results show that HIV infection is independently asso- ART, and their CD4+ T-cell count was >400 cells/mm3.This ciated with high plasma levels of ICAM-1, a biomarker of means that despite having a relatively stable infection, HIV- endothelial activation. There was no association between infected individuals still have an ongoing endothelial activation as stroke status and high levels of ICAM-1 or between HIV demonstrated by previous studies conducted in Western coun- infection and high levels of PAI-1, sTM, and VEGF (table e-1, tries.9 Therefore, ART alone might not be enough to suppress links.lww.com/NXI/A94). cardiovascular risk in people living with HIV, and additional interventions to control HIV-related inflammation might be The association between HIV infection and high levels of ICAM- needed. One potential intervention might be the use of statins as 1 reported here expands the limited literature on sub-Saharan an adjunct therapy to attenuate endothelial dysfunction and Africa6 and is consistent with findings from high-income coun- vascular inflammation. tries.2 Although other risk factors, such as diabetes, hypercholesterolemia, and ART, can contribute to endothelial activation, Unexpectedly, we showed no association between HIV and we found that this association is independent of those factors. endothelial dysfunction among patients with stroke. The most Chronicsystemicinflammation induced by HIV infection is likely explanation is that our sample size was too small, with a trigger for endothelial activation,7 which is an important only 19 HIV-positive stroke cases. Furthermore, despite component of endothelial dysfunction. In turn, endothelial restricting the analysis to ischemic stroke, the cohort remained

4 Neurology: Neuroimmunology & Neuroinflammation | Volume 6, Number 2 | March 2019 Neurology.org/NN Table 2 Univariable and multivariable analyses of factors associated with high levels of biomarkers of endothelial dysfunction

Univariable model Multivariable modelb

Biomarkers of endothelial dysfunction No. of participants with a high Crude p- Adjusted p- and participants’ clinical profilea plasma level of the biomarker (%) OR 95% CI Value OR 95% CI Value

ICAM-1

HIV-negative controls 36 (26.5) 1 1

HIV-positive controls 21 (65.6) 5.3 2.3–12.1 <0.001 3.6 1.3–10.6 0.018

HIV-negative stroke 12 (28.6) 1.1 0.5–2.4 0.789 1.0 0.4–2.4 0.978

HIV-positive stroke 7 (36.8) 1.6 0.6–4.4 0.348 1.2 0.4–4.0 0.772

PAI-1

HIV-negative controls 50 (36.76) 1 1

HIV-positive controls 11 (34.4) 0.9 0.4–2.0 0.800 0.6 0.2–1.7 0.303

HIV-negative stroke 9 (21.4) 0.5 0.2–1.1 0.069 0.4 0.1–0.9 0.037

HIV-positive stroke 6 (31.6) 0.8 0.3–2.1 0.660 0.6 0.2–2.0 0.397 sTM

HIV-negative controls 49 (36.0) 1 1

HIV-positive controls 7 (21.9) 0.5 0.2–1.3 0.132 0.3 0.1–1.2 0.084

HIV-negative stroke 18 (42.9) 1.3 0.7–2.3 0.425 1.8 0.8–4.3 0.131

HIV-positive stroke 2 (10.5) 0.2 0.1–1.2 0.042 0.2 0.0–1.0 0.050

VEGF

HIV-negative controls 45 (33.1) 1 1

HIV-positive controls 11 (34.4) 1.1 0.5–2.4 0.890 0.9 0.3–2.7 0.830

HIV-negative stroke 12 (28.6) 0.8 0.4–1.7 0.584 0.8 0.3–1.8 0.599

HIV-positive stroke 8 (42.1) 1.5 0.6–3.9 0.440 1.4 0.4–4.8 0.556

Abbreviations: ART = antiretroviral therapy; ICAM = intercellular adhesion molecule; PAI = plasminogen activator inhibitor; sTM = soluble thrombomodulin; VEGF = vascular endothelial growth factor. a Number of participants belonging to each clinical profile: HIV-negative controls (n = 136), HIV-positive controls (n = 32), HIV-negative stroke (n = 42), and HIV- positive stroke (n = 19). Only cases with ischemic stroke were included. b Adjustment variables were age, sex, hypertension, diabetes mellitus, tobacco and alcohol consumption, ART status, hypercholesterolemia, and recent infection. None of these variables had an OR significantly higher than 1. The variable “personal and family history of stroke” was omitted because of collinearity. heterogeneous, thus reducing our statistical power further and study represents a limitation and continues to be a challenge our ability to explore the varied etiologies of cerebrovascular in this field. However, there is good evidence that high diseases. However, the independent association between HIV plasma concentrations are related to increased cardiovascular infection and high levels of ICAM-1 in the control group risk.2,11,12 suggests that a cardiovascular risk does exist. Consequently, further evaluation in a larger clinical cohort is warranted. Overall, this study confirms the association between HIV infection and endothelial activation despite ART in a sub- PAI-1 is a molecule that inhibits fibrinolysis. High and low plasma Saharan African setting. Larger well-phenotyped cohort levels are expected in ischemic stroke and hemorrhagic stroke, studies are needed to clarify the contribution of HIV-related respectively. A decrease in the plasma concentration of PAI-1 endothelial activation to the long-term stroke morbidity and with increasing age has been described.10 Therefore, we suspect mortality in the ART era. that the association between levels of PAI-I and the profile “HIV- negative stroke” is due to a residual confounding effect of age. Author contributions L. Benjamin and J. Kamtchum-Tatuene conceived the study. The absence of a fixed cutoff to distinguish between low L. Benjamin acquired the samples and the clinical data. J. and high plasma levels of the biomarkers considered in this Kamtchum-Tatuene and Z. Al-Bayati performed the

Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 6, Number 2 | March 2019 5 laboratory analyses under the supervision of J. Flatley and M. on a clinical advisory board of Siemens Healthineers; received Griffiths. J. Kamtchum-Tatuene performed the statistical travel funding from World Congress of Neurology, South East analyses under the supervision of L. Benjamin and H. Asia Encephalitis, European Commission, and WHO; and Mwandumba. J. Kamtchum-Tatuene drafted the first version received research support from NIHR HPRU. L. Benjamin of the manuscript that was edited by L. Benjamin. H. received research support from GlaxoSmithKline. Full dis- Mwandumba, Z. Al-Bayati, J. Flatley, M. Griffiths, and T. closure form information provided by the authors is available Solomon revised the subsequent versions of the manuscript. with the full text of this article at Neurology.org/NN. All authors approved the final version. Publication history fl Acknowledgment Received by Neurology: Neuroimmunology & Neuroin ammation July 2, 2018. Accepted in final form October 16, 2018. The authors are grateful to (1) all the patients and their ff families for consenting to take part in this study, (2) the sta References of the Institute of Infection and Global Health and the 1. Benjamin LA, Bryer A, Emsley HC, Khoo S, Solomon T, Connor MD. HIV infection and stroke: current perspectives and future directions. Lancet Neurol 2012;11: Wellcome Trust Centre for Global Health Research for their 878–890. administrative support, and (3) the staff at Malawi-Liverpool- 2. Ross AC, Rizk N, O’Riordan MA, et al. Relationship between inflammatory markers, Wellcome Clinical Research Programme and Queen Eliz- endothelial activation markers, and carotid intima-media thickness in HIV-infected patients receiving antiretroviral therapy. Clin Infect Dis 2009;49:1119–1127. abeth Central Hospital. 3. Benjamin LA, Corbett EL, Connor MD, et al. HIV, antiretroviral treatment, hypertension, and stroke in Malawian adults: a case-control study. Neurology 2016;86: 324–333. Study funding 4. Zhang J, Defelice AF, Hanig JP, Colatsky T. Biomarkers of endothelial cell activation This research was funded by the Wellcome Trust [106829/Z/ serve as potential surrogate markers for drug-induced vascular injury. Toxicol Pathol 2010;38:856–871. 15/Z to JKT and CNR11146 to L.B.]. L.B. was also supported 5. Sereti I, Krebs SJ, Phanuphak N, et al. Persistent, albeit reduced, chronic inflammation by an NIHR Clinical Lectureship. in persons starting antiretroviral therapy in acute HIV infection. Clin Infect Dis 2017; 64:124–131. 6. Graham SM, Rajwans N, Jaoko W, et al. Endothelial activation biomarkers increase Disclosure after HIV-1 acquisition: plasma vascular cell adhesion molecule-1 predicts disease progression. AIDS 2013;27:1803–1813. J. Kamtchum-Tatuene received research support from Ge- 7. Melendez MM, McNurlan MA, Mynarcik DC, Khan S, Gelato MC. Endothelial neva University Hospital Fund for Research and De- adhesion molecules are associated with inflammation in subjects with HIV disease. Clin Infect Dis 2008;46:775–780. velopment and the Wellcome Trust. H. Mwandumba received 8. Lorenz MW, Markus HS, Bots ML, Rosvall M, Sitzer M. Prediction of clinical car- research support from the Bill and Melinda Gates Foundation diovascular events with carotid intima-media thickness: a systematic review and meta- analysis. Circulation 2007;115:459–467. and MRC. Z. AL-Bayati and J. Flatley report no disclosures. 9. de Larrañaga GF, Bocassi AR, Puga LM, Alonso BS, Benetucci JA. Endothelial M. Griffiths received travel funding from British Paediatric markers and HIV infection in the era of highly active antiretroviral treatment. Thromb Res 2003;110:93–98. Neurology Association and Royal College of Physicians of 10. Surjawan Y, Setiabudy RD, Suryaatmadja M, Ranakusuma TAS. The association of Edinburgh; received research support from Fast Track Diag- plasminogen activator inhibitor-1 level with ischemic stroke (preliminary study). Med J Indones 2009;19:158–163. nostics Research Limited, Department of Health UK NIHR, 11. Knudsen A, Katzenstein TL, Benfield T, et al. Plasma plasminogen activator inhibitor-1 MRC, European Commission, Ministry of France, and Re- predicts myocardial infarction in HIV-1-infected individuals. AIDS 2014;28:1171–1179. 12. Musselwhite LW, Sheikh V, Norton TD, et al. Markers of endothelial dysfunction, public of Indonesia. T. Solomon served on a commercial data coagulation and tissue fibrosis independently predict venous thromboembolism in safety monitoring board for the study of Ebola vaccine; served HIV. AIDS 2011;25:787–795.

6 Neurology: Neuroimmunology & Neuroinflammation | Volume 6, Number 2 | March 2019 Neurology.org/NN ARTICLE OPEN ACCESS Inﬂammatory myopathy with myasthenia gravis Thymoma association and polymyositis pathology

Naohiro Uchio, MD, Kenichiro Taira, MD, Chiseko Ikenaga, MD, Masato Kadoya, MD, Atsushi Unuma, MD, Correspondence Kenji Yoshida, MD, Setsu Nakatani-Enomoto, MD, PhD, Yuki Hatanaka, MD, PhD, Yasuhisa Sakurai, MD, PhD, Dr. Shimizu [email protected] Yasushi Shiio, MD, PhD, Kenichi Kaida, MD, PhD, Akatsuki Kubota, MD, PhD, Tatsushi Toda, MD, PhD, and Jun Shimizu, MD, PhD

Neurol Neuroimmunol Neuroinﬂamm 2019;6:e535. doi:10.1212/NXI.0000000000000535 Abstract Objective To provide evidence that idiopathic inﬂammatory myopathy (IM) with myasthenia gravis (MG) frequently shows thymoma association and polymyositis (PM) pathology and shares clinicopathologic characteristics with IM induced by immune checkpoint inhibitors (ICIs).

Methods We analyzed the clinicopathologic features of 10 patients with idiopathic IM and MG identiﬁed in 970 consecutive patients with biopsy-proven IM.

Results Seven patients (70%) had thymoma. IM and MG were diagnosed with more than 5-year time difference in 6 thymomatous patients and within 1 year in 1 thymomatous and 3 nonthymomatous patients. Seven thymomatous patients showed rhabdomyolysis-like features with respiratory failure (4/7), dropped head (3/7), cardiac involvement (2/7), and positive anti– acetylcholine receptor (anti-AChR) and anti-titin antibodies (7/7 and 4/6, respectively) but rarely showed ocular symptoms (2/7) or decremental repetitive nerve stimulation (RNS) responses (1/7) at IM diagnosis. Three nonthymomatous patients showed acute cardiore- spiratory failure with rhabdomyolysis-like features (1/3), positive anti-AChR and anti-titin antibodies (3/2 and 2/2, respectively), and fluctuating weakness of the skeletal muscle without ocular symptoms (3/3). Muscle pathology showed a PM pathology with infiltration of CD8- positive CD45RA-negative T-lymphocytes (9/9), scattered endomysial programmed cell death 1 (PD-1)–positive cells (9/9), and overexpression of programmed cell death ligand 1 (PD-L1) on the sarcolemma of muscle fibers around the infiltrating PD-1–positive cells (7/9).

Conclusion Rhabdomyolysis-like features, positive anti-AChR antibody without decremental RNS responses, and PD-L1 overexpression are possible characteristics shared by ICI-induced IM. Frequent thymoma association in patients with idiopathic IM and MG may suggest thymoma- related immunopathogenic mechanisms, including dysregulation of the immune checkpoint pathway.

From the Department of Neurology (N.U., K.T., C.I., A.U., A.K., T.T., J.S.), Graduate School of Medicine, University of Tokyo; Division of Neurology (M.K., K.K.), Department of Internal Medicine, National Defense Medical College, Saitama; Division of Neurology (Y. Shiio), Tokyo Teishin Hospital; Department of Neurology (Y. Sakurai), Mitsui Memorial Hospital; Department of Neurology (Y.H.), Teikyo University School of Medicine; and Department of Neurology (K.Y., S.N.-E.), Fukushima Medical University, Japan.

Copyright © 2018 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1 Glossary anti-AChR = anti–acetylcholine receptor; CK = creatine kinase; CTLA-4 = cytotoxic T-lymphocyte–associated protein 4; IBM = inclusion body myositis; ICI = immune checkpoint inhibitor; IM =inflammatory myopathy; MG = myasthenia gravis; MHC = major histocompatibility complex; MSA = myositis-specific autoantibody; PD-1 = programmed cell death 1; PD-L1 = programmed cell death ligand 1; PM = polymyositis; PSL = prednisolone; RNS = repetitive nerve stimulation; SFEMG = single- fiber electromyogram.

– Idiopathic inflammatory myopathies (IMs) are a heteroge- and/or regenerating fibers18 20 were required. Exclusion of neous group of muscle disorders. Myasthenia gravis (MG) muscular dystrophy by immunohistochemistry and clinical is rarely associated with IM. The reported clinical features features was also required. Inclusion body myositis (IBM) of patients with both idiopathic IM and MG included was excluded using the 188th European Neuromuscular – brachio-cervical weakness or dropped head,1 5 respiratory Centre IBM criteria.21 Sarcoid myopathy was excluded on – decompensation,3 10 muscle swelling with pain,4,11 cardiac the basis of clinical and pathologic findings.22 Patients who involvement,4,5,9 and markedly elevated serum creatine kinase developed IM as an adverse effect of drugs, including ICIs, (CK) levels.4,6,8 In addition, patients with anti–acetylcholine were excluded from this study. MG diagnosis was based receptor (anti-AChR) antibody (Ab)-positive thymomatous on clinical features of weakness with increased fatigability IM without MG symptoms showing rapidly progressive of skeletal muscles and one or more of the following 3 cri- weakness and respiratory failure with markedly elevated se- teria: (1) a positive edrophonium infusion test, (2) decre- rum CK levels12,13 have been described. Although previous mental repetitive nerve stimulation (RNS) responses, and (3) reports suggested some characteristics of idiopathic IM patients increased jitter or blocking on a single-fiber electromyogram with MG and/or thymoma, clinical features, including the (SFEMG). Thymoma diagnosis was made on the basis of CT temporal relationship between the onset of IM and MG and of the anterior mediastinum tumor and confirmed by patho- the prevalence of clinical characteristics, including thymoma logic examination. association, have not been well known because of the lack of systematical study conducted in a series of patients with IM. In recruiting patients with both IM and MG, patients with Furthermore, pathologic findings have not been studied IM who had a past or subsequent history of MG were in- systematically. cluded. Because the patients with thymoma sometimes have MG or subclinical MG or may develop MG during follow-up The rare combination of IM and MG has been recently reported periods, the patients with IM who had a past or subsequent in patients with immune-related adverse events induced by history of thymoma were also included. By these methods, immune checkpoint inhibitors (ICIs) targeting programmed among 970 consecutive patients with IM, we found 10 cell death 1 (PD-1) or cytotoxic T-lymphocyte–associated patients with both IM and MG (1.0%) and 1 non-MG thy- – protein 4 (CTLA-4).14 17 The reported clinical features of momatous patient with IM (figure 1). The 11 patients with these patients include cardiac involvement14,15 and both IM and MG included 3 patients who have been pre- rhabdomyolysis-like features with markedly elevated serum viously reported in separate reports (no. 11, Yamaguchi CK levels,14,15 suggesting similarities of the clinical features et al., 2000; no. 5, Maekawa et al., 2014; and no. 9, Suzuki – of IM between the 2 groups: ICI induced and idiopathic. et al., 2014).23 25 Therefore, we analyzed the clinicopathologic characteristics of IM associated with MG in a series of patients with biopsy- Definition of onset of IM and MG proven IM to determine whether some characteristic features IM onset was defined as the time of development of sustained are shared by ICI-induced and idiopathic patients with both IM skeletal muscle weakness with elevated serum CK levels. MG and MG. onset was defined as the earlier of 2 dates: (1) the date of development of weakness with increased fatigability of skeletal muscles (e.g., fluctuating diplopia, ptosis, or skeletal Methods muscle power) or (2) the date of detection of abnormality on RNS or an SFEMG in weakened muscles. Patients Clinical records and biopsy reports were reviewed for 970 Histochemical and consecutive patients, who were referred to our department for immunohistochemical analysis pathologic diagnosis between April 1986 and December 2017. Stored frozen muscle samples were processed into 10-μm IM diagnosis was based on the criteria proposed by Bohan and sections for routine histochemistry and immunohistochem- Peter18,19; in addition, both (1) elevated serum CK levels and istry. Immunohistochemistry were performed with a conven- (2) muscle biopsy findings of inflammatory changes with tional method, using the substrate 3,3’-diaminobenzidine major histocompatibility complex (MHC) class I expression tetrahydrochloride (DAB). Primary antibodies against MHC on non-necrotic muscle fibers and sometimes with necrotic class I (Dako, Agilent Technologies, CA), MHC class II (Dako),

2 Neurology: Neuroimmunology & Neuroinflammation | Volume 6, Number 2 | March 2019 Neurology.org/NN Results Figure 1 Flowchart of patient inclusion Clinical features of patients at IM diagnosis Table 1 shows the clinical features and histopathologic ﬁnd- ings of the skeletal muscles of 10 patients with both IM and MG; the patients’ detailed clinical features are described in appendix e-1 links.lww.com/NXI/A95.

