MG Poster Abstracts for APP upload - Update 5-10

1 | Poster Abstracts 13th International Conference on Myasthenia Gravis and Related Disorders

Abstract List

13th International Conference on Myasthenia Gravis

and Related Disorders POSTER SESSION

MAY 15, 2017 | 6:00 PM New York Marriott Downtown

Basic Science and Animal Models

1. Sonia Berrih-Aknin, DSc, Preconditioned Mesenchymal Stem Cells Treat Myasthenia Gravis in a

Humanized Preclinical Model 2. Michelangelo Cao, MD, Specificity of Shp2 Inhibition on Acetylcholine Receptor Clustering 3. Hakan Cetin, MD, Fetal and Adult Acetylcholine Receptor Function and the Impact of Receptor

Clustering 4. Leslie Jacobson, BSc, Dphil, AChR-antibody Mediated Arthrogryposis: Inhibition of Placental

Transfer in a Mouse Model Protects the Fetus 5. Linda Kusner, PhD, Preclinical Pilot Study of Alpha-fetoprotein on Moderation of MG Weakness 6. Konstantinos Lazaridis, PhD, Antigen-specific Therapeutic Immunoadsorption Results in

Amelioration of Experimental MG Symptoms 7. Christopher Meriney, BS, Lambert-Eaton Myasthenic Syndrome: Passive Transfer to Mice, Active

Zone Structure-function Relationships, and Novel Treatment Strategies 8. David P. Richman, MD, Analysis of Muscle Wasting in an Acute Severe Model of Muscle-specific

Kinase Antibody Myasthenia Gravis 9. Kazuhiro Shigemoto, MD, Mouse Model of Lrp4-MG 10. Jie Song, MD, ROCK Inhibitor Suppresses Experimental Autoimmune Myasthenia Gravis by

Inhibiting Th1, Th17 and B-cells 11. Armineh Issakhanian Stoner, MS, The Potential Role of Anti-Lrp4 Autoantibodies in the

pathogenesis of Myasthenia Gravis (MG) and Amyotrophic Lateral Sclerosis (ALS) 12. Erdem Tüzün, MD, MuSK-binding Peripheral B-cells as Biomarker for MuSK-Myasthenia Gravis


Abstract List

13. Adi Vaknin-Dembinsky, MD, PhD, Exacerbation of EAMG by NMO Antibodies or Aquaporin 4 Peptide

14. Richard Webster, PhD, Electrophysiological Characterisation of a Mouse Model of DOK7 Congenital Myasthenic Syndrome and Treatment with Salbutamol

Case Report

15. Edward W. Bahou, MD, Unusual Facial Myokymia in a Patient with Ganglionic Acetylcholine Receptor Antibodies

16. Juhyeon Kim, MD, PhD, Exacerbation of Myasthenia Gravis in Thymic Cancer Patients Treated with Pembrolizumab: Three Case Reports

17. Araya Puwanant, MD, Myasthenia Gravis and Immune Checkpoint Inhibitors: Balancing Treatment Efficacy and Complications in Cancer Immunotherapies

Clinical

18. Silvia Bonanno, MD, MuSK MG Patients Showed a Positive Response to Amifampridine Phosphate in a Randomized, Placebo-Controlled, Crossover Study

19. Elena Cortes-Vicente, MD, and Aditya Kumar, MD, Comparison of Rituximab Treatment Regimens in MuSK Antibody Positive Myasthenia Gravis

20. Mazen Dimachkie, MD, FAAN, FANA, Challenges in a Multicenter Study of Subcutaneous Immunoglobulin in Myasthenia Gravis

21. Daniel B. Drachman, MD, Pilot Trial of Abatacept in Myasthenia Gravis Patients Inadequately Responsive to Conventional Immunotherapy

22. Josep Gamez, MD, PhD, Intravenous Immunoglobulin as Monotherapy for Myasthenia Gravis during Pregnancy

23. Giulia Greco, MD, Very Late-onset Non-Thymomatous and Thymomatous Myasthenia Gravis (MG) Are Associated with Different HLA Class II Alleles

24. Jeffrey T. Guptill, MD, Comparative Effectiveness Research to Advance Treatment of Myasthenia Gravis

25. Charlene Hafer-Macko, MD, Walking Away from the Fatigue and Low Functional and Metabolic Capacity in Myasthenia Gravis

26. Kazuo Iwasa, MD, PhD, Correlations Between the Time Trends of Anti-acetylcholine Receptor Antibody Titer Variation in Myasthenia Gravis and the Incidence of Mycoplasma and Influenza Infections

27. Elzbieta Klimiec, MD, Thymus Radiological-pathological Correlations in Myasthenia Gravis — the Relevance in Clinical Practice


Abstract List

28. Arturo Leis, MD, West Nile Virus (WNV) Induces a Prolonged Pro-inflammatory State That May Promote Myasthenia Gravis (MG)

29. Xiao Xi Liu, MD, Hepatitis B Virus Reactivation in Myasthenia Gravis Patients with Immunosuppressive Treatments

30. YueXun Liu, MD, MS, Therapeutic Potential of Docetaxelpluscisplatin Chemotherapy in Myasthenia Gravis: Clinical Analysis of Patients with Thymoma-associated Myasthenia Gravis

31. Tahseen Mozaffar, MD, INSIGHTS: Analysis of Responsiveness of IVIG in Patients with Myasthenia Gravis

32. Jay S. Raval, MD, Association of Total Serum Immunoglobulin G (IgG) Concentrations during Therapeutic Plasma Exchange (TPE) Therapy and Myasthenia Gravis (MG) Persistence

33. Elisabeth Strijbos, MD, Efficacy and Safety of Tetanus Revaccination in Autoimmune Myasthenia Gravis

34. Peter Ulrichts, PhD, ARGX-113, A Novel Fc-based Approach for Antibody-induced Pathologies such as Myasthenia Gravis

35. Elisabet Westerberg, MD, Myasthenia Gravis Patients Respond with Muscular Improvement to Physical Exercise

36. Fritz Zimprich, MD, PhD, Iodinated Contrast Agents in Patients with Myasthenia Gravis: a Retrospective Cohort Study

Clinical Samples

37. Claudia Barzago, PhD, Altered MicroRNA Expression in Myasthenia Gravis Thymus: Possible Contribution to the Pathogenesis

38. Derrick Blackmore, BSc, Subcutaneous Immunoglobulin Therapy in Patients with Myasthenia Gravis Exacerbation: Results of a Prospective, Open-label, Phase 3 Study (SCIg-MG)

39. Paola Cavalcante, PhD, Presence of Epstein-Barr Virus in B-cells of Myasthenia Gravis Thymoma: Innocent Bystander or Autoimmunity Mediator?

40. Pei Chen, MD, More Mature/active Dendritic Cells in the Hyperplasia Thymuses of Myasthenia Gravis Patients

41. Mélanie A. Cron, MSc, Role of miR-150 in Autoimmune Myasthenia Gravis 42. Nadine Dragin, PhD, AhR Activation May Regulate Expression of Pro-inflammatory Factors in

Thymuses of AChR+ Myasthenia Gravis Patients 43. Alejandro Gomez, PhD, Autoantibody Capture through Barcode-based Sequencing of Single

Cells in Myasthenia Gravis


Abstract List

44. Hao Huang, MD, Distribution and Characteristics of Dendritic Cells Subtypes in Hyperplastic Thymus in Patients with MG

45. Michala Jakubíková, MD, PhD, Two-year Outcome of Thymectomy with or without Immunosuppressive Treatment in Nonthymomatous Myasthenia Gravis and its Effect on Regulatory T-cells

46. Inga Koneczny, DPhil, IgG4 Autoantibodies against Muscle-specific Kinase Undergo Fab-arm Exchange in Myasthenia Gravis Patients

47. Aditya Kumar, MD and Panos Stathopoulos, MD, PhD, Identifying the B Cell Subsets that Contribute to Autoantibody Production in Myasthenia Gravis

48. M Isabel Leite, MD, DPhil, Serum Acetylcholine-receptor Antibody Concentrations in 71 patients Included in the Randomized Trial of Thymectomy in Myasthenia Gravis (MGTx)

49. Rozen Le Panse, PhD, Skeletal Muscle Regeneration is Impaired in Response to Acute Injury in Myasthenia Gravis

50. Marina Mane-Damas, MSc, Targeting Plasma Cells with Proteasome Inhibitors for Treatment of Myasthenia Gravis

51. Angelina Maniaol, MD, PhD, Agrin Antibodies in Myasthenia Gravis — What is Their Clinical Relevance?

52. Liis Sabre, MD, PhD, Reduction of Extracellular MiR-150-5p Levels Accompanies Improved Disease Severity in Myasthenia Gravis Patients 24 Months Post-thymectomy

53. Manjisth Sengupta, PhD, MicroRNA and M-RNA Expression Study in Ectopic Germinal Centers of Myasthenia Gravis Thymus

54. Kamal Shouman, MD, Serum Creatine Kinase Levels in Myasthenia Gravis 55. Betty Soliven, MD, Impaired Regulatory B cells in Myasthenia Gravis 56. Vuslat Yilmaz, PhD, Altered Number and Functionality of Regulatory B Cells in Myasthenia

Gravis Patient Congenital Myasthenia Syndromes

57. Judith Cossins, BA, DPhil, Mutation in a Nuclear Envelope Protein Causes a Novel Congenital Myasthenic Syndrome

58. Susan Maxwell, MSc, Congenital Myasthenic Syndrome due to Mutations in Agrin 59. Mirela Daniela Panea, MSc, Pathogenic Mechanisms of RAPSN Mutations in Congenital

Myasthenic Syndromes


Abstract List

60. Pedro M. Rodríguez Cruz, MD, MSc, Mutations in GMPPB Can Combine a Myasthenic Syndrome with a Myopathy

61. Xin-Ming Shen, PhD, Novel Synaptobrevin-1 Mutation Causes Fatal Congenital Myasthenic Syndrome

Management Diagnostic 62. Sneha Alaparthi, BS and Rahul Anil, MD, Exploring Outcomes and Characteristics of

Myasthenia Gravis: Rationale, Aims and Design of Registry — The EXPLORE MG Registry 63. Paolo Emilio Alboini, MD, Cognitive Impairment in Myasthenia Gravis with MuSK Antibodies 64. Carolina Barnett-Tapia, MD, PhD, Fatigue in Patients With Myasthenia Gravis: Impact on

Quality of Life and Responsiveness to Prednisone and Immunomodulation 65. Aisling S. Carr, MRCP Neurol., PhD, A Serological and Clinical Epidemiological Study of the

Myasthenia Gravis in Northern Ireland 66. Valentina Damato, MD, Clinical and Serological Characteristics of Patients with Double

Seronegative Myasthenia Gravis 67. Robert H. de Meel, MD, Ocular Weakness Patterns in Myasthenia Gravis 68. Anna De Rosa, MD, Thymoma-associated Myasthenia Gravis: Clinical and Serological Features

of Pisa’s Cohort 69. Wei Fang, PhD, Epidemiology of Myasthenia Gravis in Southern China — Estimates Using

Hospital and Insurance Records 70. Maria Elena Farrugia, DPhil, MD, FRCP(E), Management of Ocular Myasthenia Gravis in

Glasgow 71. Asger Frost, PhD, Characteristics of Long-time Patients with Myasthenia Gravis in a Danish

Population 72. Raffaele Iorio, MD, PhD, Neurological Autoimmunity Associated with Thymoma: Beyond

Myasthenia Gravis 73. Hans Katzberg, MD, MSc, Respiratory and Sleep Disturbances in Adults and Children with

Myasthenia Gravis 74. Ikjae Lee, MD, Thymectomy Status and Quality of Life in Myasthenia Gravis Patients from the

MGFA Registry 75. Jung Hwan Lee, MD, Pregnancy Outcome in Women with Myasthenia Gravis 76. Maarika Liik, MD, PhD, Significant Regional Difference in the Perception of MG Despite

Comparable Objective Fatigue


Abstract List

77. Alexander F. Lipka, MD, Long-term Follow-up and Survival in Patients with the Lambert-Eaton Myasthenic Syndrome

78. Michelangelo Maestri, MD, Clinical and Therapeutic Characteristics of Anti-MuSK Myasthenia Gravis: Our Single-Center Experience in Pisa

79. Florit Marcuse, BSc, Reduction in Acetylcholine Receptor Antibody Levels Correlate with Clinical Improvement in Myasthenia Gravis

80. Rudy Mercelis, MD, PhD, The Predictive Role of Stimulated Single Fiber Electromyography in the Orbicularis Oculi Muscle for the Generalization of Ocular Myasthenia Gravis Symptoms

81. Yuriko Nagane, MD, Social Disadvantages Associated with Myasthenia Gravis and its Treatment: A Multicenter Cross-sectional Study

82. Trine H. Popperud, MD, Polyautoimmunity and Familial Autoimmunity in Juvenile Myasthenia Gravis

83. Stephen W. Reddel, MBBS, PhD, A Global Electronic NeuroImmunology Database for Myasthenia Gravis (eNID-MG)

84. Girija Sadalage, MBBS, MRCP (Neurology), A Prospective Clinical and Immunological Study of Late-onset Myasthenia Gravis

85. Berthold Schalke, Prof Dr., Paraneoplastic Myasthenia Gravis: Diagnostic and Therapeutic Concept for Improving Long-term Survival

86. Ha Young Shin, MD, Characteristic Pattern of Repetitive Nerve Stimulation Test in Myasthenia Gravis with MuSK Antibody

87. Kimiaki Utsugisawa, MD, PhD, Early Fast-acting Treatment Strategy Against Generalized Myasthenia Gravis

88. Stanislav Vohanka, MD, PhD, Czech National Registry of Myasthenia Gravis 89. Jianying Xi, MD, PhD, A Cohort of 50 Patients with Lambert-Eaton Myasthenic Syndrome: Long

Term Follow-up of Survival State


Case Report

POSTER ABSTRACTS

Basic Science and Animal Models

1. Preconditioned Mesenchymal Stem Cells Treat Myasthenia Gravis in a Humanized Preclinical Model

Muriel Sudres, PhD1, Marie Maurer, PhD1, Marieke Robinet, Msc1, Jacky Bismuth, MSc1, Frédérique Truffault, MSc1, Nadine Dragin, PhD1, Mohamed Attia, PhD1, Elie Fadel, MD, PhD2, Nicola Santelmo, MD3, Camille Sicsic, MSc4, Talma Brenner, PhD4, and Sonia Berrih-Aknin, DSc1 1 Center of Research in Myology, Sorbonne Université, UPMC/INSERM UMRS 974, Institute of Myology, G.H. Pitié-Salpêtrière, Paris, France; 2 Centre Chirurgical Marie Lannelongue, Le Plessis Robinson, France; 3 Hôpital Civil de Strasbourg, Strasbourg, France; 4 Department of Neurology, Agnes Ginges Center for human Neurogenetics, Hadassah Hebrew University Medical Center, Jerusalem, Israel Myasthenia gravis (MG) with anti-acetylcholine receptor (AChR) antibodies (Abs) is an autoimmune disease characterized by severe defects in immune regulation and thymic inflammation. Because mesenchymal stem cells (MSCs) display immunomodulatory features, we investigated whether and how in vitro-preconditioned human MSCs (cMSCs) could treat MG disease. We developed a new humanized preclinical model by subcutaneously grafting thymic MG fragments into immunodeficient NSG mice (NSG-MG model). Ninety percent of the animals displayed human anti-AChR Abs in the serum, and 50% of the animals displayed myasthenic-like symptoms that correlated with the loss of AChR at the muscle endplates. Interestingly, each mouse experiment recapitulated the MG features of each patient. We next demonstrated that cMSCs markedly improved MG, reducing the level of anti-AChR Abs in the serum and restoring AChR expression at the muscle endplate. Resting MSCs had a smaller effect. Finally, we showed that the underlying mechanisms involved (i) the inhibition of cell proliferation, (ii) the inhibition of B-cell related and costimulatory molecules and (iii) the activation of the complement regulator DAF/CD55. In conclusion, this study shows that a preconditioning step promotes the therapeutic effects of MSCs via combined mechanisms, making cMSCs a promising strategy for treating MG and potentially other autoimmune diseases.

2. Specificity of Shp2 Inhibition on Acetylcholine Receptor Clustering

Michelangelo Cao, MD1,2, Pedro Rodriguez Cruz, MD1, Saif Huda, MD1, Judith Cossins, PhD1, Wei Wei Liu, PhD1, Elena Pegoraro, MD, PhD2, David Beeson, PhD1, and Angela Vincent, FRCPath1 1 Nuffield Department of Clinical Neurosciences, Weatherall Institute of Molecular Medicine, University of Oxford, United Kingdom; 2 Dipartimento di Neuroscienze, Università degli Studi di Padova, Italy SH2 domain-containing phosphatase (Shp2) is a negative regulator of the AChR clustering pathway that controls -Kinase (MuSK) activity. Shp2 inhibition by NSC87877 reverses the pathogenic effects of autoantibodies in MuSK-myasthenia gravis by increasing MuSK phosphorylation and preventing the dispersal of AChR clusters. We asked whether the effects of Shp2 inhibition are exclusive to MuSK, and whether NSC87877 might also be useful in reversing the effects of mutations in MuSK or DOK7. Using CRISPR/Cas9 system, we created MuSK KO and MuSK missense mutation (p.C327R) C2C12 cells. In addition, C2C12 myoblasts were infected by retrovirus with DOK7-wild type (wt) and the DOK7 c.1124_1127dupTGCC mutation. MuSK expression and tyrosine phosphorylation were detected by


Case Report

western blotting, and the phosphorylation expressed as the ratio of MuSK phosphotyrosine to total MuSK expression. AChR clustering was studied in the presence or absence of NSC87877. MuSK expression and phosphorylation were undetectable in MuSK KO myotubes. The p.C327R mutation did not affect MuSK expression and NSC87877 had no effect on MuSK phosphorylation, and there were not AChR clusters. By contrast, NSC87877 increased MuSK phosphorylation in myotubes overexpressing DOK7-wt, as expected, but not in the myotubes overexpressing the DOK7 mutant. Consistently with the phosphorylation results, we only observed a NSC87877-induced AChR clustering in myotubes overexpressing DOK7-wt. Shp2 seems to regulate the AChR pathway exclusively at the level of MuSK but requires a functional DOK7. These observations suggest that NSC87877 might have a limited role in the treatment of congenital myasthenic syndromes, restricted to patients with mutations in the agrin/LRP4/MuSK pathway.

3. Fetal and Adult Acetylcholine Receptor Function and the Impact of Receptor Clustering Hakan Cetin, MD1,2, Richard Webster, PhD1, Judith Cossins, DPhil1, Wei W. Liu, PhD1, An Vanhaesebrouck, DVM1, David Beeson, PhD1, and Angela Vincent, MSc1 1 Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, United Kingdom; 2 Department of Neurology, Medical University of Vienna, Vienna, Austria The full significance of the developmental switch from fetal to adult acetylcholine receptors (AChR) at late gestation in humans remains elusive. Understanding the functional properties of both isoforms under different conditions (unclustered and clustered) may provide greater insight into normal function and disease mechanisms. Patch-clamping experiments were performed on cell lines expressing endogenous or transfected fetal or adult AChR. Whole-cell currents were measured in order to analyse receptor desensitisation and recovery from desensitisation. First, our findings suggest faster recovery time constants in adult compared to fetal AChR (1111 ± 71 ms and 1929 ± 120 ms, respectively; p < 0.0001). Second, experiments using chimeric AChR with exchanged gamma and epsilon M3-M4 cytoplasmic loops revealed that fast recovery from desensitisation was determined by the epsilon M3-M4 loop. Third, receptor clustering by co-transfection with rapsyn was associated with significantly faster recovery time constants. Our findings provide important clues as to the significance of the developmental switch from fetal to adult AChR. The clustering of fetal AChR at the neuromuscular junction in parallel with the increasing expression of adult AChR, would both lead to faster recovery rates from desensitisation that could be a necessary adjustment to the motor neuron firing rates that increase around the time of birth. These findings are relevant to patients with congenital myasthenic syndromes with epsilon subunit mutations where expression of adult AChR is lost but fetal AChR expression is sustained. Moreover, our observation that AChR clustering can facilitate recovery from desensitisation suggests another pathomechanism through which MuSK antibodies, by reducing AChR clustering, could impair neuromuscular transmission.

4. AChR-antibody Mediated Arthrogryposis: Inhibition of Placental Transfer in a Mouse Model Protects the Fetus

Leslie Jacobson, DPhil, Ester Coutinho, MD, DPhil, Bethan Lang, PhD, and Angela Vincent, MBBS, MSc, FRCPath, FRS Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, United Kingdom; Rare cases of arthrogryposis multiplex congenital (AMC) are caused by maternal antibodies to the fetal acetylcholine receptor (f-AChR) transferred to the fetus during mid-gestation. The antibodies cause paralysis and fixed joints due to lack of fetal movement, and immunotherapies during subsequent pregnancies can protect the fetus. AMC has been modelled in mice by injecting human f-AChR antibodies into pregnant mice from E12-E17 (Jacobson et al., JCI, 1999). Sera from mothers AMC2 and AMC3 were injected into pregnant mice dams as above. Five dams were given an inhibitor of placental IgG transfer and five a control drug; three had no additional treatment. The


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pregnancies were terminated at E17 and the fetuses observed for deformities, and blood collected for f-AChR antibodies. In AMC3-IgG-injected dams maternal serum levels of f-AChR antibody were not affected by the inhibitor but fetal levels were reduced by proximately 70% compared to results in control-drug treated mice. With the more pathogenic AMC2 serum, and higher doses of the inhibitor and control, the majority of embryos of control-treated mothers were deformed, clearly different from those in the inhibitor-treated mothers. Both maternal levels and f- AChR antibody levels were substantially reduced. These studies demonstrate that it is possible to inhibit placental transfer to protect babies from the effects of pathogenic antibodies. Since neurodevelopmental disorders can also be linked to maternal antibodies in rare cases (Coutinho, Jacobson et al., JNNP, in press), the results may have wide implications for preventing antibody-mediated diseases such as autism or intellectual disability.

5. Preclinical Pilot Study of Alpha-fetoprotein on Moderation of MG Weakness Linda L. Kusner PhD1, Manjistha SenguptaPhD1, Gabriela Aguilo-Seara1, and Igor Sherman MD2

1 George Washington University, Washington, District of Columbia, United States; 2 Alpha Cancer Technologies, Toronto, Ontario, Canada

Remission of several autoimmune disorders, including myasthenia gravis, may occur during normal pregnancy. Studies have shown that alpha-fetoprotein (AFP) can modulate antibody binding and decrease the inflammatory processes through T-cell pathways. AFP is a serum protein, produced by the fetal yolk sac and liver. We assessed a recombinant, non-glycosylated form of human alpha-fetoprotein, MM-093 for efficacy in an experimental autoimmune MG (EAMG) rat model. For EAMG, rats were induced with Torpedo AChR and weakness was monitored. On day 40 after induction, daily intraperitoneal injections of MM-093 (60 µg or 1 mg) or equivalent volume of PBS was initiated. After two weeks of treatment, EAMG rats were euthanized and samples taken. EAMG rats that received 1 mg of MM-093 demonstrated lower clinical scores and greater grip strength than control rats. MM-093 is effective in significantly reducing the weakness in EAMG. Ongoing evaluations will determine the potential mechanisms.