The mean age at IM diagnosis was 60 years (range: 47–78 years). Six patients were women. None of the patients had skin rash or interstitial lung disease. Seven patients were associated with invasive thymoma, and the other 3 had no thymoma. The temporal relationship between the onset of IM and MG are as follows: (1) 5 thymomatous patients (nos. 1, 2, and 4–6) had preexisting MG (diagnosed more than 9 years before their IM diagnosis); (2) 1 thymomatous patient (no. 7) had preexisting IM (diagnosed with MG 5 years after the IM diagnosis); and (3) 1 thymomatous patient (no. 3) and 3 nonthymomatous patients (nos. 8–10) had concurrent IM and MG (diagnosed with IM and MG within 1 year). Clinical features related to MG at IM diagnosis included fluctuating Clinical and pathologic features of 1,146 patients were evaluated based on Bohan and Peter’s myositis criteria and serum CK levels, and then 970 ocular symptoms (2/10), abnormal RNS responses on patients were identified as more than possible polymyositis or dermatomyositis with an elevated serum CK level. Among these patients, 10 patients weakened extremity muscles (4/10), positive edrophonium with a history of MG in flow A and 8 patients with a history of thymoma in infusion test (2/3), and positive anti–AChR Ab (9/10). flow B were identified. Finally, 10 patients with MG were recruited excluding the 7 overlapping patients between flow A and flow B. In addition, 1 non-MG patient with a history of thymoma was also recruited. CK = creatine kinase; Thymomatous IM patients rarely showed fluctuating ocular DM = dermatomyositis; PM = polymyositis. symptoms (2/7) and abnormal RNS responses on extremities (1/7). By contrast, 3 nonthymomatous IM patients showed abnormal RNS responses (3/3) and positive edrophonium CD4 (Dako), CD8 (Nichirei Biosciences, Tokyo, Japan), infusion test (2/2) but showed no ocular symptoms (0/3). CD20 (Abcam, Cambridge, UK), CD21 (Dako), CD45/ leukocyte common antigen (Dako), CD45RA (Dako), Before IM diagnosis, 2 patients had a history of transient CD68 (Dako), myxovirus resistance protein A (M x A; elevation of serum CK levels without limb weakness (4,209 Abcam),PD-1(Abcam),programmedcelldeathligand1 IU/L in no. 1 and 518 IU/L in no. 8). Progression of weak- (PD-L1; Abcam), and CTLA-4 (Abcam) were used. ness in IM was rapid in 5 patients. Four thymomatous patients Detection of autoantibodies with normal RNS tests on extremities presented with rapid Myositis-specific autoantibodies (MSAs), including anti–Jo-1, progression of weakness with acute markedly elevated serum – anti–PL-7, anti–PL-12, anti–Mi-2, and anti–signal recogni- CK levels (1,008 10,226 IU/L) at IM development tion particle 54-kDa protein Abs, were detected by dot- (rhabdomyolysis-like features) and required ventilator sup- blotting using recombinant proteins (Diarect AG, Freiburg, port. One nonthymomatous patient (no. 9) developed re- Germany). Anti–transcriptional intermediary factor 1 gamma spiratory muscle weakness with relatively preserved limb and anti–melanoma differentiation-associated gene 5 Abs muscle strength, cardiac failure, and acute elevation of serum were detected by immunoprecipitation, and anti–3-hydroxy- CK levels (1,140 IU/L). 3-methylglutaryl-CoA reductase Ab was detected by ELISA, as described in our earlier study.26 Anti-titin Ab was also The clinical features of the 10 patients included respiratory detected by ELISA (DLD Diagnostika, Hamburg, Germany). failure requiring ventilator support (5/10), severe weakness of extremities (4/10), dropped head (4/10), myalgia (4/10), Standard protocol approvals, registrations, dysphagia (5/9), and cardiac involvement (3/10). One thy- and patient consents momatous patient (no. 7) had initial symptoms of muscle The study was approved by the institutional ethics commit- swelling and pain in the forearms. tees of the University of Tokyo Hospital. Written informed consent was obtained from each patient at the time of biopsy The mean serum CK level was 2,712 IU/L. The serum testing and serum sampling. for autoantibodies demonstrated anti-AChR Ab (9/10), MSAs (0/9), and anti-titin Ab (6/8). A needle EMG, per- Data availability formed for 9 patients, showed spontaneous activities at rest in Anonymized data will be shared by request from any qualified most of them (8/9). Three patients (3/10) showed cardiac investigators. involvements. A cardiac muscle biopsy performed in 1 patient

Table 1 Clinical and pathologic features of IM in patients with both IM and MG

Patient 1 2 3 4 5 6 7 8 9 10

Age at IM/MG diagnosis (y) 59/50 73/56 53/52 50/38 57/43 49/36 47/52 78/78 70/70 60/60

Sex Male Female Male Female Male Female Female Male Female Female

MG diagnosis before IM +9 +17 <1f +12 +14 +13 −5<1<1<1 diagnosis (y)

Thymoma ++ ++ + + + −− −

WHO classification B2 B2 B2 ND B3 B2 B2 NA NA NA

Age at thymoma diagnosis 42 56 53 38 43 36 47 NA NA NA (y)

Thymoma status at IM Under CHT No recurrence Untreated No recurrence Recurrence No progression Untreated NA NA NA diagnosis

Disease activity of MG

Ocular symptoms/ −/− +/+ −/−f +/+ −/−−/−−/−−/−−/−−/− fluctuating weakness at IM diagnosis

Decremental responses on −− −+ −−−++ + RNS at IM diagnosis

Positive edrophonium NE NE NE NE NE NE − +NE+ infusion test at IM diagnosis

Anti-AChR Ab at MG/IM ND/2.2 11/540 100/ND 80/300 163/7 41/10 ND/12 47.7i 6.5i −i diagnosis (nmol/L)

CK level at MG diagnosis ND ND 231 ND 185 41 ND 263 1,140 282 (IU/L)

Clinical features of IM at IM diagnosis

History of transiently + −−− − − −+ −− elevated CK levels

Progression of weakness at Rapid No Rapid Chronic Chronic Rapid Rapid Chronic Rapid Chronic IM diagnosis

Limb weakness Severe No–mild Severe Moderate No–mild Severe Severe No–mild No–mild Moderate

Dropped head + − + − + −−+ −−

Dysphagia + − ++ + + −−ND −

Continued erlg.r/NNuooy erimnlg erifamto oue6 ubr2|Mrh2019 March | 2 Number 6, Volume | Neuroinflammation & Neuroimmunology Neurology: Neurology.org/NN

Table 1 Clinical and pathologic features of IM in patients with both IM and MG (continued)

Patient 1 2 3 4 5 6 7 8 9 10

Respiratory failure + (NIPPV) − + (MV) −− + (MV) + (MV) − + (MV) −

Myalgia + −−− − + + (swelling)h + −−

Fever − Low grade −− − Remittent Low grade −− −

Cardiac involvement −− −Anteroseptal − VT and diffuse −−VT and diffuse − hypokinesis hypokinesis hypokinesis

Other complications −− DG DG Alopecia, DG, DPB, − DG and DPB − Pure red cell aplasia − and AIC

Laboratory findings at IM diagnosis

CK level (IU/L) 10,226 376 1,008 341 1,059 5,102 7,315 263 1,140 292

CRP level (mg/dL) 2.6 0.06 0.04 0.07 1.83 1.04 3.32 <0.3 4.7 −

ESR (mm/h) 20 26 4 23 NE 19 73 30 NE NE

Myositis-specific −− −− − − − −− NE autoantibodies

Anti-titin Ab − NE + + − ++++NE

Spontaneous activities at ++ ++ + + + +NE − rest on nEMG

Histopathologic findings

Necrotic and/or + + + ± ± + +++ + ± + regenerating fibersa

Perimysial inflammationb ± ± +++ ++ ± ± + +++ ± ±

Endomysial inflammationb + + + ± ± + +++ + + +

CD8+ T cells surrounding/ +/+ +/+ +/+ +/− +/− +/− +/+ +/− +/− +/+ invading non-necrotic fibers

CD45RA+ cell infiltration −− +g −− − NE +g −−

MHC class I expressionc ++ +++ +++ + + +++ +++ +++ ++ +++

MHC class II expressionc ++ ++ + + + − + +++

Granulomatous lesions −− −− − − + −− −

Continued 5 6 erlg:Nuomuooy&Nuonlmain|Vlm ,Nme ac 09Neurology.org/NN 2019 March | 2 Number 6, Volume | Neuroinflammation & Neuroimmunology Neurology:

Table 1 Clinical and pathologic features of IM in patients with both IM and MG (continued)

Patient 1 2 3 4 5 6 7 8 9 10

CD20+ lymphoid −− + −− − NE + −− aggregatesd

MxA+ capillaries and fibers −− −− − − NE −− −

PD-1+ cell infiltration + + + + + + NE + + +

PD-L1 upregulation on ++ − + − ++NE++++ fiberse

CTLA-4+ cell infiltration + − ++ −−NE −− +

Classification based on Definite Definite PM Definite Probable PM Probable PM Probable PM Definite PM Probable Probable PM Definite ENMC criteria PM PM PM PM

Abbreviations: AIC = autoimmune cholangitis; CHT = chemotherapy; CK = creatine kinase; CRP = C-reactive protein; DG = dysgeusia; DPB = diffuse panbronchiolitis; ESR = erythrocyte sedimentation rate; MV = mechanical ventilation; NA = not applicable; ND = no data; NE = not examined; NIPPV = noninvasive positive-pressure ventilation; nEMG = needle electromyogram; VT = ventricular tachycardia; WHO = World Health Organization. a Necrotic and regenerating fibers in 1 fascicle: ±, sparse; +, 1 to <5; ++, 5 to <20; and +++, ≥20. b Density of inflammatory cells: −, 0; ±, 1 to <5; +, 5 to <20; ++, 20 to <100; and +++, ≥100. c MHC class I or class II antigen on the sarcolemma of non-necrotic fibers: −, 0%; +, 1% to <25%; ++, 25% to <50%; and +++, 50%–100%. d CD20+ lymphoid aggregate was defined when more than 100 CD20+ cells/place were observed. e PD-L1 upregulation on the sarcolemma of non-necrotic fibers: −, none; +, invaded fibers limitedly; and ++, invaded fibers and non-necrotic fibers around invaded fibers. f The patient who was diagnosed with ocular MG developed IM 3 months after the introduction of immunotherapy for MG. g CD45RA+ cells were observed only at perimysial lymphoid aggregates in these patients. h The patient showed myalgia with muscle swelling. i Data at IM diagnosis. (no. 6) showed abundant T-lymphocyte infiltration consistent with lymphocytic myocarditis. Figure 2 Histopathologic findings of patients with both inflammatory myopathy and myasthenia gravis Pathologic findings We observed various amounts of necrotic fibers and inflammatory cells in both the perimysium and the endomysium. Allpatientsshoweddifferent levels of overexpression of MHC class I on muscle fibers with various extent levels: 1+, n = 2; 2+, n = 2; and 3+, n = 6 (table 1). Overexpression of MHC class II on muscle fibers was observed in 9 patients (9/10). All patients showed the findings of endomysial CD8+ T-lymphocytes surrounding (10/10) and sometimes invading (5/10) non- necrotic muscle fibers expressing MHC class I antigen and therefore met the pathologic criteria of polymyositis (PM)20 (definite PM, n = 5; probable PM, n = 5; figure 2A).

Immunohistochemistry for CD45RA, which was performed in 9 frozen sample available patients, showed no endomysial CD45RA+ cells, indicating that the endomysial CD8+ T-lymphocytes surrounding and sometimes invading non- − necrotic muscle ﬁbers were all CD45RA cells (9/9). Two patients (nos. 3 and 8) showed perimysial accumulation of CD45RA+ cells (ﬁgure e-1 links.lww.com/NXI/A95).

One thymomatous patient (no. 7) showed granulomatous inflammatory cell invasion with multinucleated giant cells (figure 2B). The granulomas and giant cells were positive for CD68. Two patients (nos. 3 and 8) showed focal perimysial accumulation of mononuclear cells, many of which were positive for CD20 (figure 2, C and D).

Immunohistochemistry for PD-1 and PD-L1 showed scattered endomysial PD-1+ cells (9/9) and overexpression of PD-L1 on the sarcolemma of muscle fibers near PD-1+ cells (7/9). Of interest, 2 patients (nos. 1 and 9) showed PD-L1 (A) Immunohistochemical staining for CD8 showing CD8-positive cells invading non-necrotic muscle fibers in patient 1. (B) Hematoxylin-eosin (HE) overexpression extending on non-necrotic fibers around the staining showing granulomatous lesion with multinucleated giant cells in fibers invaded by the PD-1+ cells (figure 2, E and F). Im- patient 7. (C and D) Serial sections with HE staining and immunohisto- + chemical staining for CD20 showing perimysial aggregation of CD20-positive munohistochemistry for CTLA-4 demonstrated CTLA-4 cells in patient 8. (E and F) Serial sections with immunohistochemical fi fi staining for PD-1 and PD-L1 showing endomysial PD-1–positive cells and PD- cells in endomysial in ltration (4/9) ( gure e-2 links.lww. L1 upregulation on non-necrotic fibers in patient 9. Scale bar = 50 μm. PD-1 = com/NXI/A95). programmed cell death 1; PD-L1 = programmed cell death ligand 1. Associated thymoma The World Health Organization classifications of invasive thymoma in 7 patients were B2 (n = 5), B3 (n = 1), and no com/NXI/A95). This 51-year-old woman had moderate to data available (n = 1) (table 1). The mean age at thymoma severe weakness in her limbs and respiratory muscles re- diagnosis was 45 years (range: 36–56 years). Thymoma di- quiring ventilator support. Laboratory data showed an el- agnosis was preceded to IM diagnosis more than 12 years evated serum CK level (695 IU/L) and positive anti-AChR (range: 12–17 years) in 5 patients (nos. 1, 2, and 4–6), and and anti-titin Abs. Needle EMG showed spontaneous ac- both thymoma and IM were codiagnosed in 2 patients (nos. 3 tivities, but RNS test was normal. Chest CT showed an an- and 7). The disease statuses of thymoma at IM diagnosis were terior mediastinum tumor (1 cm in size), which was revealed as follows: no recurrence or progression without therapy (n = to be necrotic tissues. Histopathologic diagnosis of muscle fi 20 3), untreated (n = 2), recurrence (n = 1), and no progression biopsy was de nite PM. She was successfully treated with under chemotherapy (n = 1). prednisolone (PSL). Her anterior mediastinum tumor increased in size without neurologic symptoms. Five years after Thymomatous IM patient without the diagnosis of IM, the tumor was resected and pathologi- MG symptoms cally confirmed as combined B2/B3 thymoma. Her 11-year Our series included 1 thymomatous IM patient without follow-up showed no exacerbation of IM or thymoma or MG symptoms (no. 11, details in appendix e-1 links.lww. development of MG.

Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 6, Number 2 | March 2019 7 Clinical courses and response to treatments patients: none showed ocular symptoms, 1 patient showed One patient (no. 8) showed normalized serum CK levels during acute respiratory and cardiac failure with acute serum CK bed rest and refused immunotherapy. He showed no exacer- level elevation, 2 patients (2/2) showed positive anti-titin bation of symptoms for 2.6 years until final follow-up. The Ab, and all 3 patients showed PM pattern pathology, charac- remaining 9 patients, including 2 patients who showed nor- terized by CD8+ T-lymphocyte–mediated cellular autoimmu- malization of serum CK levels during bed rest, were treated with nity. PM pattern pathology is a rare pathologic finding among immunotherapy (table 2). The treatments included immuno- patients with IM29,30 and is suggested as a secondary pathologic suppressants alone in 1 patient (no. 4) and increasing doses of pattern associated with other systemic autoimmune diseases, oral PSL (20–60 mg) in the other 8 patients in combination including connective tissue diseases, viral infections, cancer, with various immunosuppressive therapies: oral immunosup- and drug toxicity.31 The overlapping of clinical, serologic, and pressants (n = 4), high-dose IV methylprednisolone (n = 4), pathologic characteristics between thymomatous and non- high-dose IV immunoglobulin (n = 2), and immunoabsorption thymomatous IM patients may suggest a common immune (n = 1). Of interest, the elevated serum CK levels normalized mechanism underlying the IM development in patients with within 3 weeks in all the treated patients, including the 3 patients both IM and MG, irrespective of thymoma association. How- with very high serum CK levels (nos. 1, 6, and 7). ever, the immune mechanical background of nonthymomatous IM patients may be heterogeneous, considering that 1 non- The mean follow-up period was 3.8 years (range: 0.5–10.9 thymomatous IM patient (no. 10) was seronegative for anti- years). During the follow-up period, 3 patients died. The AChR Ab. causes of death were sepsis following diffuse panbronchiolitis (n = 2, patients 5 and 7) and bronchopneumonitis compli- We found 1 non-MG thymomatous IM patient (no. 11) with cated by cardiac dysfunction and pure red cell aplasia (n = 1, features similar to those of thymomatous patients with both patient 9). The modified Rankin Scale scores at final follow-up IM and MG, except for the presentation of MG. A previous of the 7 patients who were alive were grade 0 (n = 1), grade 1 report described 1 non-MG thymomatous IM patient with (n = 4), and grade 3 (n = 2). similar clinical features: positive anti-AChR Ab, normal RNS responses, a very high serum CK level (7,271 IU/L), and a pathologic finding of endomysial CD8+ cells with CD68 + 13 Discussion giant cells. The clinical features in both patients supported the idea that thymoma-related immunopathogenic mecha- The prevalence of thymoma association in patients with both nisms can affect skeletal muscle toward the development of IM and MG (70%) was much more than that in patients with IM, even without MG. only MG (10%).27 Thymoma is known to be associated with various autoimmune diseases. The frequent thymoma associ- At the time of IM diagnosis, 5 patients had a preexisting ation in IM patients with both IM and MG should indicate that thymoma, and among them, 2 patients showed no signs of the association of these 2 diseases is not coincidental but in- recurrence. In the literature, we found a total of 4 patients with duced by thymoma-related immunopathogenic mechanisms. both IM and MG with preexisting thymoma for 4–17 years before IM diagnosis.4,9,32 The thymoma status of the 4 In 7 thymomatous patients with both IM and MG, ocular patients at IM diagnosis was not described, except in 1 patient symptoms or decremental RNS responses were rarely observed who was autopsied and showed no thymoma recurrence. at IM diagnosis (2/7 or 1/7, respectively). Among them, 4 However, some studies have described thymoma recurrence patients with normal RNS responses developed rapidly pro- long after thymectomy33 and microscopic thymoma (smaller gressive muscle weakness and respiratory failure with acute than 1 mm), which cannot be detected by imaging studies.34 elevation of serum CK to very high levels (rhabdomyolysis-like Considering the suggested thymoma-related immunopatho- features). Previous studies reported patients with similar clin- genic mechanisms in the development of IM, we recommend ical features: IM and anti–AChR Ab-positive thymomatous not only careful checkup for thymoma recurrence in patients MG presenting with normal RNS responses28 and rapidly with preexisting thymoma but also regular checkup for thy- progressive weakness and respiratory failure in conjunction moma development in nonthymomatous patients with both with markedly elevated serum CK levels.7,12 Our findings IM and MG. demonstrated that thymomatous patients with both IM and MG frequently showed nonsimultaneous progression of IM The presence of endomysial infiltration of T-lymphocytes and MG and often included those with severe progression and (lymphorrhage) in the skeletal muscle tissue of patients with markedly elevated serum CK levels. The nonsimultaneous MG has been known for decades, particularly in patients with progression of IM and MG suggests that distinctly different thymomatous MG.32,35 One suggestion is that the lymphocytic thymoma-related immunopathogenic mechanisms on skeletal infiltration does not represent autoimmune IM, but rather, an muscles are involved in the development of each disease. infiltration consisting of CD8+CD45RA+-naive T-lymphocytes derived from lymphocyte-rich thymoma. However, in our se- − Three nonthymomatous IM patients in our study shared ries, CD8+CD45RA cells but not CD8+CD45RA+ naive cells the following characteristic features with thymomatous IM were the lymphocytes infiltrating the endomysium, and the

Table 2 Treatment of IM in patients with both IM and MG

Patient 1 2 3 4 5 6 7 8 9 10

Treatment

Immunotherapy after IM IVMP, PSL 55 mg, PSL 30 mg PSL 50 mg and CyA 200 mg PSL 20 mg IVMP, PSL 50 mg, IVIg, IVMP, PSL 60 mg, IVIg, PB IVMP and PSL 60 mg diagnosis and TCR 3 mg TCR 3 mg and PB IAPP, and TCR 1.5 mg and MTX 12.5 mg PSL 40 mg and PB

CK normalization by rest before − +(8d) −−+ (20 d) −−+ (18 d) −− immunotherapya

Period between treatment 2 w NA ND 1 w NA <3 w 2 w NA 4 d 6 d introduction and CK normalization

Prognosis

Follow-up period (y) 1.3 3.0 2.7 2.9 1.3 0.5 6.1 2.6 0.2 17

MGFA-PIS at final follow-up MM MM Improved MM Died of MG PR Died of MG MM Died of MG PR

IM recurrence −−−−−− + −− −

MG recurrence −−−−−− NA −− −

Modified Rankin Scale change 3→01→13→32→15→65→35→62→15→62→1 (IM diagnosis→final follow-up)

Abbreviations: CK = creatine kinase; CyA = cyclosporin A; d = day; IAPP = immunoadsorption plasmapheresis; IVIg = IV immunoglobulin; IVMP = IV methylprednisolone; MGFA-PIS = Myasthenia Gravis Foundation of America postintervention status; MM = minimal manifestation; MTX = methotrexate; NA = not applicable; ND = no data; PB = pyridostigmine bromide; PR = pharmacologic remission; PSL = prednisolone; TCR = tacrolimus; w = week. a Period in brackets refers days between peak and normalization of CK levels before treatment. 9 extensive expression of MHC class I was observed in all indicates involvement of the immune checkpoint pathway patients (9/9 and 10/10, respectively), suggesting a true cell- in the development of autoimmune diseases in thymoma- mediated PM pattern of inflammation as a pathologic pheno- tous patients. type in our patients. Our study has some limitations. First, the number of patients In addition to PM pattern pathology, massive collections of with both IM and MG is small because of the rarity of si- CD20+ B-lymphocytes were observed in 2 patients and multaneous occurrence of these 2 diseases. Second, we did CD68+ granulomas in 1 patient. These pathologic findings not always follow the clinical courses in patients with IM for have already been reported in patients with both idiopathic a long time enough to detect MG association. Third, MG – IM and MG.3,9 11,13,28 Because the thymus gland is a central activity may be underestimated because SFEMG or edro- lymphatic organ that provides various immunologic functions, phonium infusion test had not been performed in all of our multifactorial autoimmune dysregulation in thymic disorders, patients. Despite these limitations, our study showed that including thymoma, has been reported.36 Further studies are idiopathic IM with MG frequently shows thymoma associ- needed to elucidate the overall immune mechanisms that ation and PM pathology. In addition, this study demon- cause these complicated pathologic features. strated that idiopathic IM with MG shares clinicopathologic characteristics with IM induced by ICIs. Further studies Some immune-related adverse events induced by antitumor should be conducted to determine the essential mechanisms therapy with ICIs have been recently reported to cause the of thymoma-related autoimmune dysregulation in patients association of MG with IM.14,16,17 A recent study of 12 with both IM and MG, particularly focusing on the roles of nonthymomatous patients who developed MG after anti- immune checkpoint molecules. tumor therapy with nivolumab (an anti–PD-1 monoclonal Ab) reported that 4 patients had IM and 3 patients had Author contributions myocarditis, including 1 patient with both IM and myocar- N. Uchio: study design and concept, acquisition of data, 15 ditis. This study also showed the following clinical features analysis and interpretation, and drafting of the manuscript. of patients with nivolumab-induced MG: markedly elevated K. Taira, C. Ikenaga, A. Unuma, M. Kadoya, and K. Yoshida: CK levels (10/12, 83%), positive anti-AChR Ab (10/12, acquisition of data. S. Nakatani-Enomoto, Y. Hatanaka, 83%), positive edrophonium infusion test (4/7, 57%), and Y. Sakurai, Y. Shiio, and K. Kaida: acquisition of data and abnormal decremental RNS responses (2/7, 29%). Patients critical revision of the manuscript. A. Kubota: critical re- withICI-inducedIMhavebeenoftenreportedtohave vision of the manuscript. T. Toda: study supervision. J. rhabdomyolysis-like clinical features with myocarditis or Shimizu: study design and concept, acquisition of data, 37 respiratory failure. Therefore, the assumption that patients analysis and interpretation, and critical revision of the with ICI-induced IM share similar immunopathogenic manuscript. mechanisms with patients with both idiopathic IM and MG should be reasonable. Moreover, we recently found PD-1+ Acknowledgment cell infiltration and extended PD-L1 (a ligand of PD-1) The authors thank Naoko Tokimura, Masako Nishizawa, and overexpression on non-necrotic fibers in patients with Yuki Ogiwara for technical assistance. anti–PD-1 Ab-induced IM.16 The similarity of clinical features between patients with ICI-induced IM and those with idiopathicIMandMGpromptedustoexaminePD-1,PD- Study funding L1, and CTLA-4 expressions in our patients. Subsequently, we This work was supported by the Health and Labour Sciences found PD-L1 overexpression on non-necrotic muscle fibers Research Grant on Rare and Intractable Diseases (Validation around PD-1+ cells in 7 patients, including 2 patients with of Evidence-based Diagnosis and Guidelines and Impact extended PD-L1 overexpression, and endomysial infiltration on QOL in Patients with Neuroimmunological Diseases) of CTLA-4+ cells in 4 patients. CTLA-4+ cells in endomysial from the Ministry of Health, Labour and Welfare of Japan fi infiltrates with invasion of muscle fibers have been reported and a Grant-in-Aid for Scienti c Research (KAKENHI; in patients with idiopathic PM or IBM.38 Both PD-1/PD-L1 15K09347) from JSPS. and CTLA-4/B7 pathways are involved in the induction of immune tolerance on activated cytotoxic T-lymphocytes, Disclosure although the 2 pathways operate at different stages of an N. Uchio, K. Taira, C. Ikenaga, M. Kadoya, A. Unuma, K. immune response.39 The expression of PD-1, PD-L1, and Yoshida, S. Nakatani-Enomoto, Y. Hatanaka, Y. Sakurai, CTLA-4 in IM pathology may suggest dysregulation of and Y. Shiio report no disclosures. K. Kaida is an associate immune checkpoint molecules in the microenvironment editor of Clinical and Experimental Neuroimmunology.A. of IM in patients with IM and MG. The backgrounds of Kubota and T. Toda report no disclosures. J. Shimizu re- the dysregulation of the immune checkpoint molecules in ceived research support from Grants-in-Aid for Scientific our patients remain unclear. However, a previous report Research. Full disclosure form information provided by has shown that genetic polymorphisms of CTLA-4 are as- the authors is available with the full text of this article at sociated with thymomatous MG patients.40 The finding Neurology.org/NN.

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Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 6, Number 2 | March 2019 11 ARTICLE OPEN ACCESS Cranial nerve involvement in patients with MOG antibody–associated disease

Alvaro Cobo-Calvo, MD, PhD, Xavier Ayrignac, MD, PhD, Philippe Kerschen, MD, Philippe Horellou, PhD, Correspondence Francois Cotton, MD, PhD, Pierre Labauge, MD, PhD, Sandra Vukusic, MD, PhD, Kumaran Deiva, MD, PhD, Dr. Marignier [email protected] Ch´e Serguera, MD, PhD,* and Romain Marignier, MD, PhD*

Neurol Neuroimmunol Neuroinﬂamm 2019;6:e543. doi:10.1212/NXI.0000000000000543 Abstract Objective To describe clinical and radiologic features of cranial nerve (CN) involvement in patients with myelin oligodendrocyte glycoprotein antibodies (MOG-IgG) and to assess the potential underlying mechanism of CN involvement using a nonhuman primate (NHP) model.

Methods Epidemiologic, clinical, and radiologic features from a national cohort of 273 MOG-IgG– positive patients were retrospectively reviewed for CN involvement between January 2014 and January 2018. MOG-IgG binding was evaluated in CNS, CN, and peripheral nerve tissues from NHP.

Results We identiﬁed 3 MOG-IgG–positive patients with radiologic and/or clinical CN involvement. Two patients displayed either trigeminal or vestibulocochlear nerve lesions at the root level, and the remaining patient had an oculomotor nerve involvement at the root exit and at the cisternal level. Additional CNS involvement was found in all 3 patients. None of the 3 patients’ sera recognized MOG expression in CN of NHP.

Conclusion Craneal nerve involvement can coexist in patients with MOG antibody disease, although the underlying pathophysiology remains elusive.

*These authors contributed equally to the manuscript.

From the Service de neurologie (A.C.-C., S.V., R.M.), sclérose en plaques, pathologies de la myéline et neuroinflammation and Centre de référence pour les maladies inflammatoires rares du cerveau et de la moelle (MIRCEM)– Hopitalˆ Neurologique Pierre Wertheimer Hospices Civils de Lyon, France; Lyon Neuroscience Research Center (A.C.-C., R.M.), U1028 INSERM, UMR5292 CNRS, FLUID Team, Lyon, France; Service de sclérose en plaques (X.A., P.L.), Hopitalˆ Universitaire de Montpellier, France; Service de Neurologie (P.K.), Centre hospitalier de Luxembourg; Inserm (P.H.), U 1184, Center for Immunology of Viral Infections and Autoimmune Diseases, Université Paris-Sud 11, CEA, DSV/iMETI, Division of Immuno- Virology, IDMIT, Facultédemédecine, Le Kremlin-Bicêtre Cedex, France; Service de Radiologie (F.C.), Centre Hospitalier Lyon-Sud, Hospices Civils de Lyon, Pierre-Bénite, France; Université Claude Bernard Lyon 1 (F.C., S.V., R.M.), F-69100 Villeurbanne, France; Lyon’s Neuroscience Research Center (S.V.), Observatoire Français de la Sclérose en Plaques, INSERM 1028 et CNRS UMR5292, Lyon, France; Service de neurologie pédiatrique (K.D.), Centre de référence pour les maladies inflammatoires rares du cerveau et de la moelle, Le Kremlin- Bicêtre, France; and INSERM US27 MIRCen (C.S.), CEA, Fontenay-aux-Roses, France.

Copyright © 2019 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1 Glossary AQP4 = aquaporin-4; CN = cranial nerve; hMOG = human MOG; MBP = myelin basic protein; MFI = mean ﬂuorescence intensity; MOG = myelin oligodendrocyte glycoprotein; MTP = methylprednisolone; NHP = nonhuman primate; NMOSD = neuromyelitis optica spectrum disorder; OCB = oligoclonal band; OFSEP = Observatoire Français de la Scl´erose en Plaques; RT = room temperature.