6. Antigen-specific Therapeutic Immunoadsorption Results in Amelioration of Experimental MG Symptoms

Konstantinos Lazaridis1, Baltatzidi Vassiliki1, Dalianoudis Ioannis2, and Socrates J. Tzartos1

1 Hellenic Pasteur Institute, Athens, Greece; 2 Thriassio General Hospital, Elefsina, Greece

In MG pathogenic autoantibodies target mainly the muscle AChR, MuSK, and less frequently LRP4 and agrin. We aim to develop an antigen-specific therapy, based on the selective extracorporeal autoantibody depletion (immunoadsorption), using sepharose-immobilized extracellular domains (ECDs) of human AChR and MuSK. We have previously described the expression of these ECDs in yeast and their use as efficient immunoadsorbents in vitro. In order to proceed with in vivo immunoadsorptions, we established and characterized a rat experimental autoimmune MG (EAMG) models based on immunization with human AChR and MuSK domains. Lewis rats immunized once with the AChR ECDs present with the typical EAMG symptoms about 45 days later and, if left untreated, remain symptomatic for at least two months thereafter. Immunized rats which reached an EAMG score of 2 were permanently catheterized and subjected to repeated immunoadsorptions (three times a week) over a period of three weeks with sepharose-ECD or sepharose-BSA. We found that approximately five days after treatment commencement the sepharose-ECD treated rats started gaining weight and showed a marked improvement of their EAMG score. Their autoantibody titers decreased progressively, and their electromyograms and muscle AChR contents improved significantly. In contrast, rats treated with sepharose-BSA did not show any signs of improvement over the entire three weeks. These results strongly support the therapeutic efficacy of


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antigen-specific immunoadsorption for MG treatment, encouraging the progression towards clinical application. Importantly, the successful development of this therapeutic approach for MG could be used as a model for other antibody-mediated autoimmune disorders as well.

7. Lambert-Eaton Myasthenic Syndrome: Passive Transfer to Mice, Active Zone Structure-function Relationships, and Novel Treatment Strategies

Rozita Laghaei, PhD2, Kristine Ojala, BS1, Christopher J. Meriney, BS1, Scott Ginebaugh, BS1,2, Andrew Puglionesi1, Tyler Tarr, PhD1, Markus Dittrich, PhD2, and Stephen D. Meriney, PhD1 1 University of Pittsburgh, Department of Neuroscience, Pittsburgh, Pennsylvania, United States; 2 Carnegie Mellon University, Pittsburgh Supercomputing Center, Pittsburgh, Pennsylvania, United States Reliability at the neuromuscular junction derives from the summed activity of numerous unreliable elements, each consisting of a synaptic vesicle and associated voltage gated calcium channels (VGCCs). Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune disease that reduces transmitter release reliability, leading to muscle weakness. LEMS autoantibody-mediated weakness is caused by the removal of some VGCCs that are critical for transmitter release, an upregulation of other VGCC types, and a disruption in organization of VGCCs within active zone transmitter release sites. We have used a combination of electrophysiological recording and MCell computer modeling to examine structure-function relationships and novel treatment strategies in the passive transfer model of LEMS in mice. We find that the organization of the transmitter release site (especially the number and distribution of VGCCs) is a critical determinant of physiological function. Further, we find that LEMS effects on physiology cannot simply be explained by a loss of VGCCs. Lastly, we have used MCell modeling to reveal the spatio-temporal dynamics of calcium ion flux into transmitter release sites during exposure to a potassium channel blocker (DAP), used alone, or in combination with a calcium channel gating modifier (GV58). We find that broadening the presynaptic action potential (using predicted DAP effects on action potential shape) increases the effectiveness of GV58 (modeled by adding drug bound states to our VGCC kinetic model). We are currently studying the dose-response details of the synergistic effect between these two drugs using our MCell model before moving to preclinical testing in animals.

8. Analysis of Muscle Wasting in an Acute Severe Model of Muscle-specific Kinase Antibody Myasthenia Gravis

Stuart W. Morell, MS1, Lucia S. Borges, PhD 1, Douglas J. Rowland, PhD1, Erik R. Wisner, DVM1, Peter Pytel, MD2, and David P. Richman, MD1 1 University of California, Davis, California, United States; 2 The University of Chicago, Chicago, Illinois, United States A unique characteristic of the muscle-specific kinase (MuSK) form of myasthenia gravis (MMG) is muscle wasting, primarily involving axial musculature. The pathogenesis of these changes in muscle bulk is unknown. A model of MMG induced in Lewis rats by a single immunization with the adult isoform of mouse MuSK, MuSK 60, is acute and severe. Histologic analysis of the neuromuscular junctions in these animals demonstrates severe injury to both presynaptic and postsynaptic components, in the absence of inflammation. This disease, experimental anti-MuSK myasthenia gravis (EAMM), mimics all aspects of the human disease, including axial muscle wasting, providing an opportunity to determine the characteristics of the wasting. Analysis of EAMM using whole animal 1.5 Tesla MRI scanning confirmed the clinical observation of wasting of axial muscles, especially the paraspinous muscles. Whole animal muscle volume was reduced by 17% compared to animals immunized with adjuvant alone, whereas, for the paraspinous muscles, volume was reduced by 30% in association with a 24% increase in average muscle fiber T2 signal intensity. High field (7 Tesla) scanning of the paraspinous muscles demonstrated major involvement of the medial compartment of this muscle group. Histologic morphometry of these muscles demonstrated 53% reduction in cross-sectional area (mean ± SEM: 2515 ± 91 μ2 vs 4744 ± 124 μ2, p < 0.001) of the individual


Case Report

muscle fibers, suggesting reduced direct trophic effects on muscle fibers in this disease, perhaps as a consequence of the combined pre- and postsynaptic injury.

9. Mouse Model of Lrp4-MG Shuuichi Mori, PhD1, Norio Motohashi, PhD1, Rumi Takashima1, Masahiko Kishi, MD2, Hiroshi Nishimune, PhD3, and Kazuhiro Shigemoto, MD1 1 Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan; 2 Toho University Sakura Medical Center, Chiba, Japan; 3 University of Kansas School of Medicine, Kansas City, Kansas, United States Recent clinical studies have cast doubt on the specificity and pathogenicity of autoantibodies (Abs) against low-density lipoprotein receptor-related protein 4 (LRP4) for MG, highlighting the need for further research. In this study, we prepared the recombinant protein of an extracellular region of human LRP4 and injected it into female A/J mice. Immunization with LRP4 protein caused mice to develop myasthenia having similar electrophysiological and histological features as are observed in MG patients with Abs against muscle-specific kinase (MuSK). Our results clearly demonstrate that active immunization of mice with LRP4 proteins causes myasthenia similar to the MG induced by anti-MuSK Abs. Further experimental and clinical studies are required to prove the pathogenicity of anti-LRP4 Abs in MG patients.

10. ROCK Inhibitor Suppresses Experimental Autoimmune Myasthenia Gravis by Inhibiting Th1, Th17 and B-cells

Jie Song, MD1, Jianying Xi, MD, PhD1, Wenbo Yu, PhD2, Lei Zhou, MD1, Jiahong Lu, MD, PhD1, Baoguo Xiao, PhD2, and Chongbo Zhao, MD, PhD1 1 Department of Neurology, Huashan Hospital, Fudan University, Shanghai, China; 2 Institute of Neurology, Huashan Hospital, Institutes of Brain Science and State Key Laboratory of Medical Neurobiology, Fudan University, Shanghai, China The Rho kinases, or ROCKs, are a family of serine-threonine kinases that act as key effectors of Rho GTPases. In recent years, the emerging involvement of ROCKs in the pathogenesis of autoimmune diseases is being uncovered. Herein, we designed this study to evaluate the therapeutic efficacy of Fasudil, a ROCK inhibitor, in experimental autoimmune myasthenia gravis (EAMG) rats and investigated the partial mechanisms focused on T, B lymphocyte subsets. Furthermore, we evaluated the effects of Fasudil on PBMCs from myasthenia gravis (MG) patients in vitro. We found that Fasudil attenuated the severity of myasthenic symptoms and reduced levels of serum-anti-acetylcholine receptor (AChR) autoantibodies in R97-116 peptide-induced EAMG rats. In vitro studies using mononuclear cells obtained from spleen and popliteal lymph nodes isolated from EAMG rats on day 15 (acute phase) and 45 (chronic phase) after primary immunization revealed that administration of Fasudil significantly suppressed the proportion of Th1, Th17, total B and memory B-cells, and down-regulated IFN-, IL-17 and antibody levels in supernatant. In particular, we found that Fasudil inhibited sorted B-cell IgG production directly. In vitro studies using primary human PBMCs from AChR-positive generalized MG patients without any immunosuppressive therapy revealed that Fasudil significantly reduced the percent of Th1, Th17, plasma cells and memory B-cells, and down-regulated cytokines IFN- and IL-17. The proportion of Treg was slightly improved. Overall, our results suggested the inhibition of ROCKs might provide potential benefit to MG patients.


Case Report

11. The Potential Role of Anti-Lrp4 Autoantibodies in the Pathogenesis of Myasthenia Gravis (MG) and Amyotrophic Lateral Sclerosis (ALS)

Armineh I. Stoner, MS, Stuart W. Morell, MS, Lucia S. Borges, PhD, Robert H. Fairclough, PhD, and David P. Richman, MD The Center for Neuroscience at the University of California, Davis, Davis, California, United States Three to 50 percent of double seronegative myasthenia gravis (DSMG) patients have anti-Lrp4 antibodies, as well as some patients who are not double seronegative (with antibodies to the nicotinic acetylcholine receptor (nAChR) or muscle-specific kinase (MuSK) in addition to Lrp4). Anti-Lrp4 antibodies have also recently been found in the serum of ALS patients. Because of this lack of specificity, it is still unclear whether anti-Lrp4 antibodies are pathogenic, or simply a secondary phenomenon that occurs because of another primary pathology. To test this, we have immunized Lewis rats with mouse Lrp4 protein, to induce anti-Lrp4 myasthenia. We have optimized our transfection protocol for mouse Lrp4 protein production in HEK cells with the use of Lipofectamine 3000 and co-transfection of Lrp4 ectodomain DNA with DNA encoding a chaperone protein. In the correct ratio (2:1 chaperone DNA: Lrp4 DNA) co-transfection has greatly helped facilitate Lrp4 secretion and production in tissue culture. In our pilot experiment, a single immunization with 200 µg of purified mouse Lrp4 ectodomain in complete Freund’s adjuvant, with at least one booster injection (in Freund’s incomplete adjuvant), induced symptoms of disease. Two days after the second booster injection, the Lrp4-immunized rats developed mild weakness, decreased mobility and significant fatigability, the latter demonstrated by performance on a Rotarod apparatus. Antibody titers for Lrp4 were determined with a dot blot assay and for the Lrp4-immunized animals, the serum Lrp4 antibody levels rose to a titer of >107. Our results suggest anti-Lrp4 antibodies may lead to the development of DSMG.

12. MuSK-binding Peripheral B-cells as Biomarker for MuSK-Myasthenia Gravis Melike Küçükerden, MSc1, Shamsher S. Saini, PhD2, Vuslat Yılmaz, PhD1, Erdem Tüzün, MD1, and

Premkumar Christadoss, MD2 1 Department of Neuroscience, Aziz Sancar Institute of Experimental Medicine, Istanbul University, Istanbul, Turkey; 2 Immune Globe Biotech, LLC, Willis, Texas, United States To characterize MuSK-binding peripheral blood B-cells, B6 mice were immunized three times with MuSK/CFA or CFA alone and flow-cytometry studies were performed with blood samples of immunized mice using Alexa Fluor 647-conjugated MuSK (produced at Immune Globe Biotech). Eight of 10 MuSK/CFA-immunized mice developed significant muscle weakness ranging between grade 1–3 and showed varying levels of serum anti-MuSK IgG, IgG1, IgG2 and IgG3 antibodies, whereas none of the CFA-immunized mice showed muscle weakness or developed serum anti-MuSK antibodies. Flow cytometry studies performed with Alexa Fluor 647-conjugated MuSK showed considerable numbers of MuSK-binding IgG+, IgG1+, IgG2+ and IgG3+ B-cells in the peripheral blood of MuSK/CFA-immunized, but not in CFA-immunized mice. None of the MuSK/CFA- or CFA-immunized mice showed appreciable reactivity with the Alexa Fluor 647-conjugated OVA used as a negative control for evaluation of non-specific binding. Notably, IgG1 was the predominant anti-MuSK IgG isotype; similarly most MuSK-reactive B-cells were IgG1+. Although serum anti-MuSK antibody levels did not show any correlation with clinical EAMG parameters, numbers of peripheral blood IgG+, IgG1+, IgG2+ and IgG3+ MuSK-binding B-cells significantly correlated with clinical grades and grip strength values of MuSK/CFA-and CFA-immunized mice. There were also significant correlations between anti-MuSK IgG, IgG1, IgG2 and IgG3 levels and MuSK-binding IgG+, IgG1+, IgG2+ and IgG3+ B-cell levels. The fluorophore-MuSK conjugate is being tested to detect the


Case Report

frequency of MuSK-binding B-cells expressing specific markers and use for diagnosis or as biomarker for MuSK autoimmune and seronegative myasthenia gravis.

13. Exacerbation of EAMG by NMO Antibodies or Aquaporin 4 Peptide Adi Vaknin-Dembinsky, MD, PhD, Tehila Mizrachi, MSc, Livnat Brill, MSc, Malcolm Rabie, MD, Yakov Fellig, MD, Mayan Zur, MSc, and Talma Brenner, PhD Department of Neurology, Hadassah Medical Center, Jerusalem, Israel Myasthenia gravis (MG) and neuromyerlitis optica (NMO) are both antibody mediated diseases. MG is caused by autoantibodies against the nicotinic acetylcholine receptor (AChR) in 85% of patients or by other neuromuscular antigens, and NMO by autoantibodies against Aquaporin 4 (AQP4). There is evidence that patients with autoimmune diseases have increased tendency to develop another autoimmune syndrome. We reported an increased prevalence of NMO in patients with MG (Archives of Neurology 2011). The present study was designed to investigate whether a related co-existing autoimmune disease may exacerbate experimental autoimmune MG (EAMG) and evaluate how active immunization with AQP4 peptides or passive transfer of NMO-Ig can affect EAMG severity. Using EAMG induced in C57bl mice by Torpedo AChR and subjecting the animals to passive transfer of NMO-IgG or to immunization with AQP4-derived peptide. We found that injection of either AQP4 peptide or NMO-Ig to EAMG or to naïve mice caused increased fatigability and aggravation of EAMG symptoms as expressed by augmented muscle weakness, decremental response to repetitive nerve stimulation, increased neuromuscular jitter, and aberration of immune responses. Thus, the increased disease severity in EAMG mice following immunization with the NMO autoantigen AQP4 or by NMO-Ig, mediated by augmented inflammatory response can explain exacerbation or increased susceptibility of patients with MG to develop additional autoimmune syndrome such as NMO.

14. Electrophysiological Characterisation of a Mouse Model of DOK7 Congenital Myasthenic Syndrome and Treatment with Salbutamol

Richard G. Webster, PhD1, An E. Vanhaesebrouck, DVM1, Susan E. Maxwell, MSc1, Judith A. Cossins, DPhil1, Yuji Yamanashi, PhD2, and David M.W. Beeson, PhD1 1 Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, United Kingdom; 2 Division of Genetics, Institute of Medical Science, University of Tokyo, Tokyo, Japan DOK7 plays a crucial co-stimulatory role in the Agrin-MuSK-Rapsyn pathway controlling clustering of acetylcholine receptors (AChR) at the neuromuscular junction (NMJ). Recessive loss-of-function mutations in DOK7 underlie a congenital myasthenic syndrome (CMS) characterized by undersized NMJ and fatigable weakness. A mouse model that expresses Dok7 containing the common CMS mutation (c.1124_1127dupTGCC) (Dok7m/m) which is lethal > 10 days was studied at P8. Dok7m/m mice were smaller than unaffected litter mates (2.7 ± 0.1 g vs 5.1 ± 0.1 g, n = 11 & 109). Miniature endplate potentials (mEPP) were found in fewer fibres in Dok7m/m mice compared with wildtype mice (35.4 ± 2.7% vs 96.7 ± 3.3; n = 9 & 5); mEPP amplitude was unaltered (2.01 ± 0.34 mV vs 1.84 ± 0.16 mV; n = 8 & 4). However, mEPP frequency was reduced in dok7m/m mice (0.88 ± 0.2 min-1 vs 3.35 ± 0.8 min-1) and fewer NMJ were detected in diaphragm whole-mount by -bungarotoxin AChR staining (2.2 ± 0.4 vs 49.9 ± 6.3 NMJ/field; n = 7).


Case Report

Dok7m/m mice were treated with salbutamol (daily 8 or 16 mg/kg or saline i.p. injections) from P4. At P8, 8 and 16 mg/kg mice were larger than saline mice (133.5 ± 4.3 & 138.0 ± 2.3 % of P4 weight vs 117.2 ± 4.7; n = 9-10). Number of fibres with detectable mEPPs was increased by 8 mg/kg salbutamol (59.1 ± 5.0% vs 40.6 ± 3.8% for saline; n = 6) as was number of NMJ per field (6.4 ± 1.2 vs 2.6 ± 0.5; n = 11-10). Salbutamol treatment did not alter mEPP frequency or amplitude. Dok7m/m mice have severely compromised neurotransmission, with a reduction in detectable NMJ. mEPP amplitude is unaffected but frequency is severely reduced. Salbutamol treatment for 4 days at 8 mg/kg improves the number of NMJ detectable by AChR staining.


Case Report

Case Report

15. Unusual Facial Myokymia in a Patient with Ganglionic Acetylcholine Receptor Antibodies Edward W. Bahou, MD and Susan C. Shin, MD Department of Neurology, Icahn School of Medicine at Mount Sinai, New York, New York, United States Antibodies to the acetylcholine receptor (AChR-Ab) of the autonomic ganglia have been implicated in autoimmune autonomic ganglionopathy (AAG), but rarely in disorders of the somatic peripheral nerve. Here, we describe a 31 year-old female who complained of bilateral involuntary facial movements that were atypical for hemifacial spasm. Magnetic resonance imaging (MRI) of the brain and cervical spine did not show evidence for a vascular loop or demyelinating disease. Surface and concentric needle electromyography (EMG) showed myokymic discharges and cramp potentials of the orbicularis oculi, corrugator, and nasalis muscles. These findings were most suggestive of a peripheral nerve hyperexcitability (PNH) disorder, which has been well-described in association with voltage-gated potassium channel autoantibodies (VGKC-Ab). Further workup revealed serum positivity for ganglionic AChR-Ab and an absence of VGKC-Ab. Computed tomography (CT) of the chest, abdomen, and pelvis did not show evidence for malignancy. Analysis of the cerebrospinal fluid demonstrated five oligoclonal bands, with normal cell count, protein, and glucose. The patient responded only partially to a trial of carbamazepine and intravenous methylprednisolone. She was then treated with onabotulinum toxin, with complete resolution of her symptoms. This case represents an unusual presentation of PNH in association with autoantibodies to ganglionic AChR, which to these authors’ knowledge, has not been previously reported.

16. Exacerbation of Myasthenia Gravis in Thymic Cancer Patients Treated with Pembrolizumab:

Three Case Reports Juhyeon Kim, MD1, Jaeho Kim, MD1, Jin Myoung Seok, MD1, Misong Choi, MD1, Ju-Hong Min, MD, PhD1, Byung-Ok Choi, MD, PhD1, Jin Seok Ahn, MD, PhD2, Myung-Ju Ahn, MD, PhD2, and Byoung Joon Kim, MD, PhD1 1 Department of Neurology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea; 2 Division of Hematology-Oncology, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, South Korea Pembrolizumab is a humanized monoclonal antibody against programmed cell death protein-1, which was recently approved for treatment of solid tumor. Because of the mechanism augmenting natural anti-tumor immune response, there are adverse effects of this drug related to autoimmune events. We report here three patients with chemotherapy-refractory thymic cancer who had exacerbation of myasthenia gravis (MG) after pembrolizumab treatment. A 40-year-old woman with a history of MG and diagnosed with thymic carcinoma with liver metastasis treated with pembrolizumab presented with progressive ptosis, diplopia and dysphagia over two weeks. Her symptoms improved after she was treated with methylprednisolone 500 mg IV daily for three days. A 48-year-old man with a history of MG and diagnosed with a malignant recurrent thymoma, treated with pembrolizumab presented with progressive ptosis and diplopia. He began pembrolizumab treatment for thymoma aggravation and ptosis aggravated and diplopia occurred. He was treated with seven cycles of plasmapheresis and IV methylprednisolone 75 mg for four weeks. A 26-year-old man with a diagnosed malignant thymoma with SVC and left innominate vein invasion, treated with pembrolizumab presented with progressive ptosis and diplopia. He had no history of MG. He was treated with IVIg, methylprednisolone 50 mg daily and Pyridostigmine 120 mg three time a day. After five days, his symptoms improved.


Case Report

Our two patients experienced exacerbation of underlying MG and one patient was newly diagnosed with MG after pembrolizumab treatment. Considering the close association between MG and thymoma, exacerbation of MG after use of pembrolizumab might be common and also occur in patients without pre-existing MG.

17. Myasthenia Gravis and Immune Checkpoint Inhibitors: Balancing Treatment Efficacy and Complications in Cancer Immunotherapies

Araya Puwanant, MD1, Natalia L. Gonzalez, MD1, Neil Busis, MD1, Angela Lu, MD1, Stanley M. Marks, MD2, Daniel Kim, MD3, and Saša A. Živković, MD1 1 Department of Neurology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, United States; 2 Pittsburgh Cancer Institute, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, United States; 3 UPMC Shadyside Hospital, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, United States In recent years, development of anticancer immunotherapies have significantly transformed the field of cancer treatment. Immune checkpoint inhibitors are potent regulators of immunologic homeostasis and prevent the development of autoimmunity and maintain self-tolerance. The most recognized immune checkpoint molecules include programmed cell dealth-1 (PD-1) and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4). Inhibitors of immune checkpoint molecules are used as immunotherapy in advanced cancers, however severe, potentially fatal, immune-related complications may be triggered from exaggerated uncontrolled immune response. We describe two patients with severe, generalized myasthenia gravis (MG), elevated creatine kinase (CK), and other immunologic complications triggered by anti-PD1 treatment: 1) A 71-year-old woman with metastatic uterine carcinosarcoma who developed generalized MG after treatment with pembrolizumab. Acetylcholine receptor and MuSK antibodies were normal. Single fiber EMG showed markedly abnormal jitters. However, elevated CK and abnormal needle EMG findings raised the possibility of myositis; and 2) a 75-year-old woman with stable seropositive ocular MG who experienced myasthenic crisis in the setting of nivolumab treatment for metastatic renal cell carcinoma. Concurrently, there was development of elevated CK, cardiac arrhythmias, and myocarditis with severe systolic dysfunction. Corticosteroids and intravenous immunoglobulin led to significant improvement in MG symptoms, however both patients passed away within three months after starting anti-PD1 treatment from cancer progression. Despite the rarity, it is essential that MG and other immunologic-related complications be recognized early and treated promptly to reduce iatrogenic complications. Additional studies are required to characterize the association of myasthenia gravis and hyperCKemia in the patients who receive anti-PD1 therapy.


Clinical

Clinical

18. MuSK MG Patients Showed a Positive Response to Amifampridine Phosphate in a Randomized, Placebo-controlled, Crossover Study

Silvia Bonanno, MD, Barbara Pasanisi, MD, Carlo Antozzi, MD, Lorenzo Maggi, MD, Francesca Andreetta, PhD, Ornella Simoncini, MSc, and Renato Mantegazza, MD Department of Neuroimmunology and Neuromuscular Diseases, Istituto Neurologico Carlo Besta, Milan, Italy MuSK-myasthenia gravis (MuSK-MG) is a subclass of the disease characterized by female predominance, early onset, prominent bulbar involvement, severe clinical course, presence of anti-MuSK antibodies, and significant resistance to treatment, particularly to cholinesterase inhibitors. An unmet medical need is to improve the therapeutic armamentarium in MuSK-MG. Amifampridine phosphate is already marketed in Europe as first-line symptomatic treatment for LEMS and some of the congenital myasthenic syndromes. Moreover, Amifampridine is known to induce improvement of neuromuscular transmission in MuSK-MG mice. Hence, we performed a 1:1 randomized, double-blind, placebo-controlled, triple crossover study, denominated MuSK-001 to evaluate safety/tolerability and clinical efficacy of Amifampridine in MuSK-MG. MuSK-001 included an open-label run-in phase, followed by sequences of one week treatment and a cross-over schedule. Main Inclusion criteria were: age > 18, MGFA class II to IV with a score of 9 on MGC. Pre-existing immunosuppressive therapies were not modified during the study. The co-primary end points (QMG changes from baseline — p = 0.0003 and MG-ADL — p = 0.0006) were statistically met as well as all the secondary end points (MGC — p < 0.0001, MGQoL15 — p = 0.0025, FSS — p = 0.0061 and Neurological Institute Carlo Besta-MG — p = 0.0014 scale changes from baseline).

19. Comparison of Rituximab Treatment Regimens in MuSK Antibody Positive Myasthenia Gravis

Elena Cortés-Vicente, MD1,2, Aditya Kumar, MD3, Sneha Alaparthi, BS3, Isabel Illa, MD, PhD1,2, and Richard J. Nowak MD, MS3 1 Neuromuscular Diseases Unit, Department of Neurology, Hospital de la Santa Creu i Sant Pau, Universitat Autònoma de Barcelona, Barcelona, Spain; 2 Centre for Biomedical Network Research on Rare Diseases (CIBERER), Madrid, Spain; 3 Program in Clinical and Translational Neuromuscular Research, Department of Neurology, Yale University School of Medicine, New Haven, Connecticut, United States Rituximab has proven to be a beneficial and durable treatment option for MuSK-myasthenia gravis (MG). However, no established infusion protocol currently exists for rituximab use in MG. We aimed to assess the clinical response of patients with MuSK-MG treated with three different rituximab protocols (A - four weekly infusions and then a monthly infusion for the next two months; B - four weekly infusions, repeated after six months; and C - four weekly infusions, repeated every six months, up to five times; dose: 375 mg/m2) in terms of clinical status (MGFA Post-intervention Status-PIS), relapse rate, durability of response, and change in prednisone dose. We found no significant difference in age at onset, sex, worst MGFA class, prednisone dose before rituximab and at last visit, re-infusion rate, time to re-infusion, follow-up time, or MGFA PIS at last visit between the groups. On Kaplan-Meier estimation, there was no significant difference in reinfusion/relapse rate between the three groups (log rank test p = 0.3413). Multivariate survival analyses showed that rituximab protocol was not


Clinical

associated with the reinfusion rate (B: HR 7.83 95% CI 0.37-165.79; C: HR 3.42 95% CI 0.29-39.79; LR chi2 = 1.98, p = 0.3716). In summary, most patients only require an induction regimen, as defined above by A and B, to achieve clinical benefit and have a durable response. Currently, there is no firm evidence to support continuous B cell depletion. In order to minimize potential for adverse events, we recommend re-treating only in case of clinical relapse.

20. Challenges in a Multicenter Study of Subcutaneous Immunoglobulin in Myasthenia Gravis

Mazen M. Dimachkie, MD1, Vera Bril, MD2, Todd Levine, MD1,3, Jaya Trivedi, MD4, Nicholas J. Silvestri, MD 5, Laura Herbelin, BSc 1, Kiley Sims, BSc1 and the MGSCIg MSG Team 1 University of Kansas Medical Center, Kansas City, Kansas, United States; 2 University of Toronto, Toronto, Ontario, Canada; 3 Phoenix Neurological Associates, University of Arizona College of Medicine, Phoenix, Arizona, United States; 4 UT Southwestern Medical Center, Dallas, Texas, United States; 5 Univ. at Buffalo Jacobs SMBS, State University of New York, Buffalo, New York, United States Background: Based on Class I studies, efficacy studies of intravenous (IV) immunoglobulin Ig in myasthenia gravis (MG) are ongoing. In routine clinical care, subcutaneous (SC) Ig might be easier to administer than IVIg. We describe recruitment challenges in this North American study evaluating efficacy and safety of SCIg in MG patients who are on stable IVIg therapy. Methods: This multi-center open label investigator-initiated study has two components: IVIg Screening Phase (ISP; Weeks -10 to -1) and Experimental Treatment Phase (ETP; Weeks 0 to 12). Our plan is to recruit 25 subjects in the ISP. At Week 0 and following three cycles of monthly IVIg, subjects with stable QMG enter the ETP. With an anticipated attrition rate of 20%, 20 subjects are enrolled into the 12-week ETP. Enrollees initially receive weekly SCIg at 1:1.20 IVIg:SCIg, which may be increased to 1:1.37 at Week 4. Primary outcome measure is the change in QMG score from Week 0 to Week 12, with other measures including MG Composite, MG-ADL, MG-QOL15, safety laboratory tests and patient satisfaction with treatment questionnaire. Site selection was performed based on feasibility questionnaires. We are reviewing site activation patterns and polling sites about recruitment challenges. Results: Out of 19 patients who participated in the ISP, 18 entered the ETP to date. We will provide an update on site activation timelines and enrollment data with an analysis of challenges. Conclusion: Conducting multicenter MG studies is challenging due to variety of reasons that go well beyond the rarity of this disease. This Investigator Initiated Research project is funded by a grant to Mazen M. Dimachkie, MD from CSL Behring.

21. Pilot Trial of Abatacept in Myasthenia Gravis Patients Inadequately Responsive to Conventional Immunotherapy

Daniel B. Drachman, MD1, Brian J. Traynor, MD, PhD2, Vinay Chaudhry, MD1, and Andrea Corse, MD1 1 Johns Hopkins School of Medicine, Baltimore, Maryland, United States; 2 National Institutes of Health, Bethesda, Maryland, United States Approximately 10% of patients with MG do not respond adequately to “conventional” immunosuppressive agents (adrenal corticosteroids, azathioprine, mycophenolate, calcineurin antagonists), cannot tolerate


Clinical

them, or require intermittent rescue with IVIg or plasma exchange. The hypothesis underlying this study is that Abatacept, which has a different mechanism of immunosuppressive action, by interfering with costimulation, will produce important clinical benefit for these myasthenic patients The pathogenesis of MG is well known to involve autoantibodies. Autoantibody production requires help from activated CD4 T-cells. Activation of the T-cells involves presentation of the antigen by antigen-presenting-cells (APCs). However, in order to activate the T-cell, antigen must be presented in association with costimulatory factors expressed by the APCs: i.e. CD80 and/or CD86, which bind to CD28 on the T-cell surface. Blockade of costimulation prevents activation of the T-cells and inhibition of antibody production. CTLA4, which is expressed on the T-cells after activation, binds CD80 and CD86, and results in turning off the T-cells. When conjugated to immunoglobulin, CTLA4-Ig (Abatacept) is soluble, and binds to CD80 and CD86, blocking their costimulatory action. Our published pre-clinical studies in rats with experimental MG have demonstrated that CTLA4-Ig powerfully immunosuppresses EAMG. Abatacept has been used effectively and safely to treat rheumatoid arthritis. This study will utilize Abatacept to treat MG patients who do not respond satisfactorily to “conventional” immunosuppressive medications, and follow them over one year to evaluate clinical benefit.

22. Intravenous Immunoglobulin as Monotherapy for Myasthenia Gravis during Pregnancy Josep Gamez, MD PhD; María Salvado, MD, Manel Casellas, MD; Susana Manrique MD, PhD;

Felix Castillo, MD, PhD, and José María Ponseti, MD, PhD Myasthenia Gravis Unit, Hospital Universitari Vall d’Hebron, Vall d'Hebron Institut de Recerca, Universitat Autònoma de Barcelona, Barcelona, Spain Pregnant women with myasthenia gravis (MG) are at increased risk of complications and adverse outcomes, including the teratogenic effects of many drugs used to treat MG women of childbearing age. The effectiveness of intravenous immunoglobulins (IVIg) on other autoimmune mediated diseases has been extensively reported in recent years, although little is known about IVIg as a monotherapy for MG during pregnancy. Five pregnant MG patients (mean age at delivery 36.4 years, SD 5.8, range 29.4–45.2) were studied in 2013–14. Their treatment was switched to monthly IVIg cycles two months before the pregnancy. Follow-up included monthly neurological QMG throughout the pregnancy and postpartum, obstetrical monitoring during monthly visits in the first two trimesters of the pregnancy, fortnightly visits between week 32 and week 36, and weekly visits after 36 weeks, and neonatal follow-up after delivery. We observed no exacerbations during pregnancy, delivery pregnancy, delivery or post-partum. The mean QMG score at baseline (before pregnancy) was 7.4 points in five women with generalized forms of MG (the MGFA class at onset of the disease was as follows: three IIa, one IIIb and one IIIb). The maximum mean value reached during pregnancy was 8.6 points. The mean pregnancy duration was 38 w + 5 d. No infant presented with transient neonatal myasthenia gravis. No congenital anomalies were identified in any of the newborns. Tolerance was excellent. These results suggest that monotherapy with IVIg during pregnancy in MG patients could be promising, although confirmation is required in studies with larger populations. Acknowledgements: PI 16/01673 FIS-FEDER.


Clinical

23. Very Late-onset Non-thymomatous and Thymomatous Myasthenia Gravis (MG) Are

Associated with Different HLA Class II Alleles

Giulia Greco1, Manuela Testi2, Giovanni Antonini3, Emanuele Rastelli1, Chiara Terracciano1, Eugenio Pompeo4, Marco Andreani2, and Roberto Massa1 1 Department of Neurosciences, University of Rome Tor Vergata, Rome, Italy; 2 Laboratory of Immunogenetics, IME Foundation, Rome, Italy; 3 Department DESMOS, University of Rome La Sapienza, Rome, Italy; 4 Department of Thoracic Surgery, University of Rome Tor Vergata, Rome, Italy A variable association of several HLA alleles with MG subtypes has been reported in different ethnic groups. Among subtypes, non-thymomatous MG with late onset is reported to be increasingly frequent. The aim of this work was to define specificities and prevalence of MG subtypes in a large series of Italian patients according to clinical (gender, age at onset, severity of symptoms, autoantibody profile and thymic abnormalities, n = 230) and immunogenetic (HLA-II alleles, n Two main peaks of incidence emerged: one with onset between 21 and 40 years of age, with overwhelming female preponderance, the second between 61 and 80 years, with higher male prevalence. This Very Late Onset subtype was largely composed by subjects with non-thymomatous, anti-AChR antibody-positive MG (VLOMG), and showed an increasing incidence. Thymoma (prevalently grade B2) was present in 23% of cases. Patients with MGFA grade V had higher anti-AChR antibody titers compared to milder cases. HLA-II allelic frequencies were compared with those of 315 healthy controls, applying the Fisher exact test. p values were corrected (pc) for multiple comparison, according to Bonferroni. A highly significant association was observed between alleles DQB1*05:02 (pc < 0.01) and DRB1*16 (pc = 0.01) and VLOMG. Conversely, the DQB1*05:01 allele showed a significant association with thymomatous MG (pc < 0.05). Our results suggest that, among Italian patients, VLOMG is by large the most frequent MG subtype and differs from thymomatous MG in terms of association with HLA-II susceptibility markers.

24. Comparative Effectiveness Research to Advance Treatment of Myasthenia Gravis Jeffrey T. Guptill, MD1,2, Shruti Raja, MD1, Pushpa Narayanaswami, MD3, and Donald B. Sanders, MD1 1 Department of Neurology, Duke University, Durham, North Carolina, United States; 2 Duke Clinical Research Institute, Durham, NC, North Carolina, United States; 3 Department of Neurology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, United States Myasthenia gravis (MG) presents serious challenges in establishing treatment efficacy through randomized clinical trials (RCTs): 1) recruitment is difficult in such a rare disease; 2) disease severity may change spontaneously; 3) novel immunosuppressive treatments are commonly used clinically in MG before regulatory testing; 4) varied MG subtypes limit the generalizability of RCTs; and 5) immunosuppressive drugs often take a long time to work, requiring long trials with high costs and frequent dropouts. Observational studies performed in real-world settings hold promise to overcome many of these limitations. Comparative effectiveness research (CER) includes observational studies that compare the benefits and harms of alternative methods to prevent, diagnose or treat a condition. Prospective observational CER is a useful process to evaluate the effectiveness of MG treatments. Observational studies performed in routine clinical settings require fewer resources and are usually less expensive than RCTs. The following recent advances have improved the feasibility of CER studies for MG: 1) the availability of MG common data elements; 2) establishment of international consensus guidance for goals of treatment; and 3) development of a web-based REDCap database that can standardize data collection and be shared. Using these


Clinical

advances, centers specializing in MG can collect long term data for longitudinal studies to compare commonly-used therapies and the usefulness of patient-centered outcome measures such as the MG-Quality of Life 15r to measure treatment responses. The major limitation of observational CER is confounding because of lack of randomization; adjustment of confounders is challenging and needs meticulous attention to obtain valid results.

25. Walking Away from the Fatigue and Low Functional and Metabolic Capacity in Myasthenia Gravis

Charlene Hafer-Macko, MD1,2, Richard Macko, MD1,2, Alice Ryan, PhD1,2, and Julia Naumes, OTD3 1 University of Maryland, School of Medicine, Baltimore, Maryland, United States; 2 Veterans Administration Maryland Health Care System, Baltimore, Maryland, United States; 3 Pacific University, Portland, Oregon, Unites States

Introduction: Weakness and fatigue in myasthenia gravis (MG) often limit participation in activities of daily living (ADL), even in relatively well-controlled MG. Physical inactivity may compound cardiovascular deconditioning. Objective: To determine whether three-month supervised exercise improves performance, fitness, ventilatory capacity, and participation. Methods: 12 adults (seven males, five females) with stable MG enrolled in three months (three days/week) of progressive exercise (walking, breathing exercises, and functional resistance training). MG scales are MG Quality of Life 15, MG ADL, Quantitative MG, MG Composite. Functional measures are timed six-minute walk, timed up-go, five sit-to-stands, stair climbs, step activity monitors, fatigue severity, and falls efficacy. Open circuit spirometry during treadmill walks determined oxygen utilization and ventilatory capacity/requirements during a) peak exercise (fitness level or V02 peak) and b) six-minute walks at preferred speed (economy of gait). Results: Baseline fitness (V02 peak, 20 + 5 ml/kg/min) is 20% below age-gender matched sedentary controls (p < 0.05), while walking metabolic cost (13 + 3 ml/kg/min) is two-fold higher, indicating that a simple six-minute walk requires 63% of peak capacity. Training improves functional capacity, fitness, gait cost, ventilatory capacity and efficiency, fatigue, falls efficacy, and community participation. Conclusion: Despite well-controlled MG, functional capacity is low. Findings reveal cardiovascular deconditioning and elevated metabolic cost of walking that may contribute to fatigue and limit ADL function in MG. Training is well tolerated, improves functional and metabolic workload, and efficiency, supporting the rationale for future randomized exercise studies that target functional capacity, fitness, efficiency, and health for adults living with MG.


Clinical

26. Correlations Between the Time Trends of Anti-acetylcholine Receptor Antibody Titer Variation in Myasthenia Gravis and the Incidence of Mycoplasma and Influenza Infections

Kazuo Iwasa, MD,PhD1, Hiroaki Yoshikawa, MD, PhD2, Tsuyoshi Hamaguchi, MD, PhD1, Kenji Sakai, MD, PhD1, Moeko Shinohara-Noguchi, MD, PhD1, Miharu Samuraki, MD, PhD1, Kazuya Takahashi, MD, PhD1,3, Daisuke Yanase, MD, PhD1,4, Kenjiro Ono, MD, PhD1,5, Chiho Ishida, MD, PhD1,3, Mitsuhiro Yoshita, MD, PhD1,6, Hiroyuki Nakamura, MD, PhD7, and Masahito Yamada, MD, PhD1 1 Department of Neurology and Neurobiology of Aging, Kanazawa University, Kanazawa, Ishikawa, Japan; 2 Health Service Center, Kanazawa University, Kanazawa, Ishikawa, Japan; 3 Department of Neurology, National Hospital Organization Iou Hospital, Kanazawa, Ishikawa, Japan; 4 Department of Neurology, Kouseiren Takaoka Hospital, Takaoka, Toyama, Japan; 5 Department of Neurology, Showa University School of Medicine, Tokyo, Japan; 6 Dementia Medical Center, Department of Neurology, and Institute for Clinical Research, National Hospital Organization Hokuriku Hospital, Nanto, Toyama, Japan; 7 Department of Environmental and Preventive Medicine, Kanazawa University Graduate School of Medical Science, Kanazawa, Ishikawa, Japan Background: The etiology and exacerbating factors of myasthenia gravis (MG) have been unknown. Genetic and environmental insults may play a role in the development and/or exacerbation of MG. Methods: 58 MG patients were followed-up from August 2006 to October 2012. We calculated adjusted anti-acetylcholine receptor antibody (Adj-AChR-Ab) titers that range showed 0–1. We used a cross-correlation function (CCF), a generalized linear model, and multiple linear regression modeling to determine the relationships among the Adj-AChR-Ab titers and the incidence of infections and the number of MG patients admitted to the hospital. Results: CCF analysis of Adj-AChR-Ab titer and incidence of infections due to Mycoplasma pneumoniae (M. pneumoniae) and influenza virus indicated the presence of significant positive correlations, with maximum coefficients of r = 0.449 (p < 0.0001; lag, -2 months) and r = 0.411 (p < 0.001; lag, 2 months), respectively. Adj-AChR-Ab titer in MG with thymoma was highly correlated with M. pneumoniae infection (r = 0.798; p < 0.0001; lag, -2 months). The relative risk for Adj-AChR-Ab titer increase (assessed using one M. pneumoniae infected patient per monitoring point) was 1.407 (95% CI, 1.193–1.661; lag, -2 months). Using 100 influenza virus infected patients per monitoring point resulted in an increase of 1.158 (95% CI, 1.071–1.253; lag, 2 months). The number of admitted patients with MG and Adj-AChR-Ab titer were significantly correlated (r = 0.518, p < 0.0001). Conclusions: Adj-AChR-Ab titer is significantly influenced by incidence of M. pneumoniae and influenza virus infection, and these agents may exacerbate MG.

27. Thymus Radiological-pathological Correlations in Myasthenia Gravis — the Relevance in Clinical Practice.

Elzbieta Klimiec, MD1, Maria Isabel Leite, MD2, Mary Quirke, RGN2, and David Hilton-Jones, MD2 1 Department of Neurology, Jagiellonian University Medical College, Krakow, Poland; 2 Nuffield Department of Clinical Neurosciences, Oxford University Hospitals, Oxford, United Kingdom Myasthenia gravis (MG) is commonly associated with thymus pathology such as follicular hyperplasia or thymoma. A recent randomized controlled trial showed that thymectomy improves clinical outcomes in certain MG subpopulations. The thymus is assessed routinely by CT or MRI imaging. The ability to identify thymoma and to distinguish between a normal gland and hyperplasia should aid clinical management and decision making. We aimed to determine the accuracy of routine imaging in predicting thymus pathology. We retrospectively analyzed records of patients from the Oxford Myasthenia Centre registry who underwent


Clinical

thymectomy, either video-assisted thoracoscopic surgery (VATS) or open surgery. Each patient received one radiological diagnosis based on the initial scan report: thymoma, hyperplasia/enlargement, or normal. Each patient received one histological diagnosis: thymoma, hyperplasia, or normal. We included 106 patients who underwent thymectomy between 1989 and 2016. Histopathological examination revealed thymoma in 40 patients (37.7%) and hyperplasia in 34 patients (32.1%). Specimens were described as normal in 32 patients (30.1%). Radiological diagnosis of thymoma was made in 39 patients (36.8%) and hyperplasia/enlargement were detected in seven patients (6.6%). The remaining 60 patients (56.6%) were classified as normal. Radiological diagnoses agreed with histopathological reports in 73 patients (68.9%). Sensitivity and specificity were calculated for each radiological diagnosis as follows: thymoma 90% and 95.5%; hyperplasia 17.6% and 98.6%; and normal 96.9% and 60.8% respectively. We conclude that the decision to offer thymectomy, in those without radiological suspicion of thymoma, should be based entirely on clinical grounds.

28. West Nile Virus (WNV) Induces a Prolonged Proinflammatory State That May Promote Myasthenia Gravis (MG)

A. Arturo Leis, MD1,2, Dobrivoje S. Stokic, MD, DSc1, Dhiraj Acharya, PhD3, Amber M. Paul PhD3, Ram B. Kuwar, PhD4, Parminder J.S. Vig, PhD5, Mark A. Ross, MD2, Gabriella Szatmary, MD, PhD6, and Fengwei Bai, PhD3 1 Center for Neuroscience and Neurological Recovery, Methodist Rehabilitation Center, Jackson, Mississippi, United States; 2 Department of Neurology, Mayo Clinic, Scottsdale, Arizona, United States; 3 Department of Biological Sciences, University of Southern Mississippi, Hattiesburg, Mississippi, United States; 4 Department of Anatomy and Neurobiology, Virginia Commonwealth University, Richmond, Virginia, United States; 5 Departments of Neurology, Neurobiology and Anatomical Sciences, and Biochemistry, University of Mississippi Medical Center, Jackson, Mississippi, United States; 6 Hattiesburg Clinic, Hattiesburg, Mississippi, United States In 2012, we reported six cases of serologically-confirmed MG that developed 3–7 months after WNV neuroinvasive disease. Since then, we, and others, have seen more cases of WNV-associated MG. Here, we offer a perspective on pathogenic mechanisms involved in WNV that may also contribute to the development of MG. WNV is quickly eradicated from the human body and there is no evidence of direct damage to the neuromuscular junction. Previously, we found pathologically elevated levels of astroglial protein S100B in cerebrospinal fluid and serum of WNV patients. We also demonstrated that S100B expression is increased in mouse astrocytes after exposure to inactivated WNV particles and that S100B activates neutrophil migration in vitro. S100B is known to bind the Receptor for Advanced Glycation End products (RAGE), an important ligand for various immunoregulatory molecules. This may be relevant because in experimental autoimmune MG, the expression of RAGE and S100B are increased and the interaction between RAGE and S100B alter T-cell balance and up-regulate AChR-specific T-cell proliferation. We also showed that WNV infection in humans induces expression of proinflammatory cytokine interleukin 17A (IL-17A), its receptor IL-17AR, and osteopontin (OPN), a multifunctional protein produced by a variety of immune cells. IL-17A, IL-17AR, and OPN play a role in many inflammatory and autoimmune diseases and remain elevated in humans long after recovery from WNV disease. The cumulative evidence suggests that WNV induces a protracted post-viral auto-inflammatory state characterized by pathological levels of S100B, IL-17A, and OPN that may promote the development of MG.