Antibodies against myelin oligodendrocyte glycoprotein anonymization techniques might result in impoverishment of (MOG-IgG) are a well-recognized cause of demyelination in data (Article 29 of Directive 95/46/EC, Opinion 05/2014 on adults and children with acute disseminated encephalomyelitis1,2 Anonymization Techniques—0829/14/EN WP 216), data – and neuromyelitis optica spectrum disorders (NMOSDs).3 6 used for this study were only pseudonymized. However, ac- New clinical phenotypes such as cortical encephalitis, brainstem cess to OFSEP data to conduct a scientific project is possible – syndromes,7 10 and fulminant cases10,11 have been more recently by following the OFSEP data access process (ofsep.org/en/ reported, suggesting that the clinical and radiologic presentation data access) and with respect to French law. of MOG antibody–associated disease could be broader than previously thought. Cranial nerve (CN) involvement in patients Autoantibody detection with serum MOG-IgG has not been described so far. All samples were examined for IgG against human MOG (hMOG) and human AQP4 by cell-based assays.12,13 Briefly, We report here clinical and radiologic features of 3 MOG- for MOG antibodies, HEK293 cells were transfected with IgG–positive patients with CN involvement. To evaluate the pEGFP-N1-hMOG plasmid (kindly provided by Markus possible underlying mechanism of MOG-IgG in the present Reindl, Innsbruck, Austria). After 48 hours, transfected cells clinical phenotype, we screened patients’ sera for reactivity in were dissociated with Accutase (Sigma-Aldrich, A6964) and the CNS, CN, and peripheral nerve from nonhuman primate incubated with phosphate-buffered saline (PBS) 8% normal (NHP) (cynomolgus macaque). goat serum (NGS) for 30 minutes at room temperature (RT). Then, patients’ sera diluted at 1:640 were incubated with transfected cells for 30 minutes at 4°C. This cutoff was selected Methods to avoid false-positive signal detected with healthy controls in previous studies.14 Cells were fixed with 1% paraformaldehyde Patients (PFA) for 15 minutes and then incubated 20 minutes at RT in Epidemiologic, clinical, and radiologic data were retrospec- the dark with a secondary antibody allophycocyanin (APC)- tively reviewed from the adult (n = 197)10 and pediatric (n = goat anti-human IgG-Fcγ fragment-specific (1:100 dilution, 76) French cohorts of MOG-IgG–positive patients diagnosed Jackson ImmunoResearch 109-136-170). between January 2014 and January 2018. For the detection of AQP4 autoantibody, HEK293 cells were MRI was performed in the clinical setting including axial and transfected with pcDNA3.1-AQP4-M23 and pEGF-C1 plasmids sagittal images of the brain and spinal cord obtained by T1-, with Lipofectamine LTX (Invitrogen 10573013). After 48 hours, T2-, fluid attenuated inversion (FLAIR), and T1-weighted cells were dissociated with Accutase (Sigma-Aldrich, A6964) and postcontrast sequences. incubated with PBS 8% NGS for 30 minutes. After blocking, cells were incubated with patients’ sera at 1:100 cells for 30 minutes at For experiments, sera from MOG-IgG–positive patients with 4°C and then fixedwith1%PFAatRTfor15minutes.HEK293 CN involvement and controls were used. As controls, we se- cells were incubated for 20 minutes at RT in the dark with lected 1 MOG-IgG–positive patient with an exclusive CNS in- a secondary antibody APC-goat anti-human IgG-Fcγ fragment- volvement, 1 healthy control, and 1 double-seronegative (MOG specific (1:100 dilution, Jackson ImmunoResearch 109-136- and aquaporin-4 [AQP4]-IgG-negative) NMOSD patient. 170). FACS analysis for MOG and AQP4-IgG was performed with the CANTO II flow cytometer (Becton Dickinson). Standard protocol approvals, registrations, and patient consents In addition, serum samples were tested for antibodies against neuronal cell surface antigens using rat brain immunohisto- The study was approved by the Ethics Committee of the chemistry, as described previously.15 University Hospital of Lyon, France. All patients provided their informed consent to participate in the study. Nonhuman primate tissue preparation and immunohistochemistry This study was conducted within the framework of Observ- atoire Français de la Sclérose en Plaques (OFSEP). Because of Nonhuman primates national confidentiality requirements, only anonymized data, Adult captive-bred 3- to 5-year-old female cynomolgus mac- not pseudonymized data, can be shared. Although aques (Macaca fascicularis) were used.16,17 Brains were

2 Neurology: Neuroimmunology & Neuroinflammation | Volume 6, Number 2 | March 2019 Neurology.org/NN sampled after intracardiac perfusion with PBS and 4% PFA. divisions) and dysmetria of the left arm. Brain MRI revealed Brains were postfixed by overnight immersion in 4% PFA and FLAIR hyperintensities of both upper cerebellar peduncles with then transferred to PBS and embedded in paraffin. For im- gadolinium enhancement (figure 1A). Left trigeminal nerve munohistochemistry analysis, 3- to 5-μm-thick paraffin sec- showed a gadolinium enhancement at the root level (figure 1B). tions from brain, cerebellum, oculomotor, trigeminal, and CSF revealed 0.52 g/L of proteins without cells or oligoclonal sciatic nerves were cut and deparaffinized. bands (OCBs). A whole-body 18-fluorodeoxyglucose PET/CT imaging showed no abnormalities. Symptoms improved spon- Immunohistochemistry taneously without treatment, and the patient did not receive any To assess myelin staining pattern in NHP tissues, IgGs were immunoactive drug. Six months later, a new right trigeminal 18 purified as previously described. Purified IgG from each pa- nerve gadolinium enhancement was observed on a routine brain tient were labeled with Alexa Fluor594(referredtoaspurified MRI without any new clinical involvement (figure 1C). Two IgG-AF594) Microscale Protein Labeling Kit (Invitrogen; years after onset, the patient remained asymptomatic and the 30008), according to the manufacturer’s instruction to prevent brain MRI showed complete resolution of lesions (figure 1D). background signal associated with the use of secondary anti- human IgG antibodies in macaque brain tissues (nonpublished Patient Id.2 observations). To ascertain that the purified IgG-AF594 from A 16-year-old girl with a medical history of idiopathic con- MOG-positive patients still bound to MOG antigen, we per- genital nystagmus presented with a 5-day history of fever, formed flow cytometry cell-based assay and compared D-mean general mental alteration, and disorientation rapidly associated fluorescence intensity (MFI), determined by the subtraction of with dizziness and vomiting, suspected from infectious menin- MFI obtained with HEK293 control cells from that obtained gitis. Neurologic examination only found an already known with HEK293 MOG–positive cells. The levels measured for the nystagmus in all directions of gaze without other oculomotor 3 patients with CN involvement were at the same level as the disturbances. Lumbar puncture was performed, and she was one obtained with the positive control patient or above (figure immediately started on IV amoxicillin and acyclovir. CSF e-1, links.lww.com/NXI/A97). showed neither cells nor OCBs, and proteins and glucose were within the normal range. All viral PCR and bacterial cultures Purified IgG-AF594 from MOG-IgG–positive patients and were negative, and IV treatment was stopped. Brain MRI revealed bilateral juxtacortical and supratentorial white matter controls were incubated with macaque tissue slices (diluted 1: fi 20) for 1 hour and 30 minutes following 30 minutes of sat- hyperintensities on FLAIR ( gure 1E). Gadolinium enhancement was observed on both third oculomotor nerves at the root uration with a block solution (BSA5%, 10% NGS). Myelin fi was also labeled with a commercial mouse monoclonal MOG exit and at the cisternal level ( gure 1F). Spinal MRI revealed a T2 cervical hyperintensity with a subtle gadolinium enhance- antibody (clone 8-18C5, MAB5680; Millipore) as a positive fi control, and a biotinylated goat anti-mouse antibody (1:200, ment ( gure 1G). Three months later, a brain MRI scan showed BA9200, Vector Laboratories, Burlingame, CA) was used as an absence of gadolinium enhancement on the third nerve and partial resolution of the brain FLAIR hyperintensities with no secondary, for 30 minutes at RT, followed by the avidin-biotin- fi peroxidase complex (Vectastain Elite ABC Kit, PK 6100, Vector gadolinium enhancement ( gure1H).Fourmonthslater,she Laboratories, Burlingame, CA). Positive antigen-antibody reac- presented with a decrease in visual acuity on the right eye to- tions were visualized by incubation with 3,3-diaminobenzidine gether with retro-orbital pain. Three days of IV methylprednisolone (MTP) (1 g/d) was started with complete recovery. After tetrahydrochloride H2O2 in 01 M imidazole, pH 7.1 for 5 minutes, followed by slight counterstaining with hematoxylin. A mouse 24 months of follow-up, the patient was asymptomatic. monoclonal anti-myelin basic protein (MBP) was used to confirm Patient Id.3 myelin staining (dilution 1/200) (LS B2231, LifeSpan Bio- A 52-year-old woman with hypertension, hypothyroidism, science), followed by a secondary goat anti-mouse antibody and chronic kidney disease was admitted for an 8-day history coupled to fluorochromes Alexa 488 (dilution 1/500) (A32731, of diplopia, vertigo, and gait instability. Neurologic examination Thermo Fisher Scientific). Labeled tissues were analyzed and revealed a “one and a half syndrome”, a positive Romberg sign, pictured with confocal microscopy. and dizziness. Blood test confirmed a renal insufficiency (cre- atinine 1.28 mg/dL and glomerular filtration rate 43 mL/min). CSF showed 27 leukocytes (65% lymphocytes), 0.51 g/L of Results proteins, normal glucose, and no OCBs. Brain MRI showed Patient clinical and radiologic characteristics asymmetrical FLAIR hyperintensities at the medulla oblongata We identified 3 (2 adult and 1 pediatric) MOG-IgG–positive and pons and in the dorsal pontine tegmentum (figure 1I). A patients with clinical or radiologic CN involvement. bilateral trigeminal and vestibulocochlear nerve gadolinium enhancement was found at the root level (figure 1, J–L). Spinal Patient Id.1 cord MRI showed no abnormalities. PET/CT imaging showed A 76-year-old man with a medical history of hypertension and no hypermetabolic foci. The patient received 5 days of IV MTP dyslipidemia developed insidiously general fatigue, and 1 month (1 g/d) with subsequent clinical recovery. After a follow-up of later a left facial numbness. Neurologic examination revealed left 12 months, the patient remained asymptomatic with no further facial hypesthesia (ophthalmic, maxillary, and mandibular treatment.

Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 6, Number 2 | March 2019 3 Figure 1 Radiologic features of human myelin oligodendrocyte glycoprotein immunoglobulin G–positive patients with cranial nerve involvement

Patient Id.1 (A–D). (A) Bilateral upper cerebellar peduncle. (B and C) Left and right trigeminal nerve gadolinium enhancement on T1W postcontrast sequence. (D) Complete resolution of the lesions on T1 postgadolinium. Patient Id. 2 (E–H). (E) White matter cerebral lesions on FLAIR sequences. (F, a–c), Bilateral oculomotor nerve gadolinium enhancement in the cisternal part of the nerve on T1W postcontrast sequence with fat saturation. (G) Focal cervical T2 lesion on spinal cord MRI. (H) Resolution of oculomotor gadolinium enhancement on T1W postcontrast sequence with fat saturation. Patient Id.3 (I–L). (I) Brainstem asymmetric FLAIR hyperintensities and dorsal pontine tegmentum hyperintensity. (J and K) Bilateral trigeminal nerve gadolinium enhancement on FLAIR postcontrast sequence. (L, a and b), Bilateral vestibulocochlear nerve gadolinium enhancement on FLAIR postgadolinium sequence.

MOG-IgG were found positive in serum samples taken at Then, we evaluated whether purified IgG-AF594 of the 3 12 months after first symptoms in patient Id.1 and at onset patients labeled NHP CN or peripheral nerves. None of the in patients Id.2 and Id.3. None of the patients had AQP4- purified IgG-AF594 showed positive staining neither in IgG or additional antibodies against neuronal cell surface proximal and distal regions of oculomotor, trigeminal, nor in antigens. distal regions of the sciatic nerve (figure 3). Equally, neither the purified IgG-AF594 from the MOG-IgG–positive patient with exclusive affection of CNS nor that of the healthy subject Tissue immunohistochemistry or the double-seronegative NMOSD control displayed any First, we analyzed by immunohistochemistry whether purified staining on trigeminal or sciatic nerves (not shown). IgG-AF594 from the 3 patients with CN involvement reacted with NHP brain slices. Purified IgG-AF594 from all 3 patients allowed myelin staining on brain slices from NHP (table, Discussion figure 2A), with similar aspect as that obtained with commercial mouse monoclonal 8-18C5 anti-MOG (figure 2B). In In this study, we report clinical and/or radiologic involvement addition, purified IgG-AF594 colocalized with the commercial of CNs in MOG-IgG–positive patients, thus enlarging the MBP antibody (figure 2C). clinical spectrum of MOG antibody–associated disease.

4 Neurology: Neuroimmunology & Neuroinflammation | Volume 6, Number 2 | March 2019 Neurology.org/NN Table Epitope recognition by human MOG among species in cell-based assays and immunohistochemistry

Cell-based assays NHP immunohistochemistry

Patient identification Human MOG (1:640) Brain Cranial nerves

Id.1 Pos MSP++ Neg

Id.2 Pos MSP++ Neg

Id.3 Pos MSP++ Neg

Control; MOG-IgG positivea Pos MSP++ Neg

Control; healthy subject Neg Neg Neg

Control; double-seronegative NMOSD Neg Neg Neg

Abbreviations: MOG = myelin oligodendrocyte glycoprotein; MSP = myelin staining pattern; Neg = negative; NHP = nonhuman primate; NMOSD = neuromyelitis optica spectrum disorder; Pos = positive. Myelin staining pattern (MSP) was classified as Neg (no staining), + (when slightly), ++ (moderate), and +++ (intense pattern). a The MOG-IgG–positive patient had exclusive involvement of the CNS.

– MOG antibody–associated disease is a relatively new entity with involved.3 10 Among our national cohort of adult and pediatric a predisposition for the optic nerve and the spinal cord, with MOG-IgG–positive patients, 3 of 273 patients (1.1%) had clinical encephalic or brainstem structures being less frequently or radiologic involvement of CNs, in addition to CNS involvement.

Figure 2 Nonhuman primate brain immunohistochemistry

(A) Purified IgG-AF594 from patients (A.a) and from controls (A.b). (B) Brain and cerebellum nonhuman primate myelin staining with commercial monoclonal MOG 8–18C5. (C) Purified IgG-AF594 from patients colocalized with commercial anti-MBP. An example of patient Id.1. hMOG = human MOG; MBP = myelin basic protein; MOG = myelin oligodendrocyte glycoprotein; NHP = nonhuman primate.

Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 6, Number 2 | March 2019 5 Figure 3 Nonhuman primate peripheral nerve immunohistochemistry

Purified IgG-AF594-IgG from patients staining in (A) trigeminal, (B) sciatic, and (C) oculomotor nerves. *Staining labeling MOG protein at the CNS region. MOG = myelin oligodendrocyte glycoprotein.