Clinical

29. Hepatitis B Virus Reactivation in Myasthenia Gravis Patients with Immunosuppressive Treatments

Wei Bin Liu, MD, PhD, and Xiao Xi Liu, MD Department of Neurology, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China Purpose: Reactivation of hepatitis B virus (HBV) is thought to be associated with immunosuppressive treatments, but insufficient information is available on myasthenia gravis (MG) patients. The purpose of this study is to evaluate the risk of HBV reactivation in hepatitis B surface antigen (HBsAg)-positive or antibody to hepatitis B core antigen (anti-HBc)-positive patients with MG under immunosuppressive treatments. Methods: MG patients (n=22) with positive HBsAg or positive anti-HBc were analyzed retrospectively. Eighteen patients received immunosuppressive treatments (corticosteroids with or without other immunosuppressant) for at least eight months. The other four patients were only treated with pyridostigmine. The main study outcome was HBV reactivation. Results: HBV reactivation occurred in six of 22 MG patients (27.2%). All of these six patients were HBsAg-positive and received immunosuppressive treatments despite antiviral medications. The earliest finding of HBV reactivation was at the third month of using immunosuppressive treatments. None of patients treated with pyridostigmine had HBV reactivation. Furthermore, all of patients who taken antiviral medications before immunosuppressive treatments did not have HBV reactivation. Conclusion: These results suggest that immunosuppressive treatments probably increase the risk of HBV reactivation in MG patients who are HBsAg-positive. Using antiviral medications in advance may prevent the occurrence of HBV reactivation in MG patients with immunosuppressive treatments.

30. Therapeutic Potential of Docetaxelpluscisplatin Chemotherapy in Myasthenia Gravis: Clinical Analysis of Patients with Thymoma-associated Myasthenia Gravis

Guoyan Qi, MM, Peng Liu, MM, Huimin Dong, MM, ShanshanGu , MM, Hongxia Yang, MM, and YinpingXue, MM Presented by YueXun Liu, MD, MS Center of Treatment of Myasthenia Gravis, First Hospital of Shijiazhuang, Shijiazhuang, Hebei Province, China Background: The present study investigated the therapeutic effect of docetaxel plus cisplatin chemotherapy on metastatic thymoma in patients with myasthenia gravis (MG). Methods: Seven MG patients with metastatic thymoma that underwent docetaxel plus cisplatin therapy were retrospectively recruited. The clinical outcome of thymoma and MG and adverse events were evaluated. Results: There were four women and three men with a median age of 49 years. Five patients were Osserman stage VI and the others were stage IIB. The median MG course was 7.17 years. The histologic types of thymoma were 3 B1, B2 and B3 in each two patients. Masaoka stage was I in one patient, II in five, and III in one patient. The median time from surgery to tumor metastasis was 5.07 years. All patients achieved partial response or stabilization of the tumors following docetaxel plus cisplatin chemotherapy. Moreover, the clinical symptoms of MG were alleviated in all seven patients, two with complete remission and the other five with marked improvement. Myelosuppression was the major adverse event observed, occurring in two patients (grade II and IV). During the median follow-up (median: 42.33 months), all patients were stabilized except one developing MG relapse.


Clinical

Conclusion: Our study presented a series of MG patients with metastatic thymoma that underwent docetaxel plus cisplatin chemotherapy. In addition to the improved/stabilized thymoma, our study showed markedly improved clinical symptoms of MG for the first time, with the tolerated adverse event, suggesting the potential role of docetaxel plus cisplatin regime in MG management. Our study may shed light on the therapeutic potential of docetaxel pluscisplatin in patients with thymoma-associated MG.

31. INSIGHTS: Analysis of Responsiveness of IVIg in Patients with Myasthenia Gravis Tahseen Mozaffar, MD1, Leslie Vaughn, RPh2, Gary Badger ,MS3, Gil Wolfe, MD4, Lara Katzin, MD5, David

Saperstein, MD6, Todd Levine, MD6, Richard Barohn, MD7, Jonathan Katz, MD8, Mazen Dimachkie, MD7, Michelle Greer, RN2, and Elissa Ritt, MAS2 1 University of California, Irvine, Orange, California, United States; 2 NuFactor Specialty Pharmacy, Temecula, California, United States; 3 University of Vermont, Burlington, Vermont, United States; 4 University at Buffalo, Buffalo, New York, United States; 5 University of South Florida, Tampa, Florida, United States; 6 Phoenix Neurological Associates, Phoenix, Arizona, United States; 7 University of Kansas, Kansas City, Kansas, United States; 8 California Pacific Medical Center, San Francisco, California, United States Objective: To determine the clinical, laboratory, and electrophysiologic criteria of myasthenia gravis (MG) patients prescribed IVIg most predictive of a response to treatment. Background: IVIg is commonly used off-label for treatment of exacerbations and maintenance therapy in MG. Due to a lack of controlled trials, there is variability in patients prescribed IVIg for MG in clinical practice. Methods: Clinical, laboratory, and electrophysiologic data was collected on 585 neuromuscular patients who were prescribed IVIg from across the country. A panel of independent, expert neuromuscular neurologists reviewed the information. Positive outcomes were independently determined based on quality-of-life measures, Patient Global Impression of Change, and clinical documentation. Results: Of the 89 MG patients who were prescribed IVIg, 44 were naive to IVIg; 68% of these patients had a positive response. 45 patients had received IVIg previously, and 73% of these patients had a positive outcome. The average dose of IVIg was 1.73 g/kg/month with a range of 0.4 g/kg/month to 4 g/kg/month. 59 patients (66%) were receiving ongoing maintenance therapy, while 30 patients (34%) received treatment for exacerbation. Positive response was most strongly associated with the independent reviewers’ determination that the patient was appropriate for IVIg (92% vs 50%, p = 0.06) based on progressive, generalized weakness. Patient’s age, sex, distribution of weakness, disease time course, and antibody status had no significant association with response. Conclusions: Overall treatment response rate was 70%, suggesting larger, controlled trials of IVIg in MG should be performed. Updated results and analysis will be presented at the time of the meeting.


Clinical

32. Association of Total Serum Immunoglobulin G (IgG) Concentrations during Therapeutic Plasma Exchange (TPE) Therapy and Myasthenia Gravis (MG) Persistence

Jay S. Raval, MD1, Rachel Kilian, MMBT2, Cynthia E. Drake, MA3, James F. Howard, MD4, and Yara A. Park, MD1 1 Department of Pathology and Laboratory Medicine University of North Carolina, Chapel Hill, North Carolina, United States; 2 Terumo BCT, Inc., Lakewood, Colorado, United States; 3 University of Colorado, Denver, Colorado, United States; 4 Department of Neurology, University of North Carolina, Chapel Hill, North Carolina, United States TPE is a common intervention to treat MG. Many patients (not-persistent MG) achieve treatment response (resolution of MG-related findings) after an acute TPE series, typically six treatments over two weeks. However, a subset of these patients requires maintenance TPE therapy to remain asymptomatic (persistent MG). There is currently no disease biomarker associated with MG-persistence after initial TPE therapy necessitating maintenance TPE. At our medical center, we routinely measure total serum immunoglobulin G (IgG) prior to each TPE procedure. In this retrospective study, we analyzed IgG during TPE therapy in not-persistent and persistent MG patients. Data from the initial treatments of patients that required TPE over a five-year period were collected. On average, not-persistent patients (n=63) experienced a 48% decrease in IgG after initial acute TPE series compared to persistent patients (n=17) with a 34% decrease (p < 0.05). Overall, 68% of not-persistent patients achieved an endpoint of IgG < 300 mg/dl (serologic response) compared to 50% of persistent patients (p < 0.05). When patients were stratified by their pre-treatment IgG into high, medium, and low tertiles, 90% of not-persistent versus 100% of persistent patients in the lowest tertiles achieved serologic response (p > 0.05). In contrast, 32% of not-persistent versus 0% (none) of persistent patients in the highest tertiles achieved serologic response (p < 0.05). We observed that baseline IgG is associated with likelihood of serologic response. The effect is apparent in both patient groups, but notably greater in persistent patients. IgG should be considered as a factor during evaluations of MG patients receiving TPE.

33. Efficacy and Safety of Tetanus Revaccination in Autoimmune Myasthenia Gravis Ellen Strijbos, MD1, Maartje G. Huijbers, PhD1,2, Inge E. van Es, BSc2, Iris Alleman, BSc3, Monique M. van Ostaijen-ten Dam, BSc4, Yvonne E. Fillie-Grijpma, BSc2, Jaap Bakker, PhD5, Erik van Zwet, PhD6, Cornelia Jol-van der Zijde, BSc4, Maarten D. van Tol, MSc, PhD4, and Jan J. Verschuuren, PhD1 1 Department of Neurology, Leiden University Medical Centre, Leiden, the Netherlands; 2 Department of Human Genetics, Leiden University Medical Centre, Leiden, the Netherlands; 3 Department of Physiotherapy, Leiden University Medical Centre, Leiden, the Netherlands; 4 Department of Paediatrics, Laboratory of Immunology, Leiden University Medical Centre, Leiden, the Netherlands; 5 Department of Clinical Chemistry and Laboratory Medicine, Leiden University Medical Centre, Leiden, the Netherlands; 6 Department of Medical Statistics and Bioinformatics, Leiden University Medical Centre, Leiden, the Netherlands Objective: To investigate the efficacy of the immune response to and safety of a tetanus revaccination in patients with autoimmune myasthenia gravis or Lambert-Eaton myasthenic syndrome. Methods: A tetanus revaccination was administered to 66 patients. Before and four weeks after revaccination a blood sample and clinical outcome scores were obtained. Anti-tetanus IgG total, IgG1 and IgG4 titers and disease-specific antibody titers (AChR, MuSK or VGCC) were measured with an ELISA or a radio-immunoprecipitation assay. A historic healthy control group was used for comparing tetanus antibody titers with that of our patients. A placebo vaccination group was used to investigate the variability of clinical outcome scores with a four weeks interval.


Clinical

Results: In 60 of 65 patients, an effective anti-tetanus antibody response was measured. Patients with immunosuppressive medication had a significantly lower pre- and post-titer compared to healthy controls, but their response was still effective. The titers of disease-specific antibodies were unchanged four weeks after revaccination. The clinical outcome scores showed no exacerbation of symptoms of the disease. Interpretation: A tetanus revaccination in patients with myasthenia gravis or Lambert-Eaton myasthenic syndrome is safe and induces an effective immune response, irrespectively of their immunosuppressive medication. We observed neither immunological nor clinical relevant exacerbations associated with the tetanus revaccination.

34. ARGX-113, A Novel Fc-based Approach for Antibody-induced Pathologies Such as Myasthenia Gravis

Peter Ulrichts, PhD, Antonio Guglietta, MD, PhD, Torsten Dreier, PhD, Hans de Haard, PhD, and Nicolas Leupin, MD argenx BVBA, Zwijnaarde, Belgium ARGX-113 is an antibody Fc-fragment modified using our ABDEG engineering technology. ABDEG refers to five mutations that we introduce in the Fc region that increase its affinity for FcRn at both neutral and acidic pH. FcRn is responsible for regulating half-life, tissue distribution and pharmacodynamic properties of IgG antibodies. ARGX-113 occupies FcRn with such high affinity that it deprives endogenous IgG antibodies, including pathogenic autoantibodies, of their recycling mechanism, leading to enhanced clearance of such antibodies by the lysosomes. We believe that this approach presents potential benefits relative to the current standard of care for MG. We have completed enrollment in a double-blind, placebo-controlled Phase 1 clinical trial in healthy volunteers to evaluate the safety, tolerability, pharmacokinetics, pharmacodynamics and immunogenicity of single and multiple doses of ARGX-113. Both single and multiple administration of ARGX-113 were associated with a reduction of circulating IgG levels (~50% for single dose; up to 85% for multiple dose), while no changes in IgM, IgA or albumin levels were observed. Multiple administrations of ARGX-113 at doses of 10 and 25 mg/kg were reported to be well-tolerated, with headache, feeling cold, chills and fatigue being the most frequently reported drug-related adverse events. All these adverse events were mild or moderate and were reported only in the highest dose group. Following this Phase 1 study, a Phase 2 study was recently launched to evaluate safety, efficacy and pharmacokinetics of ARGX-113 in generalized MG patients. Full Phase 1 study results and Phase 2 study setup will be discussed.

35. Myasthenia Gravis Patients Respond with Muscular Improvement to Physical Exercise

Elisabet Westerberg, MD, Carl-Johan Molin, MD, Johan Widenfalk MD, PhD, Sören Spörndly Ness, PhD, and Anna Rostedt Punga, MD, PhD Uppsala University, Uppsala, Sweden The benefits of physical exercise (PE) are valid for several diseases, e.g. cardiovascular disease, diabetes, rheumatoid arthritis and multiple sclerosis. Studies on PE in Myasthenia Gravis (MG) are sparse, but recent pilot studies indicate that MG patients can adhere to standard recommendations of PE without deterioration in disease activity. To develop MG disease specific recommendations of PE, further knowledge about physical activity patterns in MG patients is needed. In particular, the possibility of skeletal muscles to


Clinical

respond to PE need further characterization. In this study, we evaluated the muscle response to PE in 11 MG patients (six females; 60 ± 18 years) conducting a 12-week-training-program. First, baseline muscle parameters were compared to existing data from healthy adults. MG patients had significantly lower compound muscle action potential (CMAP) amplitudes for the biceps (6.6 ± 3.2 mV versus 8.8 ± 3.4 mV) and quadriceps (4.7 ± 2,4 mV versus 6.5 ± 1.8 mV) muscles and were weaker (25.2 ± 4.4 kg versus 39.8 ± 5.9 kg) in the proximal legs. After the 12-week-training-program, both functional and structural parameters improved in proximal leg muscles (quadriceps): CMAP amplitude increased 0.8 ± 0.7 mV, muscle thickness (ultrasound) increased 7.4 ± 5.0 mm, muscle resistance force improved with 5.1 ± 5.2 kg and 3 ± 3 more rises were performed on 30-second-chair-stand-test. Further, four out of five patients with pathological leg values on the MG Composite scale and the Quantitative MG score normalized their leg performance after the training regimen. We conclude that despite less favorable baseline muscle data compared to healthy individuals, MG patients have beneficial muscle response especially in proximal leg muscles.

36. Iodinated Contrast Agents in Patients with Myasthenia Gravis: a Retrospective Cohort Study Jakob Rath, MD1, Matthias Mauritz1, Gudrun Zulehner, MD1, Eva Hilger, MD1, Hakan Cetin, MD1,2, Gregor Kasprian, MD3, Eduard Auff, MD1, and Fritz Zimprich, MD, PhD1 1 Department of Neurology, Medical University of Vienna, Vienna, Austria; 2 Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, United Kingdom; 3 Department of Biomedical Imaging and Image-guided Therapy, Medical University of Vienna, Vienna, Austria Background: Currently, it has not been satisfactorily established, whether modern low-osmolality iodinated contrast agents (ICA) used in computed tomography (CT) studies are a risk factor for exacerbation of myasthenic symptoms. Methods: The rate of acute adverse events as well as delayed clinical worsening up to 30 days after performance of CT studies were analyzed in 73 patients with confirmed myasthenia gravis (MG) who underwent contrast-enhanced CT studies and compared to 52 patients who underwent unenhanced CT studies. Results: One acute adverse event was documented. 12.3% of MG-patients experienced a delayed exacerbation of symptoms after ICA administration. The rate of delayed severe exacerbation was higher in the contrast-enhanced group. Alternative causes for the exacerbation of MG-related symptoms were more likely than ICA administration itself in all cases. Discussion: ICA administration for CT studies in MG-patients should not be withheld if indicated but patients particularly those with concomitant acute diseases should be carefully monitored for exacerbation of symptoms.


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Clinical Samples

37. Altered MicroRNA Expression in Myasthenia Gravis Thymus: Possible Contribution to the Pathogenesis

Claudia Barzago, PhD1, Paola Cavalcante, PhD1, Stefania Marcuzzo, PhD1, Silvia Bonanno, MD1, Teresio Motta, MD2, Carlo Antozzi, MD1, Fulvio Baggi, PhD1, Pia Bernasconi, PhD1 and Renato Mantegazza, MD1 1 Neurological Institute "Carlo Besta", Milan, Italy; 2 Bolognini Hospital, Seriate, Bergamo, Italy MicroRNAs (miRNAs) are key gene expression regulators whose role in autoimmune diseases is rapidly emerging. MiRNAs have been implicated in innate and adaptive immune system modulation, inflammation, germinal center response and plasma cell differentiation. All these mechanisms are involved in myasthenia gravis (MG), but the contribution of miRNAs to the intra-thymic pathogenesis of the disease has never been thoroughly investigated. Recently, our research group provided evidence of a dysregulated miRNA signature in peripheral blood cells of MG patients, with miR-612, miR-3651, and miR-3654 found to be increased in MG compared to control samples. Herein, we aimed to investigate the possible involvement of these miRNAs — and miR-21, miR-150 and miR-146a known to be differentially expressed in MG peripheral blood — in the intra-thymic MG pathogenesis. We obtained data indicative of dysregulated miRNA expression in non-thymomatous MG compared to control thymuses. Interestingly, miR-146a, primarily known as a negative regulator of innate immunity, was decreased in MG thymuses where a chronic inflammation and innate immune activation are key players. Our findings suggest a contribution of miRNAs to the immunological alterations implicated in autoimmunity development or maintenance within MG thymus, paving the way for future miRNA-based therapeutic strategies.

38. Subcutaneous Immunoglobulin Therapy in Patients with Myasthenia Gravis Exacerbation: Results of a Prospective, Open-label, Phase 3 Study (SCIg-MG)

Derrick Blackmore, BSc


Clinical Samples

39. Presence of Epstein-Barr Virus in B-cells of Myasthenia Gravis Thymoma: Innocent Bystander or Autoimmunity Mediator?

Paola Cavalcante, PhD1, Stefania Marcuzzo, PhD1, Claudia Barzago, PhD1, Sara Franzi, PhD1, Barbara Galbardi, MSc1, Silvia Bonanno, MD1, Lorenzo Maggi, MD1, Teresio Motta, MD2, Carlo Antozzi, MD1, Fulvio Baggi, PhD1, Tommaso M. De Pas, MD3, Massimo Barberis, MD3, Pia Bernasconi, PhD1, and Renato Mantegazza, MD1 1 Neurological Institute “Carlo Besta”, Milan, Italy; 2 Bolognini Hospital, Seriate, Bergamo, Italy; 3 European Institute of Oncology, Milan, Italy Triggering factors of myasthenia gravis (MG) are still not completely known. Inflammation and innate immune activation by Toll-like receptors (TLRs), possibly linked to pathogen infections, are postulated mechanisms leading to intra-thymic anti-acetylcholine receptor autosensitization in non-thymomatous MG patients. Our previous studies showed Epstein-Barr virus (EBV) persistence and reactivation in hyperplastic MG thymuses, suggesting that EBV might contribute to B-cell dysregulation and autoimmunity in MG thymus. Herein, we investigated EBV involvement in thymoma-associated MG by searching for the presence of EBV nucleic acids and proteins in MG and non-MG thymomas. Detection frequency of EBV genome and latent EBV-encoded small nuclear RNA (EBER) 1 transcript was significantly higher in MG versus non-MG thymomas. Early BZLF1 and late gp350/220 lytic transcripts were undetectable in most samples, suggesting absence of (or rare) EBV reactivation in thymomas. Variable numbers of cells, mainly of B-cell phenotype, positive for EBERs and EBV latency proteins (EBNA1, LMP1, LMP2A) were frequently found in MG but not in non-MG thymoma tissues, revealing an association of EBV with MG rather than with thymoma. EBV-positive MG thymomas were characterized by the overexpression of EBV microRNAs. Moreover, they showed a positive correlation between TLR3 and EBER1 transcript levels, supporting a role for EBERs in activating TLR3 signaling in thymomas associated with MG. Our findings show that tumor-infiltrating EBV-infected B-cells are commonly present in thymoma of myasthenic patients, pointing out a possible contribution of EBV to B-cell-mediated autoimmunity in MG associated with this thymic tumor.

40. More Mature/active Dendritic Cells in the Hyperplasia Thymuses of Myasthenia Gravis

Patients

Pei Chen, MD, Hao Huang, MD, Yingkai Li, MD, Yan Li, MD, and Weibin Liu, MD.PhD The department of Neurology, the first affiliated hospital of Sun Yat-sen University, Guangzhou, Guangdong, China Dendritic cells (DCs) play an important role in the immune function achievement of the thymus. We wonder whether the DCs in the hyperplasia thymus of myasthenia gravis (MG) patient bear some special characteristics comparing with the normal thymus. MG patients’ thymuses and age-matched normal thymuses from patient undergoing corrective cardiac surgery were collected for further research. HE and Immunohistochemistry (IHC) staining and flow-cytometry are used to analyze the DCs in the thymus tissue or single cell suspension from thymus tissue. Both pDCs and major subtypes of cDCs: CD141+ and CD1c+ DC are present in human thymus. pDCs and cDCs are mainly located in the medulla area of the thymus and tend to be concentrated at the cortex and medulla junction. More CD11c+ cDCs are present in MG thymus medulla part, especially around the germinal centers (GC). But the density of pDCs (per mm2), whether in cortex or medulla part, presents no difference between normal or hyperplasia thymuses. Co-stimulators expression analysis by flow shows that MG patients’ cDCs express much higher CD80 and CD86, but the same level of CD40. Those results indicate that DCs in hyperplasia thymus are more active


Clinical Samples

or mature, and the increased mature DCs gathering in the medulla may promote the GC forming in MG patients’ thymuses.

41. Role of miR-150 in Autoimmune Myasthenia Gravis Mélanie A. Cron, MSc1, Solène Maillard, MSc1, Frédérique Truffault, MSc1, Ambra Vittoria Gualeni, PhD2, Annunziata Gloghini, PhD2, Elie Fadel, MD3, Sonia Berrih-Aknin, PhD1, and Rozen Le Panse, PhD1 1 UMRS 974 — UPMC Sorbonne Universities — INSERM — AIM, Center of Research in Myology, G.H. Pitié-Salpêtrière, Paris, France; 2 Department of Pathology and Laboratory Medicine, Istituto Nazionale dei Tumori, Milano, Italy; 3 Marie Lannelongue Hospital, Paris-Sud University, Le Plessis-Robinson, France Acquired myasthenia gravis (MG) is mainly mediated by autoantibodies against the acetylcholine receptor at the neuromuscular junction. In early-onset MG, the thymus is often characterized by B-cell infiltrations leading to ectopic germinal center (GC) development. MicroRNAs are small non-coding RNAs that regulate protein expression through direct interactions with mRNAs. They are involved in many physiological and pathophysiological processes, including autoimmune diseases. We previously demonstrated that miR-150-5p was upregulated in the serum of MG patients, and was significantly downregulated after thymectomy in correlation with disease improvement (Punga et al., 2014). Beyond its role as a biomarker, this study was aimed at investigating further the role of miR-150 in the physiopathology of MG. We showed that miR-150 was overexpressed in the thymus of MG patients with numerous GCs and decreased when patients were under corticoid treatment. The thymic levels of miR-150 were correlated with the expression of the B-cell marker CD19, and in situ hybridizations showed that thymic overexpression of miR-150 was due to its high expression in naive B-cells located in the mantle zone of GCs. We also demonstrated that high serum levels of miR-150 in MG could have a functional effect on PBMCs by acting on target genes. Indeed, in vitro treatment of PBMCs with miR-150 or an antagomiR-150 decreased or increased, respectively, the expression of one of its major target, c-Myb. Altogether, our data demonstrates that miR-150 could play a role in MG both at the thymic level and in periphery by modulating target gene expression.

42. AhR Activation May Regulate Expression of Pro-inflammatory Factors in Thymuses of AChR+ Myasthenia Gravis Patients

Rémi Khansa, MSc1, Jose Adolfo Villegas, MSc1, Rozen le Panse, PhD1, Sonia Berrih-Aknin, PhD1, and Nadine Dragin, PhD1, 2 1 Center of Research in Myology, Sorbonne Universités, UPMC INSERM UMRS 974, Institute of Myology, G.H. Pitié-Salpêtrière, Paris, France; 2 Inovarion, Paris, France Aryl hydrocarbon receptor (AhR) is known as a ligand-dependent transcription factor that mediates the effects of environmental pollutants. However, AhR, upon binding to its ligands, has also lately been shown as a regulator of T-cell subsets plasticity, and may favor inflammation leading to autoimmunity. Myasthenia gravis (MG) patients with anti-acetylcholine receptor antibodies (AChR+) exhibit in their hyperplastic thymuses a chronic inflammation driven by the secretion of pro-inflammatory cytokines such as interferon γ, TNF-α and Interleukin 17. This environment contributes to deregulate T-cell subsets. We wondered whether AhR might participate to the immune dysregulation observed in the thymus of the AChR+ MG patients. We first found that AhR is overexpressed in thymuses of AChR+ MG patients, suggesting a high


Clinical Samples

sensitization to AhR ligands in MG thymus. In vitro, we observed that AhR ligands, Ficz and dioxins, stimulate in thymic epithelial cells 1) expression of cytokines (IL-6, TGF-β) involved in T-cell subset differentiation; and 2) expression of CXCL13 and CCL21, chemokines known to be overexpressed in AChR+ MG thymuses. These data mimic the situation observed in MG thymus. In vivo, mice exposed to AhR ligands display an increased thymic expression of the cytokines and chemokines previously mentioned, and also an enhanced susceptibility to experimental autoimmune myasthenia gravis while a deficiency of AhR appears to be protective. Altogether, our data demonstrate that AhR activation may play a role in MG pathogenesis. One putative mechanism is through its ability to modulate mediators related to T- and B-cell development and trafficking in thymuses.

43. Autoantibody Capture through Barcode-based Sequencing of Single Cells in Myasthenia

Gravis Alejandro Gomez, PhD1, Julia Adamska, MSc1, Nithya Lingampalli, MSc1, Nora Goette, MD2, Claudio Mazia, MD2, and Bill Robinson, MD, PhD1 1 Division of Immunology and Rheumatology, Department of Medicine, Stanford University, Stanford, California, United States; 2 Neurology Division, Instituto de Investigaciones Médicas Alfredo Lanari, Universidad de Buenos Aires, Buenos Aires, Argentina Myasthenia gravis (MG) is characterized by autoantibodies against muscle antigens, mainly the acetylcholine receptor (AChR) and muscle-specific kinase (MuSK), which define two MG subtypes, AChR-MG and MuSK-MG, respectively. In addition, anti-AChR antibodies are found in patients with thymoma (Thy-AChR), a rare tumor of thymic epithelial cells that also presents autoantibodies against striational muscle proteins (STR-Abs), such as the sarcomeric protein titin. Currently, our understanding of the pathological mechanisms of specific autoantibodies, especially MuSK- and STR-Abs, remains poorly understood, largely because few monoclonal autoantibodies (mAbs) have been isolated from MG patients, and the vast majority of which are anti-AChR mAbs mainly derived from combinatorial libraries and hybridomas. Using a barcode-based method for sequencing single-cell immunoglobulin genes developed in our lab, we sequenced the repertoire of plasmablasts, which are antibody-producing cells derived from the activated B-cell clones in an ongoing immune response, from four AChR-MG, four MuSK-MG and two Thy-AChR patients. We then expressed 48 clonal-family derived mAbs from these patients (26 from Thy-AChR, seven from AChR-MG and 15 from MuSK-MG). Initial screenings for anti-AChR or anti-MuSK reactivity showed no positive hits in ELISAs or radioimmunoassays. However, some MuSK-MG mAbs showed mild reactivity in MuSK immunoprecipitation assays. Interestingly, two Thy-AChR mAbs reacted with the main immunogenic region of titin in ELISAs. One of such clones was present at multiple time-points in the patient, indicating recurrent activation. Our results suggest that sequencing of single-cell immunoglobulins is a powerful technique for the isolation and functional characterization of mAbs against MG autoantigens.

44. Distribution and Characteristics of Dentritic Cells Subtypes in Hyperplastic Thymus in

Patients with MG Hao Huang, MD, Pei Chen, MD, Chang-yi Ou, MD，Li Qiu, MD, Zhong-qiang Lin, MD, Zhi-dong Huang, MD, and Weibin Liu, MD, PhD The department of Neurology, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong, China To investigate the characteristics and distribution of different subtypes of dendritic cells (DCs) in thymus and their correlation with myasthenia gravis (MG), we collected thymic specimens of eight patients with thymic hyperplasia and 14 patients who had thymusectomy on account of cardiac surgery. Immunohistochemical methods were used to mark the two DC subtypes, plasmacytoid dendritic cell (pDC)


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and conventional dendritic cell (cDC), to observe the distribution of different subtype DCs in thymus. The amount and density of DC subtypes in different thymus structures were estimated with image analysis software. We marked pDCs and cDCs by staining with CD123 and CD11c. The two DC subtypes distribute in both cortex and medulla, and the majority is in the medulla. The density of pDC in thymus cortex and medulla of patients with MG is lower than normal controls. There is no significant difference between the density of cDCs in thymus cortex of patients with MG and normal people, however, cDCs increase in the medulla and the difference was statistically significant (p ＜ 0.05). The density of cDCs in the thymus germinal center of patients with MG increased significantly more than that in the thymic medulla (p ＜ 0.05). The density of cDCs in thymic medulla (except for germinal center) is higher than control group, but not statistically significant (p = 0.060). The density of cDCs increases significantly in the thymic medulla, especially focus on ectopic germinal center and around. It indicates that cDCs may play an important role in the formation and maintenance of disease of MG.

45. Two-year Outcome of Thymectomy with or without Immunosuppressive Treatment in Nonthymomatous Myasthenia Gravis and its Effect on Regulatory T-cells

Michala Jakubíková, MD, PhD1, Jiří Piťha, MD1, Helena Marečková, MD, PhD2, Michaela Týblová, MD1, Iveta Nováková, MD1, and Jan Schutzner, MD, PhD3 1 Department of Neurology and Clinical Neuroscience Center, 1st Faculty of Medicine of Charles University and General Teaching Hospital in Prague, Czech Republic; 2 Department of Immunology and Microbiology, 1st Faculty of Medicine of Charles University and General Teaching Hospital in Prague, Czech Republic; 3 Third Department of Surgery, 1st Faculty of Medicine of Charles University and University Hospital Motol, Czech Republic Background: Myasthenia gravis (MG) is an autoimmune disorder in which the thymus is considered the pathogenic organ. Thymectomy (TE) is a therapeutic option for MG and often ameliorates clinical symptoms. Methods: We evaluated clinical features and outcomes after TE in patients without thymoma and the influence of TE with or without concomitant immunotherapy on the CD4+CD25+ regulatory T-cell subpopulation of lymphocytes in peripheral blood in defined followed groups of non-thymomatous MG patients. Results: A total of 46 patients with generalized MG who underwent transsternal TE were identified. Neurologic outcomes after TE were favorable for the majority of patients mainly from the group treated with corticosteroids or combined immunosuppressive treatment. TEs with immunosuppressive treatment in MG patients were associated with increased percentages of CD4+CD25+ cells (p < 0.001). No significant change in the postoperative levels of CD4+CD25+ cells was observed in thymectomized patients who preoperatively only received pyridostigmine. Also their clinical response to TE after two years of follow-up was the worst of all followed groups. Conclusions: The exact mechanism by which TE ameliorates symptoms of MG is not clear yet. These observations indicate that increased percentages of CD4+CD25+ T-cells in MG may be related to disease stability and that TE and synergistic effect with concomitant, continuing immunotherapy augmented the proportion of CD4+CD25+ T-cells. On the basis of our observations TE alone is not enough to increase the number of circulating CD4+CD25+ regulatory T-cells and to establish complete stable remission.


Clinical Samples

46. IgG4 Autoantibodies against Muscle-specific Kinase Undergo Fab-arm Exchange in Myasthenia Gravis Patients

Inga Koneczny, DPhil1, Jo A. Stevens, PhD1, Anna de Rosa2, Saif Huda, MD PhD3, Maartje G. Huijbers, PhD4, Abhishek Saxena, PhD1, Michelangelo Maestri, MD2, Konstantinos Lazaridis, MD5,6, Paraskevi Zisimopoulou5,6, Socrates Tzartos, MD, PhD5,6, Jan Verschuuren, MD, PhD4, Silvère M van der Maarel, PhD4, Philip van Damme, MD6,7, Angela Vincent, FRCPath, FRS, PhD3, Marc H de Baets, MD, PhD1, Peter C Molenaar, PhD1, Roberta Ricciardi, MD2, Pilar Martinez-Martinez, PhD1, and Mario Losen, PhD1 1 Department of Psychiatry and Neuropsychology, School for Mental Health and Neuroscience, Maastricht University, Universiteitssingel 50, 6229 ER Maastricht, the Netherlands; 2 Department of Clinical and Experimental Medicine, Neurology Unit, University of Pisa, Pisa, Italy; 3 Neurology Department, Nuffield Department of Clinical Neurosciences, John Radcliffe Hospital, Oxford, United Kingdom; 4 Department of Neurology, Leiden University Medical Center, Leiden, the Netherlands; 5 Department of Neurobiology, Hellenic Pasteur Institute, Ampelokipi, Athens, Greece; 6 Neurology Department, University Hospital, Leuven, Belgium; 7 KU Leuven — University of Leuven, Department of Neurosciences, VIB — Vesalius Research Center, Experimental Neurology — Laboratory of Neurobiology, Leuven, Belgium Myasthenia gravis (MG) with antibodies to muscle-specific kinase (MuSK-MG) is hallmarked by IgG4 antibody against MuSK that inhibit a pathway that clusters the acetylcholine (neurotransmitter) receptors and leads to failure of neuromuscular transmission. A key feature of IgG4 antibodies is the ability to exchange Fab-arms with other, unrelated, IgG4 molecules, making the IgG4 molecule potentially monovalent for the specific antigen. Whether pathogenic MuSK IgG4 is mono- or divalent for their antigen is unknown. In vitro Fab-arm exchange-inducing conditions were applied to MuSK antibodies in sera, purified IgG4 and IgG1-3 sub-fractions. Solid-phase cross-linking assays were established to determine the extent of pre-existing and inducible Fab-arm exchange. Functional effects of the resulting populations of IgG4 antibodies were determined by measuring inhibition of agrin-induced AChR clustering in C2C12 cells. To confirm the results, κ/κ, λ /λ and hybrid κ/λ IgG4s were isolated and tested for MuSK antibodies. At least 50 percent of patients had IgG4, but not IgG1-3 MuSK antibodies that could undergo Fab-arm exchange in vitro under reducing conditions, but there were also MuSK antibodies in vivo that were divalent (monospecific for MuSK). Fab-arm exchange with normal human IgG4 did not prevent the inhibitory effect of serum derived MuSK antibodies on AChR clustering in C2C12 mouse myotubes. The results suggested that a considerable proportion of MuSK IgG4 could already be Fab-arm exchanged in vivo, and this was confirmed by isolating endogenous IgG4 MuSK antibodies containing both κ and λ light chains. These new findings demonstrate pathogenicity of Fab-arm exchanged antibodies.


Clinical Samples

47. Identifying the B Cell Subsets that Contribute to Autoantibody Production in Myasthenia Gravis

Panos Stathopoulos*, MD, PhD, Aditya Kumar*, MD, Richard J. Nowak, MD, and Kevin C. O’Connor, PhD Department of Neurology, Yale School of Medicine, New Haven, Connecticut, United States * Equal contributions Rituximab (anti-CD20) is increasingly used in the treatment of autoimmune MG with encouraging results. However, cellular mechanisms underlying autoantibody production and rituximab mechanism of action are not well-defined. Our proposed model for MG cellular immunopathology includes a population of autoantigen-specific CD20+ memory B cells that supplies a largely CD20- pool of autoantibody-secreting cells. Specifically, that AChR autoantibodies are mainly produced by long-lived plasma cells and, to a lesser degree, by short-lived plasmablasts, while MuSK autoantibodies being predominantly produced by plasmablasts. To test this model, we studied peripheral blood B cells from myasthenics pre- and post-rituximab treatment, and from controls. In AChR MG, cultured memory B cells produced autoantibodies that bound AChR. Although the plasmablast compartment was enlarged in a subset of AChR MG patients, AChR autoantibody production by this population was not detected. These results suggest that memory cells and long-lived plasma cells contribute to AChR autoantibody production and are in line with persistence of AChR titers post-rituximab. In MuSK MG, where rituximab dramatically decreases MuSK titers, plasmablasts were identified by two complementary approaches as MuSK autoantibody producers during post-rituximab disease relapse. We suggest that rituximab indirectly depletes short-lived MuSK autoantibody producers by eliminating their CD20+ precursors. In post-rituximab MuSK relapse, these plasmablasts re-emerge and contribute to production of MuSK autoantibodies and the associated disease relapse. Our findings identify candidate biomarkers and introduce potential mechanisms for autoantibody production as well as rituximab action in both AChR and MuSK MG.

48. Serum Acetylcholine-receptor Antibody Concentrations in 71 Patients Included in the Randomized Trial of Thymectomy in Myasthenia Gravis (MGTx)

M Isabel Leite1, Leslie Jacobson1, Camilla Buckley1, Gil I Wolfe2, Inmaculada B Aban2, Greg Minisman2, Hui-Chien Kuo3, David Hilton-Jones1, Gary R Cutter3, Henry J. Kaminski4, Angela Vincent1 1 Nuffield Department of Clinical Neurosciences and Department of Clinical Neurology, John Radcliffe Hospital, Oxford OX3 9DU, UK; 2 Dept of Neurology, JSMBS, 100 High St Buffalo, NY; 3 Department of Biostatistics, University of Alabama School of Public Health, Birmingham, Alabama; 4 Department of Neurology, George Washington University, Washington DC. Background: The multicenter, randomized trial comparing extended transsternal thymectomy plus prednisone (PredTx) to prednisone alone (Pred) showed that thymectomy improved clinical outcomes over a 3-year period (Wolfe et al JNEM 2016). We tested patients’ sera for acetylcholine-receptor antibodies (AChR-Abs) aiming to analyse whether there were associated changes in AChR-Ab concentrations. Methods: Four serum samples (baseline, 12, 24 and 36 months) per patient were tested by radioimmunoassay, using appropriate dilutions of each serum. The AChR-Ab levels in follow-up samples were expressed as a percentage of the baseline value for each patient over the 36 months of the trial. Not all samples were available and these preliminary results will be presented for 35 PredTx and 36 Pred patients. Results: Baseline QMG scores were not significantly different between PredTx and Pred patients (11.3 and 12.1), but at the final follow-up (36 months) mean QMG scores were lower in both groups compared


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to baseline and lower in the PredTx patients (4.5) than in the Pred (8.00; p=0.003) group as expected from the trial results. In this incomplete cohort, the doses of prednisone at final follow-up were not different between the groups. AChR-Ab levels fell 20% and 7% in PredTx and Pred groups, respectively, at 12 months, but by 36 months the Pred levels had increased slightly to 98% whereas the PredTx levels were 23% below baseline. Conclusions: These preliminary results need confirmation and, if possible, the remaining 55 samples will be included. Nevertheless, it is interesting that in this large subpopulation, with both groups improving clinically, at 36 months only the PredTx AChR-Ab levels were reduced from baseline levels.

49. Skeletal Muscle Regeneration is Impaired in Response to Acute Injury in Myasthenia Gravis

Mohamed Attia, PhD1,2, Marie Maurer, PhD1,2, Marieke Robinet, ES1,2,3, Fabien Le Grand, PhD1,2,3, Elie Fadel, MD4, Rozen Le Panse, PhD1,2, Gillian Butler-Browne, PhD1,2,3, and Sonia Berrih-Aknin PhD1,2 1 UPMC Paris 6 University, School of Medicine, Paris, France; 2 INSERM U974 Mixed Research Unit, Paris, France; 3 AIM, Institute of Myology, Pitié-Salpêtrière Hospital, Paris, France; 4 INSERM U999 Mixed Research Unit, Marie Lannelongue Centre, Le Plessis-Robinson, France Myasthenia gravis (MG) is a neuromuscular disease caused by anti-acetylcholine receptor (AChR) autoantibodies that impair neuromuscular signal transmission thus affecting skeletal muscle homeostasis. Myogenesis is carried out by muscle stem cells called satellite cells (SCs). However, myogenesis in MG is still unexplored. The aim of this study was to characterize the functional properties of myasthenic SCs as their role in muscle regeneration. The analysis of human muscle biopsies revealed that MG muscles displayed more SCs with higher proliferative rate than age-matched controls, as assessed by higher mRNA expression of MyoD and Ki67. Similar results were observed in the experimental autoimmune myasthenia gravis (EAMG) mouse model. To explore the potential functional consequences of the increased SC number, the muscle of EAMG and CFA-control mice were injured by cardiotoxin, and muscle regeneration was analyzed. We observed that EAMG muscles displayed a maturation delay by a decrease in fiber size and MyoG mRNA expression as well as an increase of myofibers with central nuclei, fiber number and embryonic myosin heavy-chain (embMyHC) mRNA expression, highlighting the alteration of molecular pathways associated with muscle regeneration. Altogether, our findings show for the first time functional differences between SCs from control and MG muscles. We underlined that regeneration process in MG could be impaired after muscle injury. The observed differences could be due to anti-AChR antibodies via the modulation of myogenic markers. In conclusion, the autoimmune attack in MG results in unsuspected functional changes in SCs signaling pathways and muscle regeneration.


Clinical Samples

50. Targeting Plasma Cells with Proteasome Inhibitors for Treatment of Myasthenia Gravis

Marina Mané-Damas, MSc1, Abhishek Saxena, PhD1, Gisela Nogales-Gadea, PhD2, Maarten van Beek, MSc1, Nienke Van Den Hoogen, MSc1, Peter Molenaar, PhD1, Bert Joosten, PhD1, Nick Willcox, PhD3, Pilar Martinez-Martinez, PhD1, and Mario Losen, PhD1

1 Department of Psychiatry and Neuropsychology, School for Mental Health and Neuroscience, Maastricht University, Maastricht, the Netherlands; 2 Translational Research Laboratory in Neuromuscular Diseases, Neurosciences Department, Germans Trias i Pujol Research Institute and Campus, Barcelona, Spain; 3 Nuffield Department of Clinical Neurosciences, Weatherall Institute for Molecular Medicine, University of Oxford, Oxford, United Kingdom

Autoantibodies against the muscle AChR are mainly produced by both short‐ and long‐lived plasma cells, which are resistant to standard immunosuppressive drugs (e.g., glucocorticoids). A novel therapy to eliminate plasma cells is the proteasome inhibitor bortezomib, which is used to treat patients with multiple myeloma (MM, a plasma cell malignancy). Previously, we demonstrated that bortezomib also reduced autoantibody titers in an animal model of MG (Gomez, A. M. J. Immunol. 2011). The thymus of MG patients is frequently enriched in germinal centers and contains plasma cells that produce autoantibodies in vitro, even after irradiation (which depletes B and T lymphocytes). We studied the in vitro effects of bortezomib in cultured thymus cells from MG patients undergoing therapeutic thymectomy. Treatment with a single dose of bortezomib eliminated plasma cells and thereby blocked the production of IgG, including pathogenic autoantibodies. Ultrastructural signs of apoptosis were detected in plasma cells as early as 8 h after addition of bortezomib; at 24 h, no plasma cells could be detected (Gomez, A. M. J.Immunol. 2014). Finally, we are currently testing in vitro and in vivo second‐generation proteasome inhibitors efficient in eliminating autoreactive plasma cells with special focus in investigating their side effects such as peripheral neuropathy.

51. Agrin Antibodies in Myasthenia Gravis — What Is Their Clinical Relevance?

Angelina H. Maniaol, MD, PhD1,2, Chantal ME. Tallaksen MD, PhD1, Judith Cossins, DPhil2, Mark Woodhall, PhD2, David Beeson, MD, PhD2, and Angela Vincent FRCPath2 1 Department of Neurology, Oslo University Hospital, Oslo, Norway 2 Nuffield Department of Clinical Neurosciences, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom Previous studies have detected a various proportion of agrin antibodies (Abs) in sera of myasthenia gravis (MG) patients, but evidence of their pathogenicity and clinical implication remain unclear. We established an agrin-Ab assay and determined the associated clinical features in a large MG cohort. Sera from 85 Norwegian MG patients, of whom 65 were AChR and MuSK Ab negative (double seronegative), were tested on a cell-based assay (CBA) for binding to full-length agrin (AGRN) using an Fc gamma specific anti-human IgG as a secondary antibody to avoid detection of non-specifically bound IgM. Pre-adsorption studies of positive sera were conducted to determine its specificity to agrin. We detected agrin-Abs in sera from ten MG patients (11.8 %); but only five had agrin-Abs alone, while the remaining five also had abs to AChRs and one to low-density lipoprotein receptor-related protein 4 (LRP4). The clinical characteristics were an equal male-female ratio, age of onset below 60 years and predominantly generalised weakness at onset. Two had ocular MG. Interestingly, however, the majority of cases had long disease duration and were clinically not very weak at the time samples were collected. Agrin-Ab positive MG patients were early age at onset and had long disease duration. For future studies it will be important to determine if agrin-Abs are present at onset in patients with double seronegative MG, or whether they tend to appear later during the disease when the patient might already be improved.