The 3 patients presented in this study had clinical symptoms rather than a restricted involvement of the transitional zone or or radiologic images related to the involvement of oculomo- a peripheral nerve involvement in continuation to a de- tor, trigeminal, or vestibulocochlear nerves. Two patients myelinating process from the CNS. displayed gadolinium enhancement of CNs at the transitional zone at the root level. Such region contains an overlap of We found that all patients showed a similar myelin staining central and peripheral myelin features whose extension differs pattern in the brain and cerebellar slices of NHP. Recent among CNs, over 7 and 9 mm in length in trigeminal and studies have shown that only a proportion of IgG from MOG- vestibulocochlear nerves, respectively.19,20 On the contrary, IgG–positive patients could recognize epitopes shared by the oculomotor nerve gadolinium enhancement in the third humans and rodents.7,23 Differences in MOG recognition patient extended beyond the anticipated transitional zone among species is likely the consequence of a decreased ho- between peripheral and central myelin.21 mology in amino acid sequences of MOG with 19 different amino acids of 218 between the mouse and human,23,24 but To study the underlying pathophysiology of the CN in- only 5 between macaques and humans. The most frequent volvement, we used NHP tissues to evaluate MOG-IgG recognized MOG epitope by hMOG-IgG resides at position binding in CNS, CN, and peripheral nerve structures. We 42 in the CC9 loop.25 The disparity on the type of amino acid found that purified immunoglobulins from MOG-IgG– at this position between human and rodents (proline in positive patients did not allow labeling neither in proximal nor humans and serine in rodents) is largely responsible for the in distal portions of CNs explored. Because MOG is a protein lack of epitope recognition between both species.24,25 How- expressed by oligodendroctyes in the CNS,22 the absence of ever, this amino acid discrepancy does not exist in the MOG MOG-IgG binding at the peripheral level in our NHP model protein sequence between cynomolgus macaques and human was not unexpected. However, the absence of MOG recog- who are phylogenetically closer.26 Thus, macaques are a suit- nition at the transitional zone by the serum of MOG-IgG able model to test autoantibodies from human autoimmune patients did not permit to confirm a possible role of MOG- diseases and could be potentially useful when studying dis- IgG over this region. Such CN involvement could also be eases such as MOG antibody–associated disease where the explained as the result of downstream inflammatory process difference in the structure of the protein may lead to an ab- from the intra-axial pontine lesion. We also may consider that sence of recognition by antibodies.17 the presence of other specific autoantibodies different from MOG-IgG or an unknown demyelinating process specifically Such inflammation of the peripheral nervous system has been targeting the peripheral nerve could be the cause of CN lesion reported in AQP4 antibody–positive NMOSDs. Different

6 Neurology: Neuroimmunology & Neuroinflammation | Volume 6, Number 2 | March 2019 Neurology.org/NN patterns of involvement have been described such as inflammation may persist longer in the peripheral nerve than polyneuropathy,27,28 radiculopathy,29 myeloradiculitis,30 and in the CNS.35 second motor neuron involvement.31,32 Recently, vestibulocochlear nerve lesion as a part of CN involvement has been Overall, our findings suggest that CN involvement can coexist reported for the first time in NMOSDs.33 Moreover and in patients with MOG-antibody disease. However, the un- similarly to our cases, involvement of the transitional region at derlying pathophysiology remains to be elucidated. the spinal root has also been described in a patient presenting with meningoradiculitis.30 In this case, histopathology Acknowledgment showed a loss of AQP4 expression and demyelination, sug- The authors thank Dr. Albert Saiz (Center of Neuro- gesting that the underlying pathophysiology could be immunology, Hospital Clinic, Barcelona, Spain) for having explained by the presence of AQP4 protein location at the performed different antibody testing and immunohistochem- root level, thus being a target for AQP4-IgG. In MS, radiologic istry experiments. They also thank the group of NeuroBioTec trigeminal nerve involvement has been described up to 2.9% from Hospices Civils de Lyon for supporting this study. of patients,34,35 being not always associated with clinical symptoms, as observed in our patients.35 In some cases, the Study funding lesion was also found in the cisternal region, distally from the This study was supported by a grant from the ARSEP trigeminal root entry zone.35,36 Recently, cisternal oculomo- Foundation and a grant provided by the French State and tor nerve enhancement has been described in closely relation handled by the “Agence Nationale de la Recherche”, within to the CNS demyelinating lesion.37 In all reported patients the framework of the “Investments for the Future” program, with the cisternal oculomotor or trigeminal nerve under the reference ANR-10-COHO-002 Observatoire – enhancement,35 37 an ancient or recent brainstem lesion was Français de la Sclérose en Plaques (OFSEP), by the Eugene found, which suggests an extension from the CNS in- Devic Foundation against Multiple Sclerosis (EDMUS flammation rather than a specific demyelination of the pe- Foundation), and the patient association “Déchaine Ton ripheral myelin. Some authors have pointed out that the active Coeur”.

Appendix 1 Author contributions

Author Location Role Contributions

Alvaro Cobo- Hopitalˆ Neurologique Pierre Wertheimer, Hospices Civils de Lyon, Author Study concept and design, performed experiments, Calvo, MD, Lyon acquisition of data, analysis and interpretation, and PhD writing of the manuscript

Xavier Multiple Sclerosis Clinic, Montpellier University Hospital, Author Acquisition of data and critical revision of the manuscript Ayrignac, Montpellier for important intellectual content MD, PhD

Philippe Service de Neurologie, Centre Hospitalier de Luxembourg, Author Acquisition of data and critical revision of the manuscript Kerschen, Luxembourg for important intellectual content MD

Philippe Center for Immunology of Viral Infections and Autoimmune Author Performed experiments and critical revision of the Horellou, Diseases, Division of Immuno-Virology, Le Kremlin-Bicˆetre Cedex, manuscript for important intellectual content PhD France

Francois Centre Hospitalier Lyon-Sud, Hospices Civils de Lyon, Pierre- Author Critical revision of the manuscript for important Cotton, MD, B´enite, France intellectual content PhD

Pierre Multiple Sclerosis Clinic, Montpellier University Hospital, Author Acquisition of data and critical revision of the manuscript Labauge, MD, Montpellier for important intellectual content PhD

Sandra Hopitalˆ Neurologique Pierre Wertheimer Hospices Civils de Lyon, Author Critical revision of the manuscript for important Vukusic, MD, Lyon intellectual content PhD

Kumaran Service de Neurologie P´ediatrique, Le Kremlin-Bicˆetre, France Author Acquisition of data and critical revision of the manuscript Deiva, MD, for important intellectual content PhD

Ch´e INSERM US27 MIRCen, CEA, Fontenay-aux-Roses, France Author Study concept and design, performed experiments, Serguera, acquisition of data, analysis and interpretation, and MD, PhD writing of the manuscript

Romain Hopitalˆ Neurologique Pierre Wertheimer, Hospices Civils de Lyon, Author Study concept and design, performed experiments, Marignier, Lyon acquisition of data, analysis and interpretation, and MD, PhD writing of the manuscript

Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 6, Number 2 | March 2019 7 Disclosure 12. Cobo-Calvo A,´ Ruiz A, D’Indy H, et al. MOG antibody-related disorders: common features and uncommon presentations. J Neurol 2017;264:1945–1955. A. Cobo-Calvo, X. Avrignac, P. Kerschen, P. Horellou, F. 13. Marignier R, Bernard-Valnet R, Giraudon P, et al. Aquaporin-4 antibody-negative Cotton, and P. Labauge report no disclosures. S. Vukusic neuromyelitis optica: distinct assay sensitivity-dependent entity. Neurology 2013;80: fi 2194–2200. served on the scienti c advisory boards of and consulted for 14. Mader S, Gredler V, Schanda K, et al. Complement activating antibodies to myelin Biogen, Celgene, GeNeuro, Genzyme, MedDay, Merck oligodendrocyte glycoprotein in neuromyelitis optica and related disorders. fi J Neuroinflammation 2011;8:184. Serono, Novartis, Roche, Sano -Aventis, and Teva; received 15. Boronat A, Sabater L, Saiz A, Dalmau J, Graus F. GABA(B) receptor antibodies in travel funding and/or speaker honoraria from Biogen, Gen- limbic encephalitis and anti-GAD-associated neurologic disorders. Neurology 2011; fi 76:795–800. zyme, Merck Serono, Novartis, Roche, Sano -Aventis, and 16. Bitoun S, Roques P, Larcher T, et al. Both systemic and intra-articular immunization Teva; and received research support from Biogen, Genzyme, with citrullinated peptides are needed to induce arthritis in the macaque. Front fi Immunol 2017;8:1816. MedDay, Merck Serono, Novartis, Roche, Sano -Aventis, and 17. Stimmer L, Fovet CM, Serguera C. Experimental models of autoimmune de- Teva. K. Deiva received travel funding from Biogen, Merck myelinating diseases in nonhuman primates. Vet Pathol 2018;55:27–41. Serono, Genzyme, and Novartis. C. Serguera reports no dis- 18. Marignier R, Ruiz A, Cavagna S, et al. Neuromyelitis optica study model based on chronic infusion of autoantibodies in rat cerebrospinal fluid. J Neuroinflammation closures. R. Marignier served on the scientific advisory board 2016;13:111. of MedImmune and received travel funding and speaker 19. PekerS,KurtkayaO,Uzün I, Pamir MN. Microanatomy of the central myelin-peripheral myelin transition zone of the trigeminal nerve. Neurosurgery 2006;59:354–359. honoraria from Biogen, Genzyme, Novartis, Merck Serono, 20. Pamir HA. Some histologic features of the cranial nerves. Arch Neurol Psychiatry Roche, Sanofi-Aventis, and Teva. Full disclosure form in- 1931;25:356–372. 21. Fraher JP, Smiddy PF, O’Sullivan VR. The central-peripheral transitional regions of formation provided by the authors is available with the full cranial nerves. Trochlear and abducent nerves. J Anat 1988;161:115–123. text of this article at Neurology.org/NN. 22. von Büdingen HC, Tanuma N, Villoslada P, Ouallet SL, Hauser SL, Genain CP Immune responses against the myelin/oligodendrocyte glycoprotein in experimental autoimmune demyelination. J Clin Immunol 2001;21:155–170. Publication history 23. Peschl P, Schanda K, Zeka B, et al. Human antibodies against the myelin oligoden- Received by Neurology: Neuroimmunology & Neuroinflammation drocyte glycoprotein can cause complement-dependent demyelination. October 8, 2018. Accepted in final form December 7, 2018. J Neuroinflammation 2017;14:208. 24. Mayer MC, Breithaupt C, Reindl M, et al. Distinction and temporal stability of conformational epitopes on myelin oligodendrocyte glycoprotein recognized by References patients with different inflammatory central nervous system diseases. J Immunol 1. Baumann M, Sahin K, Lechner C, et al. Clinical and neuroradiological differences of 2013;191:3594–3604. paediatric acute disseminating encephalomyelitis with and without antibodies to the 25. Marta CB, Oliver AR, Sweet RA, Pfeiffer SE, Ruddle NH. Pathogenic myelin oligo- myelin oligodendrocyte glycoprotein. J Neurol Neurosurg Psychiatry 2015;86: dendrocyte glycoprotein antibodies recognize glycosylated epitopes and perturb ol- 265–272. igodendrocyte physiology. Proc Natl Acad Sci USA 2005;102:13992–13997. 2. Brilot F, Dale RC, Selter RC, et al. Antibodies to native myelin oligodendrocyte 26. Delarasse C, Della Gaspera B, Lu CW, et al. Complex alternative splicing of the myelin glycoprotein in children with inflammatory demyelinating central nervous system oligodendrocyte glycoprotein gene is unique to human and non-human primates. disease. Ann Neurol 2009;66:833–842. J Neurochem 2006;98:1707–1717. 3. Sato DK, Callegaro D, Lana-Peixoto MA, et al. Distinction between MOG antibody- 27. Warabi Y, Yamazaki M, Shimizu T, Nagao M. Abnormal nerve conduction study positive and AQP4 antibody-positive NMO spectrum disorders. Neurology 2014;82: findings indicating the existence of peripheral neuropathy in multiple sclerosis and 474–481. neuromyelitis optica. Biomed Res Int 2013;2013:847670. 4. Höftberger R, Sepulveda M, Armangue T, et al. Antibodies to MOG and AQP4 in 28. Nagao M, Suzuki H, Ichihashi J, et al. Acute combined central and peripheral demyelination adults with neuromyelitis optica and suspected limited forms of the disease. Mult Scler showing anti-aquaporin 4 antibody positivity. Intern Med 2012;51:2443–2447. 2015;21:866–874. 29. Kim S, Park J, Kwon BS, et al. Radiculopathy in neuromyelitis optica: how does anti- 5. Kitley J, Waters P, Woodhall M, et al. Neuromyelitis optica spectrum disorders with AQP4 Ab involve PNS? Mult Scler Relat Disord 2017;18:77–81. aquaporin-4 and myelin-oligodendrocyte glycoprotein antibodies: a comparative 30. Takai Y, Misu T, Nakashima I, et al. Two cases of lumbosacral myeloradiculitis with study. JAMA Neurol 2014;71:276–283. anti-aquaporin-4 antibody. Neurology 2012;79:1826–1828. 6. Jurynczyk M, Messina S, Woodhall MR, et al. Clinical presentation and prognosis in 31. Mingbunjerdsuk P, Katirji B. Acute cervical segmental denervation in neuromyelitis MOG-antibody disease: a UK study. Brain 2017;140:3128–3138. optica spectrum disorder. J Clin Neuromuscul Dis 2014;16:90–97. 7. Sepúlveda M, Armangue T, Martinez-Hernandez E, et al. Clinical spectrum associated 32. Laemmer AB, Maihöfner C, Gölitz P, et al. Possible second motor neuron damage in with MOG autoimmunity in adults: significance of sharing rodent MOG epitopes. neuromyelitis optica. Clin Neurophysiol 2014;125:859–861. J Neurol 2016;263:1349–1360. 33. Bonnan M, Cabre P. Meningeal and vestibulocochlear nerve enhancement in neu- 8. Hamid SHM, Whittam D, Saviour M, et al. Seizures and encephalitis in myelin romyelitis optica. Eur J Neurol 2017;24:e89–e90. oligodendrocyte glycoprotein IgG disease vs aquaporin 4 IgG disease. JAMA Neurol 34. van der Meijs AH, Tan IL, Barkhof F. Incidence of enhancement of the trigeminal 2018;75:65–71. nerve on MRI in patients with multiple sclerosis. Mult Scler 2002;8:64–67. 9. Ogawa R, Nakashima I, Takahashi T, et al. MOG antibody–positive, benign, unilat- 35. da Silva CJ, da Rocha AJ, Mendes MF, et al. Trigeminal involvement in multiple eral, cerebral cortical encephalitis with epilepsy. Neurol Neuroimmunol Neuro- sclerosis: magnetic resonance imaging findings with clinical correlation in a series of inflamm 2017;4:e322. doi: 10.1212/NXI.0000000000000322. patients. Mult Scler 2005;11:282–285. 10. Cobo-Calvo A, Ruiz A, Maillart E, et al. Clinical spectrum and prognostic value of 36. Pichiecchio A, Bergamaschi R, Tavazzi E, et al. Bilateral trigeminal enhancement on CNS MOG autoimmunity in adults: the MOGADOR study. Neurology 2018;90: magnetic resonance imaging in a patient with multiple sclerosis and trigeminal neu- e1858–e1869. ralgia. Mult Scler 2007;13:814–816. 11. Di Pauli F, Höftberger R, Reindl M, et al. Fulminant demyelinating encephalomyelitis: 37. Shor N, Amador MD, Dormont D, et al. Involvement of peripheral III nerve in insights from antibody studies and neuropathology. Neurol Neuroimmunol Neuro- multiple sclerosis patient: report of a new case and discussion of the underlying inflamm 2015;2:e175. doi: 10.1212/NXI.0000000000000175. mechanism. Mult Scler 2017;23:748–750.

8 Neurology: Neuroimmunology & Neuroinflammation | Volume 6, Number 2 | March 2019 Neurology.org/NN CLINICAL/SCIENTIFIC NOTES OPEN ACCESS pRNFL as a marker of disability worsening in the medium/long term in patients with MS

Christian Cordano, MD, PhD, Bardia Nourbakhsh, MD, MAS, Michael Devereux, BS, Vincent Damotte, PhD, Correspondence Daniel Bennett, BS, Stephen L. Hauser, MD, Bruce A. C. Cree, MD, PhD, MAS, Jeffrey M. Gelfand, MD, MAS, Dr. Green [email protected] and Ari J. Green, MD, MCR

Neurol Neuroimmunol Neuroinﬂamm 2019;6:e533. doi:10.1212/NXI.0000000000000533

MS is an inﬂammatory and neurodegenerative disease of the CNS (including the retina) that leads to progressive neurologic disability. Disability correlates with the degree of axonal pathology in the disease.1,2 The clinical course of MS is unpredictable, making the search for biomarkers associated with enhanced risk of disease progression a major unmet need. Spectral domain optical coherence tomography (SD-OCT) is a potential predictor of disability worsening up to 5 years of follow-up after a single evaluation of the peripapillary retinal nerve ﬁber layer (pRNFL).3 Time domain OCT (TD-OCT) is an older technology, characterized by lower axial resolution and slower acquisition speed (400 axial scans per second) resulting in lower resolution images and more frequent motion artifacts compared with SD-OCT.4 TD-OCT was used over 10 years ago to assess retinal neurodegeneration in patients with MS but only now has enough time passed to assess its actual predictive potential for clinical progression.

To investigate the capacity of TD-OCT measures to predict disability worsening in patients with MS, we performed a retrospective evaluation of 305 patients with MS (228 relapsing- remitting, 29 secondary progressive, 32 clinically isolated syndrome, 10 primary progressive MS according to 2005 McDonald criteria,5 and 6 with MS but unclear disease course) who had undergone Stratus TD-OCT (Carl Zeiss Meditec AG, Jena, Germany) from January 2006 to December 2008, collecting the values of the pRNFL and macular volume (MV). The Com- mittee on Human Research at UCSF approved the study. All participants provided written informed consent. The baseline cohort characteristics are presented in the table. All patients had at least 1 measurement of the Expanded Disability Status Scale (EDSS) during the subsequent follow-up period. In our analyses, we used the most recent available EDSS. The median follow-up duration from the time of OCT to the most recent EDSS evaluation was 7.9 years (interquartile range 6.4–8.9 years, range 0.04–9.5 years); 91% of the cohort had >5 years of follow-up. The association between the baseline pRNFL and the subsequent EDSS was investigated using multivariable linear regression, adjusted for age and sex, taking into account the correlation of pRNFL thickness in the patient’s 2 eyes (ﬁgure). For each 1-μm decrease in the pRNFL, there was a 0.024 increase in EDSS (95% CI: 0.011–0.037; p < 0.001). In a sensitivity analysis, the results were similar (0.022 increase in the EDSS, 95% CI 0.035–0.01, p = 0.001) when analyzing only patients with >5 years of follow-up. A model adjusted for the presence of previous episodes of optic neuritis yielded similar results (a 1-μm decrease in the RNFL was associated with a 0.024 increase in the EDSS; p < 0.001). There was no association with MV. The lack of consistent association of MV with disability worsening has previously been reported3 and may relate to the lack of segmentation from whole macular thickness where some layers increase volume and others lose volume during the course of disease. In addition, TD-OCT had poorer reproducibility for volume scans because of the low speed of image acquisition. These results show that TD-OCT and a single evaluation of pRNFL atrophy is

From the Department of Neurology (C.C., M.D., V.D., D.B., S.L.H., B.A.C.C., J.M.G., A.J.G.), UCSF Weill Institute for Neurosciences, University of California, San Francisco, CA; and Department of Neurology (B.N.), Johns Hopkins University School of Medicine, Baltimore, MD.

Copyright © 2018 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1 individual level, SD-OCT is known to be more accurate. Table Demographic and baseline characteristics and the Stratus TD-OCT is no longer used in MS research because of follow-up data of the study population (number of the availability of more sophisticated OCT machines being patients = 305) able to quantify retinal measures with a much lower level of 8 Female 65.7 noise. Nevertheless, despite the eﬀect on SDs, this noise does not aﬀect the mean, and data from earlier iterations of the Average age (SD), y 42.7 (11.8) technology can be useful to indicate the association between Average disease duration in years (SD) 10 (9.2) retinal thinning and future disability. Average most recent EDSS (SD) 2.9 (1.9) Author contributions Average days from OCT to EDSS (SD) 2,696.4 (655.0) C. Cordano: study concept and design, interpretation of data, Median years from OCT to EDSS (IQR, range) 7.9 (6.4–8.9, 0.04 to and composition of the manuscript. B. Nourbakhsh: analysis 9.5) and interpretation of data. M. Devereux and V. Damotte: Average RNFL thickness (SD) 88.9 (15.2) acquisition of data and composition of the manuscript. D.

Average ON-positive RNFL (SD) (N = 193)a 82 (16.4) Bennett: acquisition of data and supervision of the manuscript. S.L. Hauser and B.A.C. Cree: study supervision and a Average ON-negative RNFL (SD) (N = 410) 91.9 (13.5) composition of the manuscript. J.M. Gelfand: analysis and % of patients using DMT at OCT visit 68.7 interpretation of data and composition of the manuscript. A.J. Green: study concept and design, analysis and interpretation Average duration of DMT use at EDSS visit in 10.3 (5.3) years (SD) of data, and composition of the manuscript.

Abbreviations: DMT = disease-modifying therapy; EDSS = Expanded Dis- Study funding ability Status Scale; IQR = interquartile range; ON = optic neuritis; RNFL = retinal nerve fiber layer. No targeted funding reported. a Seven eyes were excluded from the analysis because of poor quality (total number of eyes = 603). Disclosure C. Cordano received research support from FISM and served useful in predicting disability up to 6–9 years later in MS— as a consultant for Inception 5. B. Nourbakhsh received re- significantly longer than has been previously reported. A search support from Biogen, American Brain Foundation, limitation of the study is the absence of the baseline EDSS in National Multiple Sclerosis Society, and Patient-Centered the entire cohort, which limited our ability to consider its Outcome Research Institute. M. Devereux reports no dis- influence as a confounder in the estimate of association be- closures. V. Damotte received travel funding from ECTRIMS and ARSEP Foundation. D. Bennett reports no disclosures. tween the baseline RNFL and subsequent EDSS. Recent fi meta-analyses show that the mean RNFL loss in a population S.L. Hauser served on the scienti c advisory boards of Sym- of patients with optic neuritis is similar between TD-OCT and biotix, Annexon, Bionure, Molecular Stethoscope, Alector, SD-OCT.6,7 However, despite the similarity in means at an and Neurona; received travel funding and speaker honoraria from F. Hoffmann La Roche; receives publishing royalties from McGraw-Hill Education; received writing support from F. Hoffmann La Roche; and received research support from Figure Association of baseline RNFL with EDSS at the end of the NIH, NMSS, and Conrad N. Hilton Foundation. B.A.C. the follow-up period Cree served on the scientific advisory board of Akili and consulted for AbbVie, Biogen, EMD Serono, GeNeuro, Novartis, and Sanofi Genzyme. J.M. Gelfand served on the DSMB for an NIA-funded study; is on the editorial board of Neurology: Neuroimmunology & Neuroinflammation; received publishing royalties from DynaMed Plus; consulted for Bio- gen; received research support from MedDay and research support (to UCSF) from Genentech; is PI of an institutional clinician training award from the NMSS; received compensation for medical-legal consulting; his spouse is an associate editor of JAMA Neurology; received honoraria from UpTo- Date; and consulted for Zosano, Eli Lilly, Biohaven, and eNeura. A.J. Green served on the scientific advisory boards of MedImmune, Novartis, Inception 5 Sciences, Pipeline, and Bionure; served as an associate editor of JAMA Neurology and editorial board member of Neurology; holds a patent for RNFL = retinal nerve fiber layer. remyelination molecules and pathways; received research support from Novartis, Inception Sciences, NINDS, NIA,

2 Neurology: Neuroimmunology & Neuroinflammation | Volume 6, Number 2 | March 2019 Neurology.org/NN NIH, NMSS, Sherak Foundation, and Hilton Foundation; 2. Frohman EM, Costello F, Stüve O, et al. Modeling axonal degeneration within the anterior visual system: implications for demonstrating neuroprotection in multiple holds stock or stock options in Inception 5, Pipeline, and sclerosis. Arch Neurol 2008;65:26–35. Bionure; and served as an expert witness in Mylan Pharma- 3. Martinez-Lapiscina EH, Arnow S, Wilson JA, et al. Retinal thickness measured with optical coherence tomography and risk of disability worsening in multiple sclerosis: ceuticals v Teva Pharmaceuticals. Full disclosure form in- a cohort study. Lancet Neurol 2016;15:574–584. formation provided by the authors is available with the full 4. Leung CK, Chiu V, Weinreb RN, et al. Evaluation of retinal nerve fiber layer progression in glaucoma: a comparison between spectral-domain and time-domain op- text of this article at Neurology.org/NN. tical coherence tomography. Ophthalmology 2011;118:1558–1562. 5. Polman CH, Reingold SC, Edan G, et al. Diagnostic criteria for multiple scle- Publication history rosis: 2005 revisions to the “McDonald Criteria”. Ann Neurol 2005;58:840–846. fl Review. Received by Neurology: Neuroimmunology & Neuroin ammation July 19, 6. Petzold A, de Boer JF, Schippling S, et al. Optical coherence tomography in 2018. Accepted in final form November 8, 2018. multiple sclerosis: a systematic review and meta-analysis. Lancet Neurol 2010;9: 921–932. 7. Petzold A, Balcer LJ, Calabresi PA, et al. Retinal layer segmentation in multiple References sclerosis: a systematic review and meta-analysis. Lancet Neurol 2017;16:797–812. 1. Arnold DL, Matthews PM, Francis G, Antel J. Proton magnetic resonance spec- 8. Warner CV, Syc SB, Stankiewicz AM, et al. The impact of utilizing different optical troscopy of human brain in vivo in the evaluation of multiple sclerosis: assessment of coherence tomography devices for clinical purposes and in multiple sclerosis trials. the load of disease. Magn Reson Med 1990;14:154–159. PLoS One 2011;6:e22947.

Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 6, Number 2 | March 2019 3 CLINICAL/SCIENTIFIC NOTES OPEN ACCESS Episodic ataxia in CASPR2 autoimmunity

A. Sebastian Lopez´ Chiriboga, MD, and Sean Pittock, MD Correspondence Dr. Lopez´ Neurol Neuroimmunol Neuroinﬂamm 2019;6:e536. doi:10.1212/NXI.0000000000000536 [email protected]

A 64-year-old man developed cognitive dysfunction and generalized seizures, followed by MORE ONLINE episodes of transient speech and gait disturbances lasting several minutes, occurring multiple times per day; the events were not associated with any triggers. Neurologic examination during Video one of his episodic symptoms (video 1) showed transient ataxic dysarthria. Testing for neural autoantibodies revealed evidence of voltage-gated potassium channel complex antibodies by radioimmunoprecipitation assay; CASPR2-IgG was positive in serum and CSF by cell-based assay. Brain MRI was normal; CSF analysis showed an elevated protein level at 101 mg/dL, but it was otherwise normal. Oncologic evaluation was negative. The cognitive symptoms improved; seizures and episodic ataxia resolved after 12 weeks of treatment with IV methylprednisolone. Episodic ataxia is a manifestation of CASPR2 autoimmunity;1 patients can have normal MRI, and immunotherapy is beneﬁcial.

Author contributions A.S. Lopez-Chiriboga and S. Pittock contributed to the conception and design of the study; collection, analysis, and interpretation of the data; drafting and critical revision of the manuscript; and generation/collection of the ﬁgures. Both authors gave ﬁnal approval of the manuscript.

Study funding No targeted funding reported.

Disclosure A.S. Lopez-Chiriboga reports no disclosures. S. Pittock has a patent pending for GFAP, Septin- 5 and MAP1B autoantibodies as biomarkers of neurological autoimmumity; received research support from Grifols, MedImmune, Alexion, AEA, and NIH; had compensation for consulting activities paid to Mayo Clinic from Alexion and MedImmune; and has a financial interest with Mayo Clinic in patents that relate to functional AQP4/NMO-IgG assays and NMO-IgG as a cancer marker. Full disclosure form information provided by the authors is available with the full text of this article at Neurology.org/NN. Publication history Received by Neurology: Neuroimmunology & Neuroinflammation September 27, 2018. Accepted in final form November 8, 2018.

Reference 1. Joubert B, Gobert F, Thomas L, et al. Autoimmune episodic ataxia in patients with anti-CASPR2 antibody-associated encephalitis. Neurol Neuroimmunol Neuroinﬂamm 2017;4:e371. doi: 10.1212/NXI.0000000000000371.

From the Department of Neurology (A.S.L.C.) and Laboratory Medicine and Pathology (S.P.), Mayo Clinic, Rochester, MN.

Copyright © 2018 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1 CLINICAL/SCIENTIFIC NOTES OPEN ACCESS Anti-NMDAR encephalitis with concomitant varicella zoster virus detection and nonteratomatous malignancy

Preeti A. Prakash, BA,* Jingxiao Jin, BS,* Kanwal Matharu, MD, Tanu Garg, MD, Wendy C. Tsai, MD, Correspondence Purvesh Patel, MD, Jill E. Weatherhead, MD, MS, and Joseph S. Kass, MD, JD Dr. Kass [email protected] Neurol Neuroimmunol Neuroinﬂamm 2019;6:e537. doi:10.1212/NXI.0000000000000537

Case presentation A 50-year-old woman with a recent diagnosis of poorly diﬀerentiated nonteratomatous cervicouterine cancer presented to our hospital for breath-holding episodes resulting in perioral cyanosis. Her medical history was signiﬁcant for chickenpox at age 8 and shingles at age 15. Before her admission and 10 days after her cancer diagnosis, she was admitted to an outside hospital for confusion and intermittent catatonia. Workup at the outside hospital was unremarkable, and she was diagnosed with adjustment disorder. At home, she continued to decompensate and ultimately became incomprehensible, anorexic, and bedbound, prompting admission to our hospital.

On presentation, the patient was encephalopathic and dysautonomic. She was unresponsive to pain and exhibited diffuse hyperreflexia, episodes of generalized body tremors, and trismus to the point of transecting her tongue. Because of breath-holding spells with oxygen desaturations and the need to acquire a brain MRI, she was intubated. Her continuous EEG and brain MRI were unremarkable. Acyclovir was empirically started for 2 days but discontinued after CSF studies revealed lymphocytic pleocytosis (17 white blood cells/μL with 86% lymphocytes) with negative herpes simplex virus (HSV) PCR (figure). A 5-day course of intravenous methylprednisolone (IVMP) and intravenous immunoglobulin (IVIG) for presumed anti-N-methyl-D-aspartate receptor encephalitis (NMDAR) encephalitis was administered. CSF testing for varicella zoster virus (VZV) by PCR returned positive, prompting a 21-day course of IV acyclovir. The NMDAR antibody CSF titer later also returned positive (1:50; Dalmau Laboratory, University of Pennsylvania, Philadelphia, PA). A diagnosis of anti-NMDAR encephalitis with concomitant VZV detection was made. A week into antiviral treatment, the patient’s vital signs normalized and agitation decreased. By discharge, the patient was alert, oriented, and following simple commands but not yet at neurologic baseline.

Discussion This case report demonstrates a rare presentation of anti-NMDAR encephalitis with concomitant VZV detection in the setting of a nonteratomatous gynecologic tumor.1,2 In a seminal case series published in 2008, about 60% of patients with anti-NMDAR encephalitis had a tumor, most often an ovarian teratoma.3 Anti-NMDAR encephalitis has also been linked with tumors of endocervical and endometrial origin.4 These tumors express NMDA receptor 1 (NR1), as well as synaptophysin, neuron-speciﬁc enolase, CD56, and chromogranin. Resection of tumors that express NRs can shorten recovery time.5 Our patient’s tumor expressed synaptophysin, but NR1 testing was not performed. Thus, it is unknown whether her cervicouterine tumor demonstrates a true association with NMDAR immunoreactivity.

*These authors contributed equally to this work.

From the Department of Internal Medicine (P.A.P., K.M.), Department of Neurology (J.J., T.G., J.S.K.), Department of Anesthesia (W.C.T.), and Department of Pulmonary, Critical Care, and Sleep Medicine (P.P.), Baylor College of Medicine; and National School of Tropical Medicine (J.E.W.), Baylor College of Medicine, Houston, TX.

Copyright © 2019 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1 Neurotropic viruses, most frequently HSV, have been linked Figure Time course of the patient’s diagnostics and treatment to anti-NMDAR encephalitis.6 One proposed mechanism for this association is that these viruses may lyse neurons, re- leasing antigens that sensitize IgG antibodies to the NR and triggering autoimmune encephalitis.1,2 Another hypothesis is that CNS inﬂammation from anti-NMDAR encephalitis may cause shedding of latent viral DNA or a viral reactivation leading to secondary encephalitis.7

To date, VZV has been documented twice in the setting of anti- NMDAR encephalitis.1,2 One patient presented with cranial nerve palsies and improved signiﬁcantly after treatment with IV acyclovir and immunoadsorption. The other patient had a teratoma and presented with confusion and behavioral changes after a viral-like illness. She was refractory to treatment with teratoma resection, acyclovir, IVIG, and plasma exchange.

Our patient completed the full ﬁrst-line therapy for both anti- NMDAR and VZV encephalitis. Although her improvement in symptoms correlated temporally with antiviral therapy, the eﬀects of the IVMP and IVIG treatment cannot be discounted because the response to immunotherapy often lags behind its completion. Because she was treated for both disease processes, it is unclear whether VZV triggered our patient’s autoimmune condition or was present in the CSF as a result of latent viral shedding. Her presenting symptoms were more consistent with anti-NMDAR encephalitis than VZV encephalitis. Never- theless, we recommend antiviral treatment in cases of anti- NMDAR encephalitis with concomitant VZV detection because the immunosuppressive regimen for anti-NMDAR encephalitis may worsen an underlying VZV infection.

Previous literature suggests that anti-NMDAR encephalitis should be considered in any person aged 50 years or younger who presents with acute psychotic changes of less than 4 weeks’ duration, especially in the setting of a viral prodrome or abnormal movements.5 Current standard of care for patients with suspected anti-NMDAR encephalitis is to treat with acyclovir until HSV PCR testing is complete. Our case is part of a growing body of evidence that suggests providers should continue antiviral treatment until VZV PCR testing has resulted negative as well. Additional research could investigate the optimum treatment course and prognosis when anti-NMDAR encephalitis presents with concomitant viral detection.

Acknowledgment The authors thank Dr. Jennifer Chu for her invaluable help in interpreting the patient’s EEG studies.