Clinical Samples

52. Reduction of Extracellular MiR-150-5p Levels Accompanies Improved Disease Severity in Myasthenia Gravis Patients 24 Months Post-thymectomy

Liis Sabre, MD, PhD1,2, Carl Johan Molin, MD1, and Anna Rostedt Punga, MD, PhD1 1 Uppsala University, Uppsala, Sweden; 2 University of Tartu, Tartu, Estonia The circulating microRNAs (miRs) miR-150-5p and miR-21-5p have a disease specific signature in myasthenia gravis (MG), in which thymus is considered to play an important role. Since the recent randomized trial provided evidence on positive outcome post-thymectomy in MG patients (1), we analyzed circulating microRNAs in sera of MG patients before and after thymectomy. We analyzed serum samples from 40 patients (31 women) who underwent extended transsternal thymectomy and received prednisone at enrollment (baseline), and then 12, 24 and 36 months after thymectomy. Both miR-150-5p and miR-21-5p were normalized to reference miR-191-5p. Paired t-test and Wilcoxon signed rank test were used. Patients aged 18 to 63 (mean 35.8 ± 14.1) years had generalized MG with disease duration <5 years at enrollment. The prednisone dose was maintained until minimal-manifestation status was obtained, with maximal dose 6 months after thymectomy. Mean levels of miR-150-5p were significantly reduced at 24 months after thymectomy (5.1 ± 1.1) compared to baseline (6.2 ± 1.3; p<0.0001) whereas miR-21-5p levels were not. A similar pattern of improved clinical disease activity by reduced quantitative MG (QMG) score levels was seen at 24 months after thymectomy (median: 4 points) compared to baseline (median: 11 points; p<0.0001). Our study indicates longitudinal reduction of miR-150-5p in MG patients 24 months after thymectomy, which accompanies a significant reduction in clinical QMG score. This adds novel insights into the effects of thymectomy on circulating disease specific miRNAs and further supports the role of miR-150-5p as a potential biomarker for the future in MG. Reference: Wolfe GI, Kaminski HJ, Aban IB, Minisman G, Kuo HC, Marx A, et al. Randomized Trial of Thymectomy in Myasthenia Gravis. The New England Journal of Medicine. 2016;375(6):511-22. Acknowledgements: The work was done with the support of NIH grant U01 NS 42685 and the MGTX study group.

53. MicroRNA and M-RNA Expression Study in Ectopic Germinal Centers of Myasthenia Gravis Thymus

Manjistha Sengupta, PhD1, Bi-Dar Wang, PhD1, Norman Lee, PhD1, Alex Marx, PhD2, Linda Kusner, PhD1, and Henry Kaminski, MD1 1 George Washington University, Washington, District of Columbia, United States; 2 University Medical Centre Mannheim, University of Heidelberg, Mannheim, Germany A characteristic pathology of myasthenia gravis (MG) is thymic hyperplasia with ectopic germinal centers (GC). However, the mechanisms that trigger and maintain thymic hyperplasia are poorly characterized. Dysregulation of microRNAs (miRNAs) and their target genes has been identified in the pathology of several autoimmune diseases. Here we assessed the miRNA and mRNA profiles of the MG thymus and have investigated their role in GC formation and maintenance. MG thymus samples were assessed by histology and grouped based upon the appearance of GC; GC positive and GC negative. Thirty-four mature miRNAs and forty-eight annotated mRNA transcripts were differentially expressed between the two groups (>1.5 fold change, ANOVA p<0.05). The miRNAs identified are involved in immune response pathways, other autoimmune diseases and cancer pathways. The cellular and molecular functions of the mRNAs involve cell death and cell survival, cellular proliferation, cytokine


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signaling and extra-cellular matrix reorganization. Seven mi-RNA and mRNA pairs are reciprocally regulated. The Regulator of G protein Signalling 13 (RGS13), known to be involved in GC regulation, was identified in specimens with GC and was paired with downregulation of miR-452-5p and miR-139-3p.The interactions were validated in RGS13 expressing Raji cell line. Our study indicates a distinct miRNA and mRNA expression pattern in ectopic GC in MG thymus. These miRNAs and mRNAs are involved in regulatory pathways common to inflammation and immune response, cell cycle regulation and anti-apoptotic pathways suggesting their involvement in support of GC formation in the thymus.

54. Serum Creatine Kinase Levels in Myasthenia Gravis Kamal Shouman, MD and Robert P. Lisak, MD Wayne State University School of Medicine, Detroit, Michigan, United States Serum creatine kinase (CK) is frequently tested in the basic metabolic profile and in evaluation of patients with motor weakness. We were interested to see if CK elevations were found in patients with myasthenia gravis (MG) and if so were there correlations with severity of disease, particularly at the time of elevated CK, medications, including for other disorders, MG-associated antibody status, thymoma and/or other associated diseases. We reviewed records of 91 patients with MG, CK was tested on one or more occasions on 74 patients and elevations of CK detected in 10. One patient had a benign thymoma, one had thymic hyperplasia and 3 had thymectomies with pathology not available. Four were on statins at the time of CK elevation. In two statins were discontinued but elevation persisted after discontinuation. One patient had hypokalemia and reaction to a medication. Serum CK returned to normal when the problem was resolved. Three had hypothyroidism adequately treated in two. One patient had concomitant rippling muscle disease, seronegative for VGKC antibodies and normal CT of the chest. One had idiopathic necrotizing myopathy at biopsy. There was no obvious association with MG severity including at the time of elevated CK. One patient had muscle specific kinase (MuSK) antibodies, one was double seronegative and the others had acetylcholine receptor (AChR) antibodies. Although several patients had no other explanation for elevated CK, the presence of an elevated CK should lead to consideration of a concomitant disorder when seen in patients with proven MG.

55. Impaired Regulatory B cells in Myasthenia Gravis

Jian Rong Sheng, MD, PhD, Kourosh Rezania, MD, and Betty Soliven, MD Department of Neurology, The University of Chicago, Chicago, Illinois, United States Autoimmune diseases such as myasthenia gravis (MG) are often precipitated by impairment in immunoregulatory mechanisms. B cells play a central role in MG by producing pathogenic autoantibodies, but some B cell subsets such as IL-10 producing B cells are capable of suppressing immune responses. We have previously shown that regulatory B cells (Bregs) attenuate the severity of experimental autoimmune myasthenia gravis (EAMG) in an interleukin-10 (IL-10)-dependent manner. The goal of this study was to investigate the role of human Bregs in seropositive MG focusing on CD19+CD1dhi CD5+ and CD19+CD24hiCD38hi subsets. Peripheral blood samples were obtained from 18 patients (10 males, 8 females) with clinically definite MG (aged 18 to 86) and from 13 healthy controls aged 18 to 80. We found that MG patients exhibited a decrease in the frequency of both Breg subsets and IL-10 producing B cells within each subset, which correlated with disease severity. In addition, there was impaired suppression of Th1 polarization in MG. There was no correlation between serum AChR antibody levels and % Bregs. These findings, taken together with EAMG data, indicate that Bregs play an important role in regulating the severity of MG.


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56. Altered Number and Functionality of Regulatory B Cells in Myasthenia Gravis Patients Vuslat Yilmaz, PhD1, Solène Maillard, MSc2, Frédérique Truffault, MSc2, Bruno Eymard, MD3, Jean-François Regnard, MD4, Sonia Berrih-Aknin, PhD2, and Rozen Le Panse, PhD2 1 Department of Neuroscience, Aziz Sancar Institute of Experimental Medical Research (DETAE), Istanbul University, Istanbul, Turkey; 2 UMRS 974 - UPMC Sorbonne Universities - INSERM - AIM, Center of Research in Myology, Paris, France; 3 Neuromuscular Disease Centre, AIM, Pitié - Salpêtrière Hospital, APHP, Paris, France; 4 Department of Thoracic Surgery, Cochin Hospital of Paris, APHP, René Descartes University, Paris, France To date, B cells have been considered to play a pathogenic role in myasthenia gravis (MG) due to their ability to produce autoantibodies targeting mainly the acetylcholine receptor (AChR). However, a small percentage of B cells possess regulatory properties (Breg cells). We investigated the proportion and function of Breg cells in AChR+ MG patients and analyzed the influence of MG treatments. PBMCs were analyzed for CD19+CD24++CD38++ Breg cells by flow cytometry. We observed a significant decrease of Breg cells in MG patients. Analyzing patients before and after thymectomy, we showed a restoration of the proportion of Breg cells after thymectomy. In parallel, we observed the presence of Breg cells in thymic germinal centers. In contrast, in MG patients treated with corticosteroids, a more severe decrease of Breg cells was observed. Similarly dexamethasone reduced in vitro the number of Breg cells. Breg cell function is based on their ability to produce IL-10. Upon PBMCs stimulation with CpG or CpG/CD40, a significant increase of CD19+IL-10+ cells and of the mean fluorescence intensity (MFI) for IL-10 was observed with control PBMCs. Using PBMCs from MG patients, the increase of CD19+IL-10+ cells was still observed but to a lesser extent and was not associated with an increase in IL-10 MFI. In this study, we clearly demonstrated a decrease in the proportion and function of Breg cells in MG suggesting altered immunomodulatory mechanisms. If thymectomy restores the percentage of Breg cells, the effect of corticosteroids seemed detrimental for Breg cells despite their well-known anti-inflammatory properties.


Congenital Myasthenia Syndromes

Congenital Myasthenia Syndromes 57. Mutation in a Nuclear Envelope Protein Causes a Novel Congenital Myasthenic Syndrome

Judith Cossins, DPhil1, Pedro Rodriguez Cruz, MD1,2, Susan Maxwell, MSc1, Gareth Llewelyn, MD3, Robin Kennett, MD4, Ravi Knight, MD4, Simon McGowan, PhD1, Jacqueline Palace, MD2, and David Beeson, PhD1 1 Neurosciences Group, Nuffield Department of Clinical Neurosciences, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom; 2 Nuffield Department of Clinical Neurosciences, John Radcliffe Hospital, Oxford, United Kingdom; 3 Department of Neurology, University Hospital of Wales, Heath Park, Cardiff, United Kingdom; 4 Neurosciences, John Radcliffe Hospital, Oxford, United Kingdom Congenital myasthenic syndromes are a heterogeneous group of inherited disorders in which neuromuscular transmission is compromised, leading to fatigable muscle weakness. More than 25 genes have been implicated in the disorders. Using whole-exome sequencing on an affected individual with no known causative mutation, we identified a homozygous frameshift mutation within a gene which encodes an inner-nuclear membrane protein. A sibling, previously undiagnosed, was found to have the same homozygous mutation and was also affected, thus demonstrating mutation segregation with disease. Both affected individuals had normal early developmental milestone and the index case presented at 7 years. Clinical features include proximal weakness, with mild distal weakness in finger extension and ankle dorsiflexion. Mild wasting of shoulder-girdle and medial gastrocnemius was also noted. Serum creatine kinase was normal. A muscle biopsy gave no specific diagnostic indicators. Repetitive nerve stimulation in anconeous muscle showed 40% decrement, and single fibre EMG in extensor digitorum communis was abnormal (36% increased jitter; 64% blocking). Both siblings show a positive response to pyridostigmine. The frameshift mutation is located in exon 1, and the protein is not detected in the index case’s muscle nuclei. The function and pathogenic mechanism of the gene is unknown, but we hypothesised that it may indirectly regulate acetylcholine receptor (AChR) cell surface expression. To investigate this, the gene was knocked out in C2C12 myotubes using CRISPR/Cas9 technology. AChR cell surface expression was quantified using 125I-alpha-bungarotoxin, but was unchanged in the knockout myotubes compared with controls. Other possible pathogenic molecular mechanisms are being investigated.

58. Congenital Myasthenic Syndromes Due to Mutations in Agrin Wei Wei Liu, PhD1, Susan Maxwell, MSc1, Katsiaryna Belaya, PhD1, Judith Cossins, PhD1, Sarah Finlayson, MD1, Heinz Jungbluth, MD3, Jacqueline Palace, DM2, and David Beeson, PhD1 1 Neurosciences Group, Nuffield Department of Clinical Neurosciences, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, United Kingdom; 2 Department of Neurology, John Radcliffe Hospital, Oxford, United Kingdom; 3 Evelina London Children’s Hospital, St. Thomas’ Hospital, Westminster Bridge Road, London, United Kingdom Congenital myasthenic syndromes (CMS) are genetic disorders that affect neuromuscular transmission. Agrin, released by the motor neurons, stimulates the rapid phosphorylation of MuSK (Muscle Specific Kinase), leading to MuSK activation and acetylcholine receptor (AChR) clustering on the muscle under the nerve terminal. We report two CMS kinships in which respective homozygous missense variants, c.5300G>A, p.(Gly1767Asp) and c.5722T>C, p.(Trp1908Arg) in AGRN were identified. Full length Agrin harbouring these variants was expressed in HEK293 cells and then used to induce AChR cluster formation in differentiated C2C12 myotubes. The expression levels of p.(Gly1767Asp) in HEK293 cells was similar compared to wild type, but p.(Trp1908Arg) was markedly reduced. Using purified agrin in functional assays, activity of both p.(Gly1767Asp) and p.(Trp1908Arg) mutations caused the number of AChR clusters induced in cultured C2C12 myotubes to be significantly reduced compared to wild type. Time course analysis



showed a significant difference in the rate of degradation of agrin containing the p.Trp1908Arg mutation, which appears markedly less stable than wild type or p.(Gly1767Asp) agrin. By contrast, although stable the p.(Gly1767Asp) agrin led to a pronounced reduction in MuSK phosphorylation that was not seen for p.(Trp1908Arg). Thus, it is likely that these two cases of AGRN-CMS are caused by differing underlying molecular mechanisms, p.(Gly1767Asp) through loss of functional activity and p.(Trp1908Arg) through loss of intrinsic protein stability. It is of note that the two individuals homozygous for p.(Trp1908Arg) have a relatively mild phenotype, whereas the case homozygous for p.(Gly1767Asp) is severely affected.

59. Pathogenic Mechanisms of RAPSN Mutations in Congenital Myasthenic Syndromes

Jonathan Cheung, PhD1, Mirela D. Panea, MSc, Richard Webster, PhD Judith Cossins, PhD, David Beeson, PhD Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, United Kingdom Rapsyn (RAPSN) is essential for clustering acetylcholine receptors (AChR) at the neuromuscular junction (NMJ). Congenital myasthenic syndrome (CMS) due to RAPSN mutations cause a deficiency of AChR at the NMJ, and are characterized by acute episodes of respiratory distress in infancy that tend to diminish with age. RAPSN-CMS is autosomal recessive and most patients harbor mutation p.Asn88Lys. Mutations in the RAPSN-gene promoter region are also relatively common. Here we investigate the molecular mechanisms that underlie impaired function in affected individuals with RAPSN-CMS who do not harbor either c.264C>A, p.(Asn88Lys), or a RAPSN promoter mutation. Rapsn expression in the C2C12 mouse muscle cell line was ‘knocked out’ using CRISPR/Cas9 technology. RAPSN harboring respective mutations was re-introduced using retroviral expression systems. Cells were differentiated to form myotubes and AChR clusters formed following incubation with human AGRN. AChR cluster number and size, AChR cell surface expression, RAPSN expression and RAPSN stability were determined. RAPSN mutations c.133G>A, p.(Val45Met), c.272G>T, p.(Arg91Leu) and c.457G>A, p.(Ala153Thr) were identified in affected individuals either as homozygotes or in combination with a null allele (truncation). These mutations were studied and compared to either wild type or c.264C>A, p.(Asn88Lys) RAPSN. All mutations reduced the number and area of AChR clusters formed compared to wild type. RAPSN containing p.(Val45Met) is rapidly degraded, whereas p.(Arg91Leu) and p.(Ala153Thr) markedly increased AChR cluster disassembly. Due to the phenotypic variability of RAPSN-CMS we are not in a position to correlate the genotype with phenotype but with these assays can define if particular RAPSN variants are pathogenic.



60. Mutations in GMPPB Can Combine a Myasthenic Syndrome with a Myopathy Pedro M Rodríguez Cruz, MD, MSc1,2, Katsiaryna Belaya, DPhil2, Wei Wei Liu, PhD2, Susan Maxwell, BSc2, Maria Farrugia, MD, DPhil3, Richard Petty, MD3, Timothy J. Walls, MD4, Maryam Sedghi, MSc5, Keivan Basiri, MD6, Janice L Holton, MD7, Robin Kennett, MD1, Francesco Muntoni, MD8, Jacqueline Palace, MD, DM1, and David Beeson, PhD1 1 Nuffield Department of Clinical Neurosciences, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom; 2 Nuffield Department of Clinical Neurosciences, John Radcliffe Hospital, Oxford, United Kingdom; 3 Department of Neurology, Institute of Neurological Sciences, Southern General Hospital, Glasgow, United Kingdom; 4 Department of Neurology, Royal Victoria Infirmary, Newcastle upon Tyne, United Kingdom; 5 Medical Genetics Laboratory, Alzahra University Hospital, Isfahan University of Medical Sciences, Isfahan, Iran; 6 Neurology Department, Neuroscience Research Centre, Isfahan University of Medical Sciences, Iran; 7 Department of Molecular Neurosciences, UCL Institute of Neurology, London, United Kingdom; 8 Dubowitz Neuromuscular Centre & MRC Centre for Neuromuscular Diseases, UCL Institute of Child Health, London, United Kingdom Congenital myasthenic syndromes (CMS) are disorders caused by mutations in genes encoding proteins that are essential for maintaining the integrity of neuromuscular transmission. In recent years we have identified a connection between glycosylation defects and CMS by using next generation sequencing. Here, we review CMS patients with mutations in the GDP-mannose pyrophosphorylase B (GMPPB) to identify the associated clinical, neurophysiological, pathological and laboratory features. Presentation was delayed beyond infancy with proximal muscle weakness and minimal or absent craniobulbar manifestations such as ptosis, ophtalmoplegia or facial weakness. Electrophysiological testing showed selective abnormalities of neuromuscular transmission predominantly in the affected muscles, and myopathic changes. Muscle biopsy showed dystrophic features and reduced labeling for of α-dystroglycan glycosylation. In addition, myopathic changes were present on muscle MRI. Serum CK was significantly increased compared to other CMS subtypes within the Oxford CMS cohort. Patients were responsive to pyridostigimine alone or combined with 3,4-diaminopyridine and/or salbutamol. Patients with GMPPB-CMS have a similar phenotype to other CMS subtypes harboring mutations within the early stages of the N-glycosylation pathway. Additional characteristics shared with the dystroglycanopathies include myopathic features and raised CK levels. This confirms that CMS do occur in the absence of classic myasthenic manifestations such as ptosis, ophthalmoplegia or facial weakness, and links myasthenic disorders with dystroglycanopathies. These clinical features make them easily misdiagnosed as myopathies or muscular dystrophies. The recognition of these disorders is important since they can be treated symptomatically with cholinesterase inhibitors.

61. Novel Synaptobrevin-1 Mutation Causes Fatal Congenital Myasthenic Syndrome

Xin-Ming Shen, PhD1, Rosana H. Scola, MD2, Paulo J Lorenzoni, MD2, Cláudia S.K. Kay, MD2, Lineu C. Werneck, MD2, Joan Brengman, BS1, Duygu Selcen, MD1, and Andrew G. Engel, MD1 1 Mayo Clinic, Rochester, Minnesota, United States; 2 Hospital de Clínicas, (UFPR), Curitiba, Brazil The proband born to consanguineous parents was severely hypotonic, areflexic, and weak since birth. By age 10 years, she had spine deformities and limited eye movement but sat up unaided. Her speech was slurred but she could swallow. She did not respond to pyridostigmine, was worsened by respiratory infections, and died at age 14 of respiratory failure. EMG studies showed a decremental response at 2 Hz stimulation and several fold potentiation of the evoked muscle action potential on high frequency nerve stimulation pointing to a presynaptic defect. Exome sequencing identified a homozygous c.340delA frameshift mutation in synaptobrevin 1 (SYB1), one of the three SNARE proteins essential for synaptic



vesicle exocytosis. Analysis of both human spinal cord gray matter and normal human muscle revealed expression of the SYB1A and SYB1D isoforms, predicting expression of one or both isoforms in the motor nerve terminal. The identified mutation elongates the intravesicular C- terminus of the A isoform from 5 to 71, and of the D isoform from 4 to 31, residues. Transfection of either mutant isoform into bovine chromaffin cells markedly reduced depolarization-evoked exocytosis, and transfection of either mutant isoform into HEK cells significantly decreased expression of either mutant compared to wild-type. The mutation is pathogenic because elongation of the intravesicular C-terminus of SYB1 of the A and D isoforms increases the energy required to move their C-terminus into the synaptic vesicle membrane, a key step for fusion of the synaptic vesicle with the presynaptic membrane, and because it is predicted to reduce expression of either isoform in the nerve terminal.


Management Diagnostic

Management Diagnostic 62. Exploring Outcomes and Characteristics of Myasthenia Gravis: Rationale, Aims and Design

of Registry — The EXPLORE MG Registry Rahul Anil, MD, Aditya Kumar, MD, Sneha Alaparthi, BS, Joan Nye, BS, and Richard J. Nowak, MD, MS

Program in Clinical and Translational Neuromuscular Research, Department of Neurology, Yale University School of Medicine, New Haven, Connecticut, United States Myasthenia gravis (MG) is a prototypical autoimmune disease with an estimated prevalence of 40-180 per million people worldwide. Though much information exists about the diagnosis, treatment, and epidemiology, a comprehensive data registry is critical to better understand disease mechanisms, treatment outcomes, and the impact of targeted treatment strategies. We aimed to conduct a detailed study of such factors among myasthenics followed at our center by creating the “Exploring Outcomes and Characteristics of Myasthenia Gravis (EXPLORE MG) Registry.” This retrospective and prospective registry will record data on clinical characteristics, diagnostic tools, management strategies and their outcomes, comorbidities, and health care utilization. We have collected data through February 28, 2017, and have completed an interim analysis on 232 MG patients (mean age 60 years, range 17-99; Female:Male, 1.03:1) comprising 166 with antibodies against acetylcholine receptor (AChR), 20 with antibodies against muscle-specific kinase (MuSK), and 46 seronegative patients. This cohort consists of 64 patients with ocular disease, 168 patients with generalized disease, and 64 patients post-thymectomy, including 20 with thymoma. Additionally, we have the unique opportunity to study a large cohort of refractory MG patients. This registry is strengthened by a complementary biorepository. Identification of key clinical features that may predict responsiveness to treatment or provide additional insight into patient outcomes is essential to improved patient care. As current research focuses on the development of patient-tailored, targeted-treatment regimens, this registry can help provide important clinical/epidemiological data from a large modern patient cohort with long-term follow-up. We anticipate launching this registry nationally, soon.