The patient initially presented to an outside hospital for altered behavior Study funding and was discharged home with a psychiatric diagnosis. When she continued No targeted funding reported. to deteriorate, she presented to our hospital with breath-holding spells, encephalopathy, and dysautonomia. The patient completed a 5-day course of IVMP and IVIG for presumed anti-NMDAR encephalitis. She was initially Disclosure started on broad-spectrum antibiotics and acyclovir, which were discontinued after a negative HSV PCR. Once her CSF returned positive for VZV, P.A. Prakash and J. Jin report no disclosures. K. Matharu owns she was started on a 21-day course of acyclovir. HSV = herpes simplex virus; IVIG = intravenous immunoglobulin; IVMP = intravenous methylpredniso- stock in Johnson and Johnson. T. Garg, W.C. Tsai, P. Patel, lone; LP = lumbar puncture; VZV = varicella zoster virus. and J.E. Weatherhead report no disclosures. J.S. Kass received travel funding and/or speaker honoraria from the National

2 Neurology: Neuroimmunology & Neuroinflammation | Volume 6, Number 2 | March 2019 Neurology.org/NN Board of Medical Examiners, American Academy of Neurol- 2. Sol´ıs N, Salazar L, Hasbun R. Anti-NMDA Receptor antibody encephalitis with concomitant detection of Varicella zoster virus. J Clin Virol 2016;83: ogy, BrainPac; is an associate editor for Continuum and Con- 26–28. tinuum Audio; received publishing royalties from Elsevier; and 3. Dalmau J, Gleichman AJ, Hughes EG, et al. Anti-NMDA-receptor encephalitis: case series and analysis of the effects of antibodies. Lancet Neurol 2008;7: has been a paid speaker for Pri-Med. Full disclosure form 1091–1098. information provided by the authors is available with the full 4. Hara M, Morita A, Kamei S, et al. Anti-N-methyl-D-aspartate receptor encephalitis associated with carcinosarcoma with neuroendocrine differentiation of the uterus. text of this article at Neurology.org/NN. J Neurol 2011;258:1351–1353. 5. Dalmau J, Lancaster E, Martinez-Hernandez E, Rosenfeld MR, Balice-Gordon R. Publication history Clinical experience and laboratory investigations in patients with NMDAR enceph- Received by Neurology: Neuroimmunology & Neuroinflammation alitis. Lancet Neurol 2011;10:63–74. September 26, 2018. Accepted in final form November 30, 2018. 6. Nosadini M, Mohammad SS, Corazza F, et al. Herpes simplex virus-induced anti-N- methyl-d-aspartate receptor encephalitis: a systematic literature review with analysis of 43 cases. Dev Med Child Neurol 2017;59:796–805. References 7. Linnoila J, Binnicker M, Majed M, Klein C, McKeon A. CSF herpes virus and auto- 1. Schäbitz WR, Rogalewski A, Hagemeister C, Bien CG. VZV brainstem encephalitis antibody profiles in the evaluation of encephalitis. Neurol Neuroimmunol Neuro- triggers NMDA receptor immunoreaction. Neurology 2014;83:2309–2311. inflamm 2016;3:245. doi: 10.1212/NXI.0000000000000245.

Appendix 1 Author contributions

Name Location Role Contribution

Preeti A. Prakash, BA Baylor College of Medicine, Houston, TX Author Conceptualized the study; conducted literature review; interpreted data; and drafted and revised the manuscript for intellectual content

Jingxiao Jin, BS Baylor College of Medicine, Houston, TX Author Conceptualized the study; conducted literature review; interpreted data; created figure; and revised the manuscript for intellectual content

Kanwal Matharu, MD Baylor College of Medicine, Houston, TX Author Conceptualized the study; interpreted data; and revised the manuscript for intellectual content

Tanu Garg, MD Baylor College of Medicine, Houston, TX Author Conceptualized the study; interpreted data; and revised the manuscript for intellectual content

Wendy C. Tsai, MD Baylor College of Medicine, Houston, TX Author Interpreted data and revised the manuscript for intellectual content

Purvesh Patel, MD Baylor College of Medicine, Houston, TX Author Interpreted data and revised the manuscript for intellectual content

Jill E. Weatherhead, MD Baylor College of Medicine, Houston, TX Author Interpreted data and revised the manuscript for intellectual content

Joseph S. Kass, MD, JD Baylor College of Medicine, Houston, TX Author Interpreted data and revised the manuscript for intellectual content

Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 6, Number 2 | March 2019 3 CLINICAL/SCIENTIFIC NOTES OPEN ACCESS A fatal case of daclizumab-induced liver failure in a patient with MS

Mark Stettner, MD, PhD,* Catharina C. Gross, PhD,* Anne K. Mausberg, PhD, Refik Pul, MD, Correspondence Andreas Junker, MD, Hideo A. Baba, MD, Andreas Schulte-Mecklenbeck, PhD, Heinz Wiendl, MD, Dr. Stettner [email protected] Christoph Kleinschnitz, MD,* and Sven G. Meuth, MD, PhD*

Neurol Neuroimmunol Neuroinﬂamm 2019;6:e539. doi:10.1212/NXI.0000000000000539

Daclizumab (DAC; Zinbryta) is a humanized monoclonal antibody directed against the IL-2 receptor alpha chain and was approved for treatment of relapsing-remitting MS (RRMS) in May 2016. In March 2018, DAC has been withdrawn from the market, after the fatal case of severe liver failure described here and 12 other cases (3 of them fatal) of serious inﬂammatory brain disorders including encephalitis and meningoencephalitis. Notably, most of the cases – occurred in Germany,1 4 most likely because DAC received only a third-line approval by the US Food and Drug Administration.

Here, we present the fatal case of a 26-year-old woman with severe liver failure after a 4-month therapy with DAC that had been initiated 5 months after the first diagnosis of RRMS during which the patient presented severe and unusually high disease activity (expanded disability status scale [EDSS] score of 5.5). On first diagnosis, the presence of thyroid peroxidase (92 U/L) and thyroglobulin antibodies indicated a comorbidity with Hashimoto disease. Although liver enzyme counts including aspartate transaminase (AST, 42 U/L) and alanine transaminase (ALT, 84 U/L) doubled within the 2.5 weeks prior DAC initiation, values normalized prior to first administration of the drug and remained stable during the treatment. A decreased EDSS score of 4.5, assessed after 4 months, confirmed the patient’s response to treatment.

Three weeks after receiving the fourth dose of DAC, the patient developed fatigue and jaundice with significantly increased AST (3,041 U/L; reference <35 U/L), ALT (4,760 U/L; reference <35 U/L), lactate dehydrogenase (628 U/L; reference <215 U/L), and total bilirubin (21.5 mg/dL; reference <1.1 mg/dL) values. Two days later, she was hospitalized in a som- nolent state with jaundice, arterial hypotension, and lactate acidosis. Diagnosis of acute irre- versible liver failure prompted a high urgency liver transplantation that was performed the following day, after receiving approval by the ELAC (Eurotransplant Liver Advisory Com- mittee) audit. One day after transplantation, the patient developed circulatory instability and catecholamine dependency with continuous peaks of transaminases (AST: 8,568 U/L; ALT: 4,207 U/L). Despite intensified treatment, the patient died 4 days later because of systemic inflammatory response syndrome, septic shock, and multiple organ failure.

We suspected DAC-induced hepatic (DIH) injury possibly owing to natural killer (NK)-cell hyperactivation, because drug reaction with eosinophilia and systemic symptoms (DRESS) and other causes of hepatic injury, including alcohol and drug abuse or viral hepatitis, could be excluded. A contribution of tizanidine, which was administered as co-medication after the fourth cycle of DAC treatment and has also been shown to induce serious liver injury as

*Authors contributed equally to this work.

From the Department of Neurology (M.S., A.K.M., R.P., C.K.), University Hospital Essen; Clinic of Neurology with Institute of Translational Neurology (C.C.G., A.S.-M., H.W., S.G.M.), University Hospital Munster,¨ University Munster;¨ Institute of Neuropathology (A.J.), University Hospital Essen; and Institute of Pathology (H.A.B.), University Hospital Essen, Germany.

(A) Immunohistochemical detection of leukocyte infiltration in the liver of the fatal case patient with DAC-induced hepatic injury (DIH) compared to the case of a 39- year-old RRMS patient with AIH but no further relevant medical conditions. At timepoint of biopsy, this patient received glatiramer acetate without any further relevant co-medications. Seven-micrometer paraffin embedded sections were stained, with anti-CD16 (macrophages, neutrophils, NK cells), anti-CD3 (T cells), anti-CD4 (T- helper cells), anti-CD8 (cytotoxic T cells), anti-NKG2A (NK cells), anti-CD19 (B cells), and granzyme B (GranB) and matching fluorochrome-labeled secondary antibodies (Alexa Fluor 488, green; Alexa Fluor 555, red; Alexa Fluor 647, purple) as indicated; nuclei were visualized using 4,69diamidino-2-phenylindole (DAPI, blue); scale bar 100 μm. (A.a): Representative image of CD16 + macrophages localized around T-cell infiltration zones. (A.b): Distribution of CD4+ T-helper cells and cytotoxic CD8+ T cells − within T-cell infiltrates. (A.c): CD19 + B cells within T-cell infiltration zone. (A.d): NKG2A + CD3 NK cells within T-cell infiltration zone. (A.e and A.f) Granzyme B expression of NKG2A + NK cells (A.e) and CD3+ T cells (A.f) on Sequential sections. (B) PBMC derived from patients with MS (N = 4) before (DAC BL [B.a]) and after 3 months of DAC therapy (DAC 3M [B.b]) in comparison to the index case (DIH [B.c]) as well as the patient with autoimmune hepatitis (AIH [B.d]) were stained with lineage-specific (B.a–B.D) CD1c, CD3, CD4, CD8, CD14, CD16, CD19, CD56) and B-cell subset-specific (B.e–B.H) CD19, CD20, CD21, CD23, CD24, CD27, CD38, IgD, IgM) fluorochrome- conjugated antibodies and acquired by flow cytometry. Lymphocytes and B cells were electronically selected and resulting data were normalized before submitting equal numbers of randomly selected cells to unsupervised cluster analysis by viSNE software. Barnes–Hut Stochastic Neighbor Embedding (bh-SNE) algorithm was used for dimensionality reduction followed by automated identification of lymphocyte (B.a–B.d) and B-cell subsets (B.e–B.H), respectively, by phenograph algorithm. (C) Median fluorescence intensity (MFI) of DNAM-1 expressing CD56dim, CD56bright, and regulatory T cells (Treg). (D) Intracellular expression of GM-CSF in CD4 and CD8 memory T cells following stimulation with leukocyte activation cocktail (PMA, ionomycin, brefeldin A). (E) Percentage of CD56bright NK cells expressing CCR5, or secreting cytolytic vesicles (CD107a+), and expressing GM-CSF as a consequence of co-incubation with HepG2 liver cells. (F) Proportion and MFI of CCR5 of monocytes with a proinflammatory M1-like phenotype.

2 Neurology: Neuroimmunology & Neuroinflammation | Volume 6, Number 2 | March 2019 Neurology.org/NN a monotherapy,5,6 could not be excluded. Interestingly, tiza- Leibniz Research Centre for Working Environment and Human nidine was the common denominator between this case and Factors at TU Dortmund, for his suggestions regarding the another fatal case of DIH injury in the SELECT trial.5,7 hepatocyte co-culture assays. This project was funded by the German Research foundation (SFB/Transregio CRC128 Immunohistochemistry of the explanted liver tissue of the projects A09 to HW and CCG and Z2 to HW) and the patient with DIH revealed CD3+ T-cell infiltrates surrounded German Ministry for Education and Research (BMBF; by damaged zones of CD16+ macrophages. The same project 01GI1603A to HW and CCG). observations were made for the liver tissue of a 39-year-old woman diagnosed with autoimmune hepatitis (AIH) 7 months Study funding after first diagnosis of MS (figure 1A) as an example of an This project was funded by the German Research foundation autoinflammatory disease of both CNS and liver. While CD4+ (SFB/Transregio CRC128 projects A09 to HW and CCG T-helper cells dominated in the patient with DIH, equal and Z2 to HW) and the German Ministry for Education and numbers of T-helper and cytotoxic CD8+ T cells were observed Research (BMBF; project 01GI1603A to HW and CCG). in the AIH liver tissue. Despite the fact that autoimmune liver disease has a wide pathophysiologic spectrum, more B-cell Disclosure infiltrates were detected within the T-cell zones of the patient M. Stettner served on the scientific advisory board for UCB with DIH compared to AIH. Enrichment of granzyme and Biogen Idec; received travel funding and/or speaker B-expressing NKG2A+ NK-cells in the DIH liver tissue sug- honoraria from Biogen, Genzyme, Novartis, Sanofi-Aventis, gests an active role of NK cells in hepatic injury. UCB, Grifols, TEVA, and Bayer; and served as an associate editor for European Journal of Medical Research. C.C. Gross In accordance with enriched B-cell infiltrates in the DIH liver, received speaker honoraria and travel funding from Biogen, immune cell profiling of the blood revealed significantly en- Euroimmun, Genzyme, Mylan, Novartis, and Bayer; served as hanced circulating B cells (figure 1B), consisting mainly of review editor for Frontiers in Neuroimmunology; and received naive B cells and increased proportions of autoreactive anti- research support from ReSTORE German Research Foun- body expressing complement receptor (CD21) negative dation, BMFB, and Innovative Medical Science. A.K. Maus- B cells.8 Despite a DAC-induced increase of CD56bright NK berg reports no disclosures. R. Pul served on the scientific cells, the DIH case exhibited significantly decreased pro- advisory board for Merck, Biogen, Bayer, and Novartis; re- portions of CD56dim NK cells.9,10 Diminished cell-surface ceived travel funding and/or speaker honoraria from Merck, expression of the activating receptor DNAM-1 (DNAX Biogen, Mylan, TEVA, and Sanofi-Genzyme; served as guest Accessory Molecule-1), which has been recently identified as editor for Frontiers in Neurology, Cells; and received research fi a crucial player in NK- and regulatory T(Treg)-cell mediated support for TEVA, Novartis, Merck, Sano -Genzyme. A. 6 7 fi control of T-cell activity, on NK and Treg ( gure 1C), Junker, H.A. Baba, and A. Schulte-Mecklenbeck report no suggests impaired immune regulatory function. Accordingly, disclosures. H. Wiendl served on the scientific advisory board proinflammatory granulocyte-macrophage colony-stimulating for Bayer, Biogen, Sanofi-Genzyme, Merck Serono, Novartis, factor (GM-CSF) production was increased in circulating Roche, and TEVA; received travel funding and/or speaker T cells of the patient with DIH (figure 1D). Increased ex- honoraria from Bayer Vita, Bayer Schering, Bioven, CSL pression of the liver homing receptor C-C chemokine receptor Behring, EMD Serono, Fresenius Medical Care, Sanofi- type 5 (CCR5) on CD56bright NK cells and enhanced cytotoxic Genzyme, Merck Serono, Omni-Mad, Novartis, TEVA, function as well as GM-CSF production in response to liver GlaxoSmithKline, and GW; served as an editorial board cells (figure 1E) indicated expanded liver invasion and hepa- member for PLoS One, Neurotherapeutics, Recent Patents on totoxicity of this subset. Finally, enhanced GM-CSF levels Inflammation & Allergy Drug Discovery; consulted for Bioge, resulting from T- and NK-cell hyperactivation may drive the Merck Serono, Novartis, OmniaMed, Roche, and Sanofi- differentiation of CCR5-expressing proinflammatory M1-like Genzyme; and received research support from Bayer monocytes leading to the highly increased frequencies observed HealthCare, Bayer Vital, Biogen, Merch Serono, Novartis, in the blood of the patient with DIH (figure 1F).11 Overall, our Sanofi-Genzyme, Sanofi, TEVA, German Ministry for Edu- findings indicate DIH injury resulting from innate immune cell cation and Research, Interdisciplinary Centre of Clinical activation and impaired immune regulation. Although in- Research, PML Consortium, German Research Foundation, volvement of the liver frequently occurs in DRESS, this case Else Kröner-Fresenius Foundation, Hertie Foundation, RE does not fulfill critical criteria for DRESS such as hyper- Children’s Foundation. C. Kleinschnitz receives honoraria eosinophilia, rashes, and reactivation of herpes viruses,12,13 thus, for lecturing and travel expenses for attending meeting differing from the recently described DAC-induced DRESS from Amgen, Bayer HealthCare, Bristol-Myers Squibb, cases. Boehringer Ingelheim, Biogen, Biotronik, CSL Behring, Daiichi Sankyo Genzyme, Desitin, Eisai, Ever Pharma, Acknowledgment MedDay Pharmaceuticals, Merck Serono, Mylan, Novartis, The authors would like to thank Steffi Hezel, Daniela Rooster- Pfizer, Roche, Sanofi-Aventis, Siemens, Stago, and Teva; re- man, Lena Schüneman, and Julia Sundermeier for their excellent ceived research funding from the German Ministry for Edu- technical assistance. The authors thank Prof. Carsten Watzl, cation and Research (BMBF), Corona Foundation, Deutsche

Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 6, Number 2 | March 2019 3 Forschungsgemeinschaft (DFG), Deutsche Stiftung Neuro- Publication history logie (DSN), Else Kroener-Fresenius Foundation, European Received by Neurology: Neuroimmunology & Neuroinflammation August Union, and Bayer Healthcare, Biogen, CSL Behring, Gen- 6, 2018. Accepted in final form December 7, 2018. zyme, Merck Serono, Novartis and Teva; served on the editorial board for PLoS One and Stroke; and holds a patent for References ff fi 1. Devlin M, Swayne A, Newman M, et al. A case of immune-mediated encephalitis E ectivity of speci c FXII/FXIIa inhibitors, particularly rHA- related to daclizumab therapy. Mult Scler 2018:1352458518792403. Infestin-4 used to treat neuroinflammatory diseases. S. Meuth 2. Luessi F, Engel S, Spreer A, Bittner S, Zipp F. GFAPalpha IgG-associated encephalitis upon daclizumab treatment of MS. Neurol Neuroimmunol Neuroinflamm receives honoraria for lecturing and travel expenses for 2018;5:e481. attending meeting from Almirall, Amicus Therapeutics 3. Rauer S, Stork L, Urbach H, et al. Drug reaction with eosinophilia and systemic Germany, Bayer HealthCare, Biogen, Celgene, DiaMed, symptoms after daclizumab therapy. Neurology 2018;91:e359–e363. 4. Scheibe F, Metz I, Radbruch H, et al. Drug reaction with eosinophilia and systemic Genzyme, MedDay Pharmaceuticals, Merck Serono, Novar- symptoms after daclizumab therapy in MS. Neurol Neuroimmunol Neuroinflamm tis, Novo Nordisk, ONO Pharma, Roche, Sanofi-Aventis, 2018;5:e479. 5. de Graaf EM, Oosterveld M, Tjabbes T, Stricker BHC. A case of tizanidine-induced Chugai Pharma, QuintilesIMS, and Teva; received research hepatic injury. J Hepatol 1996;25:772–773. funding from the German Ministry for Education and Re- 6. Gill D, Allam F, Boyle J. Importance of medication reconciliation: tizanidine-induced search (BMBF), Deutsche Forschungsgesellschaft (DFG), hepatitis. Am J Ther 2017;24:e623-e624. 7. Giovannoni G, Gold R, Selmaj K, et al. Daclizumab high-yield process in relapsing- Else Kröner-Fresenius Foundation, German Academic Ex- remitting multiple sclerosis (SELECTION): a multicentre, randomised, double-blind change Service, Hertie Foundation, Interdisciplinary Center extension trial. Lancet Neurol 2014;13:472–481. 8. Isnardi I, Ng YS, Menard L, et al. Complement receptor 2/CD21- human naive B cells for Clinical Studies (IZKF) Munster, German Foundation contain mostly autoreactive unresponsive clones. Blood 2010;115:5026–5036. Neurology and Almirall, Amicus Therapeutics Germany, 9. Bielekova B, Catalfamo M, Reichert-Scrivner S, et al. Regulatory CD56(bright) Biogen, DiaMed, Fresenius Medical Care, Genzyme, Merck natural killer cells mediate immunomodulatory effects of IL-2Ralpha-targeted therapy (daclizumab) in multiple sclerosis. Proc Natl Acad Sci U S A 2006;103: Serono, Novartis, ONO Pharma, Roche, and Teva; served 5941–5946. on the editorial board for PLoS One; and holds patents for 10. Gross CC, Schulte-Mecklenbeck A, Runzi A, et al. Impaired NK-mediated regulation ff fi of T-cell activity in multiple sclerosis is reconstituted by IL-2 receptor modulation. E ectivity of speci c FXIIa inhibitors/FXIIa inhibitorsa Proc Natl Acad Sci U S A 2016;113:E2973–E2982. inhibitors, particularly rHA-Infestin-4 used to treat neuro- 11. Verreck FA, de Boer T, Langenberg DM, et al. Human IL-23-producing type 1 inflammatory diseases, Diagnosis of a novel autoimmune macrophages promote but IL-10-producing type 2 macrophages subvert immunity to (myco)bacteria. Proc Natl Acad Sci U S A 2004;101:4560–4565. disease, “NR2B selective NMDA-receptor Antagonists for 12. Cacoub P, Musette P, Descamps V, et al. The DRESS syndrome: a literature review. Treatment of immune-mediated inflammatory diseases.” Full Am J Med 2011;124:588–597. 13. Tohyama M, Hashimoto K, Yasukawa M, et al. Association of human herpesvirus 6 disclosure form information provided by the authors is reactivation with the flaring and severity of drug-induced hypersensitivity syndrome. available with the full text of this article at Neurology.org/NN. Br J Dermatol 2007;157:934–940.

Appendix 1 Author contributions

Name Location Role Contribution

Mark Stettner Department of Neurology, University Author Designed and conceptualized the study; analyzed and interpreted data; and Hospital Essen, Essen, Germany drafted the manuscript for intellectual content

Catharina C. Clinic of Neurology, University Hospital Author Designed the study; analyzed and interpreted data; and drafted the manuscript Gross Munster,¨ Munster,¨ Germany for intellectual content

Anne K. Department of Neurology, University Author Played a major role in the acquisition and interpretation of data Mausberg Hospital Essen, Essen, Germany

Refik Pul Department of Neurology, University Author Designed the study; played a major role in the acquisition of data; and revised Hospital Essen, Essen, Germany the manuscript

Andreas Junker Institute of Neuropathology, University Author Played a major role in the acquisition of data and revision of the manuscript Hospital Essen, Essen, Germany

Hideo A. Baba Institute of Pathology, University Author Played a major role in the acquisition of data and revision of the manuscript Hospital Essen, Essen, Germany

Andreas Clinic of Neurology, University Hospital Author Played a major role in the experimental design, acquisition, and analysis of flow Schulte- Munster,¨ Munster,¨ Germany cytometry data; designed figures; and revised the manuscript Mecklenbeck

Heinz Wiendl Clinic of Neurology, University Hospital Author Interpreted data and revised the manuscript for intellectual content Munster,¨ Munster,¨ Germany

Christoph Department of Neurology, University Author Designed the study; analyzed and interpreted data; and drafted the manuscript Kleinschnitz Hospital Essen, Essen, Germany for intellectual content

Sven G. Meuth Clinic of Neurology, University Hospital Author Conceptualized the study; analyzed and interpreted data; and drafted the Munster,¨ Munster,¨ Germany manuscript for intellectual content

4 Neurology: Neuroimmunology & Neuroinflammation | Volume 6, Number 2 | March 2019 Neurology.org/NN CLINICAL/SCIENTIFIC NOTES OPEN ACCESS Rituximab during pregnancy in neuromyelitis optica: A case report

Jahir Miranda-Acuña, MD, Erica Rivas-Rodr´ıguez, MD, Michael Levy, MD, PhD, Mustafa Ansari, MS, Correspondence Roslynn Stone, PharmD, PhD, Vivek Patel, MD, and Lilyana Amezcua, MD, MS Dr. Amezcua [email protected] Neurol Neuroimmunol Neuroinﬂamm 2019;6:e542. doi:10.1212/NXI.0000000000000542

Neuromyelitis optica spectrum disorder (NMOSD) is a rare autoimmune disorder of the CNS, which predominantly targets the optic nerves and spinal cord leading to blindness and paral- ysis.1 There is a 6–9:1 ratio aﬀecting females far more often than males, and there is an increased risk of relapse during and immediately after pregnancy.2 B-cell depletion therapy has been reported to prevent rebound in postpartum relapses.3,4 In this report, we describe the safety and clinical eﬀect of B-cell depletion in both the patient and her infant baby.

Clinical report At 1-month postpartum, a 33-year-old Japanese American female developed a lower thoracic sensory level and lower extremity motor weakness from a longitudinally extensive transverse myelitis from T2 to T4. She developed bilateral optic neuritis 2 months later and tested seropositive for the aquaporin-4 (AQP4) antibody with a titer >160 U/mL by ELISA followed by cell-based assay and performed by ATHENA diagnostics. She was diagnosed with NMOSD, and IV rituximab infusions were started using standard dosing of 1,000 mg infused twice with 2 weeks between doses. She remained clinically stable and conceived 3 months after last rituximab infusion with corresponding %CD19 + count <1% and an NMO titer of 14.1 U/mL (ﬁgure, A). Because of the severity of her postpartum relapses and personal experience, a physician-patient informed decision was made to continue rituximab throughout pregnancy.

Pregnancy course B-cell depletion was maintained essentially at zero throughout pregnancy (ﬁgure, A). Optical coherence tomography was performed during pregnancy and followed very same principles emphasized in the OSCAR-IB methodology as were prior measures.5 Subclinical thinning of the ganglion cell-inner plexiform (GCIP) layer compared to baseline was also noted in the left eye (ﬁgure, B) during her neuro-ophthalmologic visit before infusion. Infusion of rituximab of 1,000 mg was given at 24 weeks of pregnancy. The rest of her pregnancy was clinically unremarkable under the care of a high-risk obstetrician and she delivered a healthy male at 38- week gestation via vaginal delivery.

Infant and maternal outcome Appearance, Pulse, Grimace, Activity, Respiration scores were normal and the infant’s %CD19 + cells were 1% at birth. At 2 months, the infant’s %CD19 + count rose to 23%. As a result, no change in the infant’s vaccination schedule was made and standard vaccinations were given. No

From the USC MS Comprehensive Care Center (J.M.-A., E.R.-R., M.A., L.A.), Department of Neurology, Keck School of Medicine, University of Southern California, Los Angeles, CA; Department of Neurology (M.L.), Johns Hopkins University, Baltimore, MD; Department of Neurology and Pharmacology (R.S.), University of Southern California; and Department of Ophthalmology (V.P.), Roski Eye Institute, University of Southern California, Los Angeles, CA.

Copyright © 2019 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1 Figure (A) Longitudinal measurements of NMO AQP4 antibody titer and CD19 cell counts before, during, and after pregnancy demonstrating declining AQP4 antibody titer and near complete B-cell depletion, along with rituximab infusions from 2015 to 2018

(B) Using optical coherence tomography, the thickness of the GCIP layers of both eyes were followed before, during, and after pregnancy (2015–2018). We noted subclinical thinning within this layer in her better eye during pregnancy, which returned to baseline thickness after delivery. GCIP = ganglion cell-inner plexiform; NMO = neuromyelitis optica.

infections, normal development, and normal B-cell counts Previous studies of rituximab exposure during pregnancy in were reported at 6-month follow-up. AQP4 antibody testing other autoimmune or hematologic conditions have shown in the infant’s serum was negative. that %CD19 + levels in the newborn are less than 1% at birth and tend to recover to normal levels after 6–8 weeks.8 As for the mother, post-delivery follow-up revealed her to be In our case, %CD19 + cells in the newborn were consistent well and her physical examination to be stable. She was con- with prior reports with rapid recovery seen by 3 months.8 tinued on rituximab. No neurologic or infectious sequelae at In addition, no infections or other hematologic abnormal- the 6-month follow-up were reported. GCIP thickness of her ities were reported in the newborn. Because of the rapid left eye returned to baseline when evaluated at 4 months recovery of the %CD19 + cell counts, infants exposed to postpartum (ﬁgure, B). rituximab in utero are expected to have a normal vaccination response.9

Discussion This case illustrates a favorable outcome in NMOSD and newborn well-being after gestational exposure to rituximab. We describe the clinical outcome of using rituximab in a patient We suggest that rituximab can be considered during preg- with NMOSD during pregnancy, which appeared safe and well nancy when there are increasing concerns and clinical history tolerated both to mother and infant. Treatment with B-cell suggesting a high risk for gestational and postpartum relapse. depleting therapy in NMO during pregnancy is controversial We also recommend that a multidisciplinary approach should because of scarce data.2,3 Nevertheless, as prior studies have be taken involving neurology, neuro-ophthalmology, high- shown, the annualized relapse rate of 1.8 in patients with risk obstetrics, and pediatrics to follow the patient and infant postpartum NMOSD is substantially higher than preceding closely. pregnancy.6 The decision to continue the patient on rituximab through pregnancy was made to reduce the risk of relapse. Interestingly, progressive thinning of the GCIP thinning in Author contributions NMOSD has been described in prior studies, which was also J. Miranda-Acuña: drafting of the manuscript, design, editing fi noted in this case.7 Because this patient’sGCIPhadbeenstable nal text, and review of the literature. E. Rivas-Rodriguez: fi over time, a thinning of the GCIP during pregnancy could drafting of the manuscript editing nal text and review of the fi represent changes associated with inflammation and less likely literature. M. Levy: editing, drafting, and reviewing nal text. fi degeneration, because recovery to pre-existing baseline was M. Ansari: editing nal text and review of the literature. R. fi seen after pregnancy. The GCIP thinned slightly during the Stone: editing nal text and review of the literature. V. Patel: fi time of her pregnancy and recovered its prepregnancy caliber editing, drafting, and reviewing nal text. L. Amezcua: drafting fi after our patient gave birth. We also found this finding to be of the manuscript, design, editing nal text, and review of the interesting and postulate that the transient GCIP thinning literature. could be a result of alterations in the immune environment during pregnancy. Whether GCIP could be used as a marker in Study funding pregnancy in NMOSD is unknown. No targeted funding reported.

2 Neurology: Neuroimmunology & Neuroinflammation | Volume 6, Number 2 | March 2019 Neurology.org/NN Disclosure provided by the authors is available with the full text of this J. Miranda-Acuña received travel funding from ACTRIMS; article at Neurology.org/NN. E. Rivas-Rodriguez reports no disclosures; M. Levy served on the scientific advisory board for Asterias, Chugai, Alexion, Publication history fl served on the editorial board for Multiple Sclerosis and Received by Neurology: Neuroimmunology & Neuroin ammation September 16, 2018. Accepted in final form December 10, 2018. Related Disorders, holds patents for Aquaporin-4 sequence that elicits pathogenic T-cell response in animal model of References neuromyelitis optica, patent covers use of peptide for di- 1. Wingerchuk DM, Banwell B, Bennett JL, et al. International consensus diagnostic agnostic and therapeutic developments, consulted for criteria for neuromyelitis optica spectrum disorders. Neurology 2015;85:177–189. 2. Shosha E, Pittock SJ, Flanagan E, Weinshenker BG. Neuromyelitis optica spectrum Guidepoint Global, Gerson Lehrman Group, Cowen Group, disorders and pregnancy: interactions and management. Mult Scler 2017;23:1808–1817. received research support from Viropharma/Shire, Acorda, 3. Ringelstein M, Harmel J, Distelmaier F, et al. Neuromyelitis optica and pregnancy fi during therapeutic B cell depletion: infant exposure to anti-AQP4 antibody and ApoPharma, Sano ,Genzyme,Alnylam,Alexion,Terumo prevention of rebound relapses with low-dose rituximab postpartum. Mult Scler 2013; BCT, NINDS, and Guthy-Jackson Charitable Foundation; 19:1544–1547. 4. Das G, Damotte V, Gelfand JM, et al. Rituximab before and during pregnancy: M. Ansari received speaker honoraria for MS Views and a systematic review, and a case series in MS and NMOSD. Neurol Neuroimmunol News, received research support from USC Keck Medical Neuroinflamm 2018;5:e453. doi: 10.1212/NXI.0000000000000453. 5. Schippling S, Balk LJ, Costello F, et al. Quality control for retinal OCT in multiple Center, National MS Society; R. Stone and V. Patel reports sclerosis: validation of the OSCAR-IB criteria. Mult Scler 2015;21:163–170. no disclosures; L. Amezuca served on the scientific advisory 6. Shimizu Y, Fujihara K, Ohashi T, et al. Pregnancy-related relapse risk factors in fi women with anti-AQP4 antibody positivity and neuromyelitis optica spectrum dis- board for Celgene and Genzyme, participated in a short- lm order. Mult Scler 2016;22:1413–1420. documentary about MS in the Hispanic community, con- 7. Oertel FC, Havla J, Roca-Fernandez A, et al. Retinal ganglion cell loss in neuromyelitis optica: a longitudinal study. J Neurol Neurosurg Psychiatry 2018;89:1259–1265. sulted for Celgene and Genzyme, received research support 8. Chakravarty EF, Murray ER, Kelman A, Farmer P. Pregnancy outcomes after maternal from MedDay, Biogen, NIH NINDS, Guthy-Jackson Char- exposure to rituximab. Blood 2011;117:1499–1506. 9. Klink DT, van Elburg RM, Schreurs MW, van Well GT. Rituximab administration in itable Foundation, National MS Society, and California third trimester of pregnancy suppresses neonatal B-cell development. Clin Dev Community Foundation. Full disclosure form information Immunol 2008;2008:271363.

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