63. Cognitive Impairment in Myasthenia Gravis with MuSK Antibodies Paolo E. Alboini, MD, Camillo Marra, MD, and Amelia Evoli, MD Institute of Neurology, Catholic University, Rome, Italy Cholinergic pathways in central nervous system (CNS) are crucial for memory and learning. MuSK is expressed in the hippocampus and it is thought to be involved in synaptic plasticity and memory formation. We evaluated cognitive performances in MuSK-MG patients in comparison with age- and gender-matched AChR-MG patients and healthy controls. Sixty-eight subjects (26 MuSK-MG, 21 AChR-MG patients and 21 controls) underwent an extensive neuropsychological test battery including the Mini Mental State Examination and an extended version of the Mental Deterioration Battery, comprehensive of Rey’s Auditory Verbal Learning Test, visual and digit span backward-forward, Multiple Features Targets Cancellation, Copying Drawings free and joining landmarks, phonological and semantic word fluency, naming nouns and verbs, Raven’s Colored Matrices, Stroop test, trail making test and Free and Cued Selective Remanding Test. Subjects with psychiatric illnesses or comorbidities that may affect neuropsychological performances were excluded. Statistical analysis was performed by “One-Way MANOVA.” At the time of the study, all patients were in good disease control, three MuSK-MG patients and a single AChR-MG patient were not taking immunosuppressive treatment, five MuSK-MG and nine AChR-MG patients were under cholinesterase-inhibitors. MuSK-MG patients scored worse in linguistic tasks, such as naming objects and verbs (p<0.005) and in memory recall after semantic cueing (p=0.03). These findings suggest a selective temporal-hippocampal



dysfunction in MuSK-MG patients. Cognitive performances were unrelated to disease severity (p=0.37). The mechanisms of MuSK activation in the CNS are still unclear. The occurrence of cognitive impairment in MuSK-MG patients warrants confirmation in further studies.

64. Fatigue in Patients with Myasthenia Gravis: Impact on Quality of Life and Responsiveness to Prednisone and Immunomodulation

Carolina Barnett, MD, PhD, Ari Breiner, MD, MSc, Hans Katzberg, MD, MSc, and Vera Bril, MD, FRCP Division of Neurology, Department of Medicine. University Health Network and University of Toronto, Toronto, Canada

We studied the impact of fatigue in the quality of life (QoL) of patients with myasthenia and the responsiveness of NeuroQol-fatigue short form in patients receiving prednisone or immunomodulation. 257 patients enrolled in a study on MG outcomes were assessed with the NeuroQol-fatigue, the Myasthenia Gravis Impairment Index (MGII), the MG-QOL15, the SF-36 and the EQ5D. 95 patients receiving prednisone, IVIG or PLEX were reassessed 3-4 weeks after treatment. At follow-up, patients were assessed with the NeuroQol-fatigue, MGII, MG-QOL15 and EQ5D and patient impression of change (PIC). We compared fatigue scores across different MGFA classes by ANOVA and through correlations with other measures. We built regression models for the MG-QOL15, SF-36 and EQ5D including clinical variables, MGII and fatigue scores. Responsiveness was assessed by paired t-test and standardized response mean. The minimal important difference (MID) was calculated using an anchor-based approach. 137 patients were female and the mean age was 58±15. The fatigue score was significantly higher with increasing MGFA class (Remission:4 ±10, Minimal Manifestations:10±15, I:13±20, II:39±27, III:59±27, IV:83±24, ANOVA p<0.0001). Fatigue was highly correlated with the MGII, QOL15, EQ5D and the SF36 (r: 0.66, 0.77, -0.72 and 0.71 respectively, p<0.001). In all regression models, fatigue was a main driver of QoL. Fatigue scores significantly changed with prednisone and immunomodulation (-1.8 ±4 and -3.6±7; paired t-tests p<0.05). The MID for groups was -4.0 points and for individuals -1.5 points. This study shows that fatigue has major impact on MG patients’s QoL and that it can improve with available MG treatments.

65. A Serological and Clinical Epidemiological Study of the Myasthenia Gravis in Northern

Ireland  Aisling Carr, MRCP Neurol., PhD1,2,3, Peter McCarron3, Melissa Jones4, Chris Maguigan4, Niall Tubridy4, Judith Cossins5, Jacqueline Palace5, David Beeson5, Maria Leite5, Angela Vincent5, John McConville2,6  1 Centre for Neuromuscular Diseases, National Hospital of Neurology and Neurosurgery, London, United Kingdom; 2 Department of Neurology, Belfast Health and Social Care Trust, Royal Victoria Hospital, Grosvenor Road, Belfast, Northern Ireland, United Kingdom; 3 Department of Epidemiology and Public Health, Queens University Hospital, Royal Victoria Hospital Site, Grosvenor Road, Belfast, Northern Ireland, United Kingdom; 4 Department of Neurology, St. Vincent’s University Hospital,  Elm Park,  Dublin, Ireland  5 Neurosciences Groups,  University of Oxford, West Wing,  John Radcliffe Hospital,  Oxford, United Kingdom; 6 Department of Neurology, Southeastern Health and Social Care Trust, Ulster Hospital, Upper Newtownards Road, Dundonald, Belfast, Northern Ireland, United Kingdom Genetic, serological and thymus pathology differences in autoimmune MG indicate the existence of distinct subgroups within the disease spectrum which overlap and are inconsistently defined in previous epidemiological studies. We conducted a population-based epidemiological study using multiple ascertainment sources and capture-recapture analysis to establish optimal ascertainment in Northern Ireland, a region of the UK with a population of 1.7 million.  We set out to define potential disease sub-groups, including antibody negative MG. Detailed demographic, clinical and thymic histology data were



collected and all sera were examined for antibodies to acetylcholine receptor, rapsyn-clustered acetylcholine receptor, MuSK, LRP4 and skeletal muscle. 342 cases of autoimmune myasthenia gravis were alive in Northern Ireland on the prevalence date (31/12/2009), Prevalence Rate: 202.4 (181.0,223.9) cases per million. 212 incident cases presented in nine years, Incidence Rate: 13.9 (12.2,15.8) cases per million person-years. We report sub-group information on incidence, prevalence and demographics of clustered AChR, MuSK, LRP4 and antibody negative MG and the AChR antibody positive subgroups: EOMG (Early-Onset MG), LOMG and MG associated with thymoma. We show that skeletal muscle antibodies aid the definition of LOMG and identify cases with onset <50 years with clinical and demographic characteristics of LOMG. We show how the different age-related epidemiology of thymoma-associated and LOMG explains why skeletal muscle antibodies are predictive of thymoma in MG with onset <50 years. Notably, we detected a 7-fold increase in LOMG incidence over the study period. Of 25 AChR-negative cases (11.8% of incident population), 8 had antibodies to rapsyn-clustered AChR (3.8%), but none had antibodies to LRP4 or MuSK. 15 (7.1%, 3 untested) cases of clinically defined autoimmune MG remained seronegative on all assays.  This is the first population-based examination of MG with exhaustive clinical and serological characterisation of the disease allowing the definition of seronegative MG and the description of MG subgroup frequency, demographics and associations. Based on our findings, we propose novel subgroup definitions for future work. 

66. Clinical and Serological Characteristics of Patients with Double Seronegative Myasthenia Gravis

Valentina Damato, MD1,2, Amelia Evoli, MD1, Thomas Smith, MSc2, Mark R. Woodhall, PhD2, Paolo E. Alboini, MD1, Sarosh R. Irani, MD, DPhi2, and Angela Vincent, FRCPath2 1 Institute of Neurology, Catholic University, Rome, Italy; 2 Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, United Kingdom Patients without AChR and MuSK antibodies by radioimmunoprecipitation assay (RIPA) are referred to as having seronegative myasthenia gravis (SNMG). Cell-based assays (CBAs) for detection of clustered AChR, MuSK and LRP4 antibodies in SNMG patients have been established but their frequencies in well studied clinical cohorts are not clear. Here, we measured clustered antibodies in serum samples of 52 well characterized Italian SNMG patients (35 women, medium age at onset 40 years, medium follow-up 11.5 years, 48 patients with generalized disease). Twenty-eight/52 (54%) patients were untreated at sampling time. The sera, not all from disease onset, were retrospectively tested by CBAs using HEK 293 cell-lines transfected to express clustered AChR, full-length MuSK or LRP4. From RIPA-negative MG patients, antibodies to clustered AChR and MuSK were detected in 11.5% (6/52) and 6% (3/52) respectively. Patients with clustered AChR-antibodies (four men/two women) were aged between 18-64 years at onset, with a mild disease course; two had thymic hyperplasia. They responded well to cholinesterase inhibitors, but immunosuppressants were required in four patients. Patients with MuSK antibodies were all female and had a typical MuSK phenotype with bulbar symptoms, with a less severe course than RIPA-positive MuSK patients and good response to prednisone. The 43 (83%) patients seronegative for MuSK, AChR, and LRP4-Abs had age of onset, distribution of weakness, disease severity and treatment responsiveness similar to RIPA positive AChR-MG. Full evaluation of the diagnostic and clinical use of CBAs will depend on applying these tests to sera at onset and before immunotherapies.



67. Ocular Weakness Patterns in Myasthenia Gravis

Robert H. de Meel, MD1, Wouter F. Raadsheer, BSc1, Erik W. van Zwet, PhD2, Martijn R. Tannemaat, MD, PhD1, and Jan J. Verschuuren, MD, PhD1 1 Department of Neurology, Leiden University Medical Center, Leiden, the Netherlands; 2 Department of Biostatistics, Leiden University Medical Center, Leiden, the Netherlands

Introduction: Myasthenia gravis (MG) is characterized by fatigability and fluctuating weakness that usually begins in ocular muscles. Even though MG is a systemic disease, ptosis is reported to be typically asymmetrical. Furthermore, different reports have found distinct patterns of diplopia in MG. In our study we want to further elucidate ocular weakness patterns in MG.

Methods: We analyzed ptosis characteristics in a retro- and prospective cohort and investigated diplopia patterns in a prospective cohort by systematically examining extra-ocular muscle (EOM) fatigability in eight gaze directions for 60 seconds.

Results: We included 301 MG patients, of which 103 prospectively, and analyzed 1525 visits. Ptosis and eye closure weakness were initially asymmetric in 88.5% and 5.3%, respectively. 78% of patients with initial symmetric ptosis developed asymmetric ptosis within 3 visits. The EOM were affected unilaterally in 76%. The most commonly affected EOM were the m. rectus lateralis (66%) and the m. obliquus superior (59%). Non-spontaneous ptosis and diplopia manifested within 30 seconds in 63% and 82%, respectively. 81% of patients with diplopia manifested this by combining the top left and bottom right gaze directions for 30 seconds, whereas with the QMG diplopia item (center right gaze direction for 60 seconds) only 68% manifested diplopia. Discussion: Ptosis and diplopia were asymmetrical in the majority of patients. Initial symmetric ptosis often became asymmetric over time. Diplopia most often manifested within 30 seconds. Combining two gaze directions for 30 seconds may result in a higher yield than that of the current QMG diplopia item.

68. Thymoma-associated Myasthenia Gravis: Clinical and Serological Features of Pisa’s

Cohort Anna De Rosa, MD1, Roberta Ricciardi, MD1,2, Michelangelo Maestri, MD1, Melania Guida, MD1, Marco Lucchi, MD2 , Alfredo Mussi, MD2, and Ubaldo Bonuccelli, MD1 1 Department of Clinical and Experimental Medicine, Neurology Unit, University of Pisa, Pisa, Italy; 2 Division of Thoracic Surgery, Cardiothoracic and Vascular Surgery Department, University of Pisa, Pisa, Italy TAMG (Thymoma-associated MG) represents one of the subtypes of MG associated with autoantibodies directed against the acetylcholine receptor (AChR-Ab). Approximately 10–20% of MG patients have a thymoma and about 30% of thymoma patients have thymoma-associated MG. The recurrence of thymoma is rare and the rate ranges according to different reports. Our aim was to analyze our large cohort of MG patients with thymoma and relapsed thymoma according to: age of MG onset, age of thymectomy, surgical approach, oncological features (according to histological classifications: WHO and Masaoka-Koga), post-thymectomy status, current MG clinical status and AChR-Ab serum titres. We selected 388 MG patients with AChR-Ab and thymoma: 360 with TAMG and 28 (7.2%) who experienced one or more recurrences of thymoma. Patients with recurrent neoplastic disease were usually younger than those with thymoma, with a mean age of MG onset of 39.5 ±12.1 years. There was not a significant sex prevalence and bulbar symptoms were relevant in this subtype of MG; compared to the thymoma group, the current MG clinical status was not



related to the severity of neurological disease before thymectomy. The average neoplastic disease-free time was 3.7 years. Antibody-titres usually declined after thymectomy and chemio/radiotherapy whilst recurrences of thymoma were not associated with significant variations in AChR-Ab serum titres or clinical status of the patients. Compared to non-thymomatous MG, TAMG clinical presentations tended to be severe at onset and immediately after thymectomy but, in the long-term, pharmacological and complete stable remission was achieved in a high percentage of patients.

69. Epidemiology of Myasthenia Gravis in Southern China — Estimates Using Hospital and Insurance Records

Wei Fang, PhD1, Yan Li, MD, PhD2, Rong Mo, MD2, Li Qiu, MD2, Changyi Ou, MD2, Zhidong Huang, MD2, Zhongqiang Lin, MD2, Weizhi Wang, MD, PhD3, Lihua Wang, MD, PhD3, JianjianWang, MD, PhD3, and Weibin Liu, MD, PhD2 1 Queens College, City University of New York, New York, United States; 2 Sun Yat-Sen University, the First Affiliated Hospital, Guangzhou, China; 3 Harbin Medical University, the Second Affiliated Hospital, Harbin, China An epidemiological study of myasthenia gravis (MG) was conducted in China using hospital and insurance records. Data were solicited from clinical records of 21 hospitals in Guangzhou (a representative city of southern China), 5 hospitals in Harbin (a representative city of northern China) and insurance records from two systems: job-based (JBI) and residence-based (RBI) in Guangzhou. Incidence rates of MG were estimated based on insurance records and prevalence rates of MG were estimated based on both data sources during 2000-2014. The annual incidence rate (/100,0000) of MG in Guangzhou city was estimated to be 3.452.70 for JBI (2002-2014) and 1.550.53 for RBI (2009-2014). It had a steady increase (from 0.88 to 6.36, R2=0.75) for JBI and little fluctuation for RBI. The annual prevalence rate (/100,0000) of MG in Guangzhou city was estimated to be 11.073.67 for JBI (2009-2014) and 2.190.95 for RBI (2008-2014). During this period, annual prevalence of MG in Guangzhou had a significant increase (from 4.78 to 14.37) with a female bias (4.441.59 for male vs 6.652.08 for female) for JBI, and had little fluctuation with no gender bias (1.030.48 for male vs 1.160.48 for female) for RBI. The proportion of hospitalized MG patient (/100,000) was 38.3424.99 in Guangzhou (n=15) and 12.8012.84 in Harbin (n=4). Hospitals receiving a large number of secondary and tertiary MG patients were excluded to avoid overestimation. The proportion of hospitalized MG patients was higher in the south than in the north, probably due to various genetic, environmental and social/cultural factors.



70. Management of Ocular Myasthenia Gravis in Glasgow Maria Elena Farrugia, DPhil, MD, FRCP(E)1, Marie Cleary PhD, DBO2; and Caroline Carmichael, BA1 1 Neurosciences Department, Institute of Neurological Sciences, Glasgow, United Kingdom; 2 Orthoptic Department, Gartnavel Hospital, Glasgow, United Kingdom We retrospectively studied all patients with ocular myasthenia (OMG) and with positive acetylcholine receptor antibodies. We identified 93 patients (64 men, 29 women). The mean age at disease onset was 63y (SD 17; minimum 16, maximum 90y). Mean follow-up was 6.2y (SD 6.1y). The majority (67, 72%) experienced ptosis with diplopia and 18 (19%) patients experienced ptosis alone, while 7 (7.5%) patients complained of diplopia without ever experiencing ptosis. Initial treatment consisted of pyridostigmine in 64, prednisolone in 4, pyridostigmine and prednisolone in 1, pyridostigmine and azathioprine in 1, no treatment in 8 and unknown in 15. Sixty-nine patients continued to use pyridostigmine for symptom management (36 patients using this in exclusion). At final review, management consisted of pyridostigmine alone in 22, pyridostigmine and prednisolone in 6, prednisolone only in 17, prednisolone plus azathioprine in 8, azathioprine only in 9, mycophenolate mofetil (MMF) alone in 1, prednisolone plus MMF in 1, methotrexate in 2, ciclosporin in 1, no medication in 21 and unknown in 4. Ptosis repair surgery was performed in 7 patients. Twenty patients had prisms fitted to their glasses and one had blenderm occlusion of their lens. Outcomes in patients who received pyridostigmine or no treatment were compared against those who received immunosuppression (with or without pyridostigmine). The former had milder symptoms at final review, but this did not reach statistical significance. In conclusion, a high proportion of OMG patients continued with pyridostigmine treatment. Older age-groups and background comorbidities were often limiting factors to aggressive immunotherapy.

71. Characteristics of Long-time Patients with Myasthenia Gravis in a Danish Population Asger Frost, PhD The National Rehabilitation Centre for Neuromuscular Diseases, Aarhus, Denmark During the past 4 – 5 decades, due to more efficient medical treatment, myasthenia gravis (MG) has developed from being a life threatening disease to one with social implications. Register data reveals an increased risk (>factor 5) for MG patients in their active working age of losing their attachment to the labor market 2 years after being diagnosed. However little is known about long-term characteristics for patients living with MG. The National Rehabilitation Centre for Neuromuscular Diseases carried out a cross sectional survey in which information from 93 Danish MG patients (age: 18 to 65 and diagnosed between 1997 and 2013) was analyzed. The survey was divided into 3 groups according to how long the patients had lived with MG: Group 1, > 24 and < 76 month (N=39). Group2, ≥ 76 and < 128 month (N=35) and group 3, ≥ 128 and < 180 month (N=19). Baseline characteristics on gender, age at diagnosis, serological profile, neurologic evaluation, and treatment were outlined, and status on grade of progression and remission, refractory to treatment, experienced fatigue, quality of life and labor marked attachment (LMA) of the three groups were presented, adjusted, and scrutinized. Preliminary results indicate that for a majority of MG patients, the variables investigated are stabile in the three groups. However, even when neurological symptoms are reduced considerably through pharmaceutical treatment, complete or pharmacologic remission or absence of fatigue are seldom, and LMA remains at a considerable lower level than one year before the time of diagnosis.



72. Neurological Autoimmunity Associated with Thymoma: beyond Myasthenia Gravis Raffaele Iorio, MD, PhD, Valentina Damato, MD, Paolo Emilio Alboini, MD, Gregorio Spagni, MD, and Amelia Evoli, MD Institute of Neurology, Fondazione Policlinico Universitario “A. Gemelli,” Catholic University, Rome, Italy Thymic malignancy is often associated with autoimmune neurological diseases with myasthenia gravis (MG) being the most frequent accompaniment. Recently, novel autoantibodies have been identified that may expand the serological profile of thymoma. The aim of this study was to investigate the neurological manifestations either isolated or associated with MG and the frequency of neural Immunoglobulin G (IgG) autoantibodies in patients with thymoma. We retrospectively identified and classified 269 patients with histopathologically confirmed thymoma seen in our Institution. Patients were divided into 3 groups: (1) isolated MG (n = 244); (2) MG plus additional autoimmune neurological manifestations (n = 15); and (3) neurological disorders other than MG (n = 10). Groups 2 and 3 are the object of this study. Four patients from group 2 were excluded because no serum was available to test. Antibody testing was performed by immunohistochemistry on mouse brain sections, by cell-based assays (CBA) using HEK293 cells transfected with neuronal plasma membrane antigens and by immunocytochemistry on rat hippocampal neurons. Nine patients were females, the mean patient age was 53 years. In group 2, neuromyotonia was diagnosed in 6/11 (55%) patients, encephalitis in two, psychiatric disturbances in two, and Morvan’s syndrome in single cases. In group 3 the final diagnoses were: encephalitis (4 patients), neuropathy (4), psychosis (1) and cerebellar ataxia (1). Indirect immunofluorescence assays on mouse brain sections revealed IgG in the serum of 16/21 patients binding to the brain neuropil. CASPR2-IgG, Lgi1-IgG and GABAAR-IgG were the most frequent antibodies detected. In 9 patients IgG binding to currently unclassified antigens were observed.

73. Respiratory and Sleep Disturbances in Adults and Children with Myasthenia Gravis Hans Katzberg, MD, MSc1, Clodagh Ryan, MD2, Heba Qashqari, MD3, Kevin Vezina, MD3, Carolina Barnett Tapia, MD, PhD1, Abdullah Khayatt3, Nicolas Chrestien4, Jiri Vajsar, MD3, Indra Narang, MD3, and Vera Bril, MD1 1 Division of Neurology, Department of Medicine, University Health Network and University of Toronto, Toronto, Canada; 2 Division of Respirology, Department of Medicine, University Health Network and University of Toronto, Toronto, Canada; 3 Department of Pediatrics, Department of Medicine, Hospital for Sick Children and University of Toronto, Toronto, Canada; 4 Department of Pediatrics, Department of Medicine, Laval University, Quebec City, Canada Respiratory and sleep disturbances occur in both adults and children with myasthenia gravis, however, the extent of these disturbances in the outpatient setting is unknown. In order to further study this, adult patients with generalized MG attending the UHN Neuromuscular Clinic were recruited for office-based respiratory assessment sequentially and all patients with MG seen at the Hospital for Sick Children were studied with pulmonary function tests and sleep studies. Adult patients were administered the Epworth Sleepiness Scale, UCSD Shortness of Breath Questionnaire (UCSD-SOBQ) and MRC Dyspnea Scale (MRC-DS). Supine and seated FVC was performed on all participants and children also underwent polysomnography. 86 patients (35 males and 51 females, 8 children and 78 adults) ages 11-82 and mean disease duration 1 month-41 years were enrolled in the study. 26% of patients had abnormal upright FVC and 35% abnormal supine FVC. In the adult patients, 21% were found to have excessive daytime sleepiness, mean UCSD-SOBQ was 25 out of 120 and median MRC-DS of 1 out of 4. Two of 8 children with purely ocular MG and mild generalized MG were found to have mild to moderate obstructive sleep apnea. Although additional results are expected, to date it appears that supine FVC may be more frequently



abnormal than seated FVC in adults and children with MG, excessive daytime sleepiness is common in adults with MG and that obstructive sleep apnea can occur even in the setting of mild or purely ocular MG in children.

74. Thymectomy Status and Quality of Life in Myasthenia Gravis Patients from the MGFA

Registry Ikjae Lee, MD1, Henry Kaminski, MD2, Haychang Xin, PhD3, and Gary Cutter, PhD4 1 Department of Neurology, University of Alabama at Birmingham, Birmingham, Alabama, United States; 2 Department of Neurology, The George Washington University, Washington, District of Columbia, United States; 3 Department of Health Care Organization and Policy, University of Alabama at Birmingham, Birmingham, Alabama, United States; 4 Department of Biostatistics, University of Alabama at Birmingham, Birmingham, Alabama, United States Background: Myasthenia gravis (MG) is an antibody mediated autoimmune disorder affecting the neuromuscular junction leading to various degree of fatigable muscle weakness causing disability and impaired quality of life. Method: The MG patient registry is a patient-driven, nationwide database with MG patients participating across the United States supported by the Myasthenia Gravis Foundation of America (MGFA). Total 1315 patients age >=18 who were diagnosed with MG and registered between 7/1/2013 and 6/30/2016 were included. Patients were grouped into Thymectomy and No-thymectomy groups based on thymectomy status. Basic demographic information, disease related history, and MG-QOL15 score were compared between the groups. Results: Total 1315 patients were included from the MG patient registry, 406 patients had thymectomy and 909 patients did not. Thymectomy group was significantly younger (48.4 vs 57.2), more predominantly female(78% vs 56%), younger age at symptom onset (34.8 vs 50.3), more likely to have thymoma (32% vs 0.6%), prior ICU admission (45% vs 25%) and feeding tube placement(15% vs 9%) compared to No-thymectomy group. Age, gender, years from symptom onset to diagnosis were identified as confounding variables. Multivariable analysis adjusted for the above confounding variables showed lower MG-QOL15 score (better) in thymectomy group (20.2 vs 23.7, p=0.002). Further analysis showed the benefit was associated with female gender (21.3 vs 26.2) but not with male gender (21.2 vs 21.0). Conclusion: Quality of life reported by myasthenia gravis patients was better in the thymectomy group and the benefit was seen predominantly in women.

75. Pregnancy Outcome in Women with Myasthenia Gravis Jung Hwan Lee, MD, Ha Young Shin, MD, Ilnam Sunwoo, MD, PhD, Sei Kwang Kim, MD, PhD, Young-Chul Choi, MD, PhD, and SeungMin Kim, MD, PhD Yonsei University College of Medicine, Seoul, Korea Myasthenia gravis (MG) is a chronic autoimmune disorder of neuromuscular junction (NMJ) characterized by varying weakness and easy fatigability of skeletal muscles. The disease is twice as common in females, especially with childbearing age. So, we investigated clinical course of MG and complications during pregnancy and puerperium in women with myasthenia gravis in Korea. This study is one center oriented, retrospective and case-control study. We included 38 definite MG mothers treated due to delivery or abortion in our hospital, from January 1, 2005 to June 30, 2013. We described clinical course of MG during pregnancy, delivery, and puerperium, and investigated several factors about clinical course of MG and compared adverse pregnancy outcomes between MG mothers and age-matched normal controls without



any previous medical history. Among total 49 pregnancies, there were 8 abortions and 41 live birth deliveries, including 20 vaginal deliveries and 21 cesarean sections. In those 41 cases, 10 cases showed worsening course of MG disease during pregnancy and there were more patients having ocular symptom at onset in not-worsening group (25, 80.6%) rather than worsening group (4, 40.0%) (p=0.036). The incidence of adverse pregnancy outcomes between MG mothers and normal control groups did not show statistically significant difference except preterm birth (4.9% vs. 29.3% [p=0.014], favorable outcome in MG mothers). We suggested that when MG patient are pregnancy, we should care more for patients, whose symptoms at onset were limb or bulbar symptoms. Furthermore, For MG patient during pregnancy, we can decide to undergo routine obstetrical management.

76. Significant Regional Difference in the Perception of MG Despite Comparable Objective Fatigue

Liis Sabre, MD, PhD1,2, Elisabet Westerberg, MD1, Maarika Liik, MD, PhD2, and Anna Rostedt Punga, MD, PhD1 1 Uppsala University, Uppsala, Sweden; 2 University of Tartu, Tartu, Estonia In this study, we aimed at evaluating MG patients between comparable regions in two Baltic sea countries, Estonia and Sweden. This cross-sectional study included southern counties in Sweden and Estonia of comparable size. All patients with a confirmed MG diagnosis were asked to answer two questionnaires including demographic and disease specific data, life-style issues and mental fatigue (fatigue severity scale; FSS). Clinical fatigue was assessed objectively through the Quantitative Myasthenia Gravis Score (QMG). The prevalence rate standardized to European standard population was 172.6 (95% CI 131.1 – 214.1) in Estonia and 148.1 (95% CI 152.2 – 245.6) in Sweden. The current age and age at disease onset were similar in these regions but overall female to male ratio was different (2:1 in Estonia vs 1:1 in Sweden). QMG score did not differ; however, subjectively Estonian patients judged their symptoms more expressed (5.6 ± 2.8) compared to the Swedish patients (3.4 ± 2.3, p=0.0005). Estonian patients also had significantly higher FSS scores (5.0 ± 1.7) than Swedish patients (3.5 ± 1.6) (p=0.001), and they were physically less active (regular physical activity 29.1% in Estonia and 74.2% in Sweden, p=0.004). Treatment regimen was generally similar but more patients were on azathioprine treatment in Estonia (p=0.004) and they used higher daily doses of pyridostigmine (367 ± 199 mg in Estonia vs 231 ± 161 mg in Sweden, p=0.007). These data indicate that despite comparable objective fatigue, significant regional differences in the perception of MG severity exist.



78. Clinical and therapeutical characteristics of anti-MuSK Myasthenia Gravis: our single-center experience in Pisa

Roberta Ricciardi, MD1,2, Michelangelo Maestri, MD1, Anna De Rosa, MD1, and Melania Guida, MD1 1 Department of Clinical and Experimental Medicine, Neurology Unit, University of Pisa, Pisa, Italy; 2 Division of Thoracic Surgery, Cardiothoracic and Vascular Surgery Department, University of Pisa, Pisa, Italy Background: Myasthenia gravis (MG) with autoantibodies to muscle specific tyrosine kinase (MuSK) represents a distinct type of disease as compared with AchRAb positive and double seronegative patients. Methods: In this retrospective study, we reviewed the clinical and therapeutical characteristics of 101 MuSK-MG patients followed in our Center in Pisa. Results: Seventy-nine percent (81#) were female, with disease onset at 40±7.7 yrs. Anti-MuSK antibody titres vary from 0.15 to 2.62 nmoli/l, while ACAB were negative. As concerns MGFA classification, 2 were class 1, 1 2A, 23 2 B, 47 3B, 22 4B and 6 class 5. Dosage of antiMuSK titre were higher in class 4-5 patients. Most of patients were under acetylcholinesterase inhibitor therapy at our first observation. Complete discontinuation of this therapy leads to a clinical improvement and resolution of side effects in all patients. Steroid therapy and immunomodulation (mainly with IVIG) were the most used and efficacious treatments. Thymectomy was performed before the diagnosis of this subtype of MG in 15 patients without any change in clinical status. At the end of follow up, 48 patients achieved pharmacological remission and 5 complete stable remission (52,5% in total). Conclusions: MuSK-MG has peculiar features characterized by a predominant oculo-bulbar symptoms and characteristic therapeutic response. The use of anticholinesterase inhibitors led to severe side effects and thymectomy was not followed by a clinical response. Thus, both therapeutic approach should be avoided. With an appropriate long term immunomodulant therapy, majority of the patients could achieve remission also in this subtype of MG.



79. Reduction in Acetylcholine Receptor Antibody Levels Correlate with Clinical Improvement in Myasthenia Gravis

Florit Marcuse, BSc1, Mario Losen, PhD2, Pilar Martinez-Martinez, PhD2, Jan Damoiseaux, PhD3, Janneke Hoeijmakers, MD, PhD4, Jos Maessen, MD, PhD1, Marc De Baets, MD, PhD2 1 Department of Cardiothoracic surgery, Maastricht University Medical Center, the Netherlands; 2 MHENS, School for Mental Health and Neuroscience, Maastricht University, the Netherlands; 3 Department of Cellular Diagnostics & Cell Therapy, Maastricht University Medical Center, the Netherlands; 4 Department of Neurology, Maastricht University Medical Center, the Netherlands Background: There is no consensus about a correlation between clinical severity of disease and the levels of anti-acetylcholine receptor antibody (AChR) in patients with myasthenia gravis (MG). Previous research showed no association with a follow up for several months. The aim of this 10-year-retrospective study was to investigate the possible correlation between the patients’ clinical symptoms and anti-AChR-levels measured with a specific quantitative immunoprecipitation assay (RIA). Methods: We retrospectively analyzed 49 patients with MG, who were treated at the Maastricht University Medical Center. Inclusion criteria were defined based on positive anti-AChR-levels (>0.25 nmol/L) and the use of minimal one immunosuppressive therapy. The anti-AChR-levels were measured by RIA with serial serum dilutions (IBL, Germany). Clinical severity of disease was measured by using the Myasthenia Gravis Foundation of America (MGFA) protocol. We analyzed the MGFA-scores and anti-AChR-levels at onset of disease, and subsequently after 6 months, 1 year, 3 years, 5 years and 10 years.

Results: The mean anti-AChR-levels and the mean MGFA-score of the 49 patients decreased continuously during follow up. A drop of 25% and 50% of the anti-AChR-levels was measured at a mean time of 21 months and 32 months respectively. Conclusions: The correlation coefficient r = 0.98 was found between clinical severity of disease and the decrease of anti-AChR levels in patients with MG. On average it took more than two years to achieve a 50% reduction of anti-AChR-levels. Anti-AChR-levels are useful as a marker for the evaluation of immunotherapy and have to be followed for several years.



80. The Predictive Role of Stimulated Single Fiber Electromyography in the Orbicularis Oculi Muscle for the Generalization of Ocular Myasthenia Gravis Symptoms.

Roxanne Berghs and Rudy Mercelis, MD, PhD University of Antwerp, Antwerp, Belgium and Antwerp University Hospital, Edegem, Belgium Myasthenia gravis (MG) starts with symptoms of diplopia or ptosis in the majority of patients. At present, it is impossible to predict if the disease will stay limited to the ocular muscles or expand to other body regions. Common tests like the titer of acetylcholine receptor (AChR) antibodies or the results of electromyographic (EMG) tests of neuromuscular transmission including single fibre EMG in the extensor digitorum communis muscle failed to have a good predictive value. We reviewed the data of patients who underwent stimulated single fibre EMG (SSFEMG) of the orbicularis oculi muscle for suspicion of myasthenia gravis with only ocular complaints at the time of the examination. We isolated the cases where the diagnosis of MG could be confirmed by the presence of AChR antibodies or by fluctuating clinical findings with a clear response to cholinesterase inhibitors or steroids, with a minimal clinical follow-up of three years. 57 MG patients could be included in the study, SSFEMG was abnormal in 42 and normal in 15. Of the 42 patients with abnormal SSFEMG, 15 had generalized disease at the last follow-up and 27 remained purely ocular. Only two of the 15 patients with normal SSFEMG developed generalized disease. The predictive value of a normal SSFEMG was however not statistically significant. Of the 40 patients who remained ocular after at least three years follow-up only 17 were treated with steroids or immunosuppressives, in contrast with 12 of the 17 patients with generalization.

81. Social Disadvantages Associated with Myasthenia Gravis and Its Treatment: A Multicenter Cross-Sectional Study

Yuriko Nagane, MD1, Hiroyuki Murai, MD2, Tomihiro Imai, MD3, Daisuke Yamamoto, MD3, Emiko Tsuda, MD3, Naoya Minami, MD4, Yasushi Suzuki, MD5, Tetsuya Kanai, MD6, Akiyuki Uzawa, MD6, Naoki Kawaguchi, MD7, Masayuki Masuda, MD8, Shingo Konno, MD9, Hidekazu Suzuki, MD10, Masashi Aoki, MD11, and Kimiaki Utsugisawa, MD1 1 Department of Neurology, Hanamaki General Hospital, Hanamaki, Japan; 2 Department of Neurological Therapeutics, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan; 3 Department of Neurology, Sapporo Medical University Hospital, Sapporo, Japan; 4 Department of Neurology, Hokkaido Medical Center, Sapporo, Japan; 5 Department of Neurology, Sendai Medical Center, Sendai, Japan; 6 Department of Neurology, Chiba University School of Medicine, Chiba, Japan; 7 Chiba Neurology Clinic, Chiba, Japan; 8 Department of Neurology, Tokyo Medical University, Tokyo, Japan; 9 Department of Neurology, Toho University Oh-hashi Medical Center, Tok yo, Japan; 10 Department of Neurology, Kinki University School of Medicine, Osaka, Japan; 11 Department of Neurology, Tohoku University Graduate School of Medicine, Sendai, Japan To clarify the social disadvantages associated with myasthenia gravis (MG), we evaluated 917 consecutive cases of established MG seen at 13 neurological centers in Japan. All patients completed a questionnaire on social disadvantages resulting from MG and its treatment at study entry. Clinical severity at the worst condition, and that at the current condition was determined. Maximum dose and duration of dose ≥20mg/day of oral prednisolone during the disease course were obtained from the patients’ medical records. Achievement of the treatment target (minimal manifestation status with prednisolone at ≤5 mg/day) was determined at 1, 2, and 4 years after starting treatment and at study entry. We found that 27.2% of the patients had experienced unemployment, 4.1% had been unwillingly transferred, and 35.9% had experienced a decrease in income, 47.1% of whom reported that the decrease was ≥ 50% of their previous total income. In addition, 49.0% of the patients reported feeling reduced social positivity. Factors promoting social disadvantages were severity of illness, dose and duration of prednisolone, long-term treatment, and



a depressive state and change in appearance after treatment with oral steroids. Early achievement of the treatment target was a major inhibiting factor. MG patients often experience unemployment, unwilling job transfers, and a decrease in income. In addition, many patients report feeling reduced social positivity. To inhibit such social disadvantages, a focus needs to be placed on helping MG patients resume a normal lifestyle as soon as possible by achieving the treatment target.

82. Polyautoimmunity and Familial Autoimmunity in Juvenile Myasthenia Gravis Trine H. Popperud, MD1,2,3, Magnhild Rasmussen, MD3,4, and Emilia Kerty, MD, Prof1,2

1 Department of Neurology, Oslo University Hospital, Norway; 2 Institute of Clinical Medicine, University of Oslo, Norway; 3 Unit for hereditary and inborn neuromuscular disorders, Department of Neurology, Oslo University Hospital, Oslo, Norway; 4 Department of Clinical Neuroscience for Children, Section for Child Neurology, Oslo University Hospital, Oslo, Norway Background: Autoimmune disorders (AID) are multifactorial in origin and affect at least 5% of the World’s population. Polyautoimmunity, the presence of a second AID in an individual, is a well-known phenomenon also reported among adult myasthenia gravis (MG) patients where it occurs in 13-22%. Familial autoimmunity, the presence of diverse autoimmune disorders in members of a nuclear family, is another described characteristic of AID. We aimed to assess the frequency of poly-autoimmunity, AID associated autoantibodies and familial autoimmunity in a juvenile MG cohort in Norway. Patients and Methods: Patients were identified from January 2012 to April 2016, through multiple strategies. A total of 75 patients with MG onset ≤ 18 years of age were identified. 63 subjects gave written consent and were included. Retrospective clinical data were collected by means of medical chart and a questionnaire. 51 patients gave new blood samples for autoantibody analysis. Females constituted 84%. Onset was prepubertal (< 12 years) in 33%. AChR ab were present in 73%, and 50% underwent thymectomy. Results: We found that a total of 20 (32%) juvenile MG patients had at least one additional AID, in all but one with onset after MG diagnosis. In addition, 21 cases had autoantibodies associated with an AID without any verified AID diagnosis other than MG. Familial autoimmunity was recorded in 29 (46%). Details will be presented. Conclusion: We show that polyautoimmunity and familial autoimmunity are common among juvenile MG cases in Norway, as well as the presence of AID associated autoantibodies.

83. A Global Electronic NeuroImmunology Database for Myasthenia Gravis (eNID-MG). Katherine A Buzzard, MB, BS, PhD1, Carolina Barnett, MD, PhD2, Ted Burns, MD3, Helmut Butzkueven, MBBS, PhD4, D. Sean Riminton, MBBS, PhD5, and Stephen W. Reddel, MBBS, PhD6. 1 Department of Neurosciences, Eastern Health, Box Hill Department of Neurology, Royal Melbourne Hospital, Parkville, Melbourne, Australia; 2 Division of Neurology- Department of Medicine, University Health Network and University of Toronto, Toronto, Canada; 3 Department of Neurology, University of Virginia, Charlottesville, Virginia, United States of America; 4 Department of Medicine, University of Melbourne, Melbourne, Australia; 5 Department of Immunology, Concord Hospital, University of Sydney, Sydney, Australia; 6 Department of Neurology, Concord Hospital, University of Sydney, Sydney, Australia The nervous system is vulnerable to inflammatory damage caused by the immune system with a variety of



neuroimmunological diseases. These are often chronic, generally treatable, and associated with significant disability. Although individually rare, collectively neuroimmunological diseases including myasthenia gravis (MG) affect a significant number of individuals worldwide and cause a disproportionately heavy health care burden. Lack of epidemiological and evidence-based outcome information poses significant problems in neuroimmunology. Disease registries can be powerful and cost-effective instruments for expanding our knowledge of diseases and treatments. They are particularly useful in rare diseases where the opportunity to pool ‘real world’ data reaches sufficient size for epidemiological and clinical research. The electronic NeuroImmunology Database (eNID) has been developed as an extension to MSBase, a successful international registry for patients with Multiple Sclerosis, with >40,000 patients globally and numerous publications. A single common structure has governance, cost and usability advantages while permitting cross-disease comparisons. MG as a canonical condition with clear definitions and outcome measures, will be the first disease subregistry to be developed, followed by CIDP, autoimmune encephalitis and neurosarcoidosis. By intention, eNID and MSBase use simple outcome measures, are user friendly and can aid clinics, with a graphical display of the patient’s course, therapies and outcomes over time. It is simple to recruit and to input data in clinic, with deidentified data freely accessible to researchers. MG outcome measures include clinical (MG composite), patient reported (MGII), and general (employment / activity and modified Rankin). A global MG database will significantly advance myasthenia research.

84. A Prospective Clinical and Immunological Study of Late Onset Myasthenia Gravis

Girija Sadalage, MRCP1,2,3,4, Saiju Jacob, DPhil3, David Hilton-Jones, MD4, Camilla Buckley, DPhil4, Angela Vincent, FRS, FRCPath5, and Paul Maddison, MD1,2 1 University of Nottingham, Nottingham, United Kingdom; 2 Queen’s Medical Centre, Nottingham, United Kingdom; 3 Queen Elizabeth Hospital Birmingham, Birmingham, United Kingdom; 4 John Radcliffe Hospital, Oxford, United Kingdom; 5 University of Oxford, Oxford, United Kingdom Introduction: Epidemiological studies conducted in several countries have shown that the incidence of myasthenia gravis in the older population (>50 years) is increasing. We were interested in establishing if there were different clinical and immunological features between early (EOMG) and late onset myasthenia gravis (LOMG). Methods and Results: We recruited 150 patients making this the largest prospective cohort study in MG to date with 100% recruitment rates in the Trent region. Most (72.7%) patients had LOMG. Comparison between MG composite scores shows that disease severity at diagnosis was greater (mean of 9.37 Vs 5.74, p = 0.0339) in the LOMG group compared to EOMG. Purely ocular presentation was observed at similar rates between the two patient groups (48% EOMG, 36% LOMG, p = 0.24) but acetylcholine receptor antibody titres were higher (median titres of 177.1 Vs 23.83 x 10-10 mol/L, p = 0.007) in LOMG. There was a significant fall in titres with time, in keeping with improved MG composite scores (median score of 7 Vs 0, p < 0.0001), both in patients who were and were not given immunosuppression. 41 of our 150 patients had some form of immunosuppression in the first 12 months of the study, including 15 patients who were immunosuppressed at recruitment. At their 12 month follow up, 4 of 19 (who had been seen for their annual review out of a total of 41) i.e. 21% of the EOMG patients were in remission. None of them had been on any form of treatment. 44 of the LOMG patients (out of 109) had been seen for their 12 month follow up. 28 out of the 44 (63.6%) were asymptomatic: 7 had not been on any form of MG treatment (15.9%), 5 were on pyridostigmine alone (11.4%), and 16 were on pyridostigmine along with some form of immunosuppression during the 12 months since recruitment (36.3%). A delay in diagnosis does not seem to affect the disease severity (p=0.4423).



Conclusion: Our prospective cohort study confirms that the incidence of LOMG is higher than EOMG with greater disease severity in LOMG patients at diagnosis. Immunological studies are ongoing and should add to the clinical data already collected.

85. Paraneoplastic Myasthenia Gravis: Diagnostic and Therapeutic Concept for Improving Longterm Survival

Lukas Kirzinger Dr1, Micha Ried, PD, Dr2, Okka Hamer, Prof, Dr3, Andrea Goetz, Dr3, Joerg Marienhagen, PD,Dr4, Reiner Neu, Dr2, Gerhard Schuierer, Prof, Dr1, Hans-Stefan Hofmann, Prof, Dr2, Alexander Marx Prof, Dr5, and Berthold Schalke, Prof, Dr1 1 University of Regensburg, Neurology at District Hospital/medbo, Regensburg, Germany; 2 University of Regensburg, Medical School, Thoracic Surgery, Regensburg, Germany; 3 University of Regensburg, Medical School, Radiology, Regensburg, Germany; 4 University of Regensburg, Medical School, Nuclear Medicine, Regensburg, Germany; 5 University of Heidelberg/University Medical Centre Mannheim, Pathology, Mannheim, Germany Paraneoplastic Myasthenia gravis (TMG) is a thymoma induced disorder. Long-term survival is determined by the tumor. CT with contrast medium is the gold standard for initial tumor detection. If acetylcholine receptor antibody is positive and MG is verified, biopsy is not meaningful because the probability of a thymoma is nearly 100%. Complete surgical resection still is the standard therapeutic procedure. Primary operability depends on localization, size and mainly on the boundaries between thymoma and great vessels, heart, lung and thoracic wall. Cine Magnetic Resonance Imaging may help to improve the accuracy of preoperative staging of advanced thymoma. If a primary operation is not possible because of size or local infiltration, neoadjuvant therapy either by conventional chemotherapy (e.g. PAC) or with octreotide + prednisolon. Treatment with octreotide has the capability to reduce tumor volume to enable secondary resection. Thymomectomy should be done as an open trans sternal procedure to avoid dissemination of tumor cells in the chest by contamination of the instruments used for minimal invasive procedures. If R0 resection is not possible because of infiltration of adjoining organs, extended surgical resection including pleurectomy followed by local therapy e.g. hyperthermic intrathoracic chemotherapy plus subsequent systemic chemotherapy plus radiotherapy upgrade the therapeutic options. Patients with TMG should always be treated by an integrated team which is experienced to apply such a multimodality treatment regime including treatment of MG which can deteriorate either by such treatment procedures or after thymomectomy. We present a flow chart to exemplify methods to improve care of TMG patients.

86. Characteristic Pattern of Repetitive Nerve Stimulation Test in Myasthenia Gravis with MuSK

Antibody Ha Young Shin, MD, Seung Woo Kim, MD, Jung Hwan Lee, MD, and Seung Min Kim, MD Department of Neurology, Yonsei University College of Medicine, Seoul, Korea Responses to repetitive nerve stimulation (RNS) in patients with muscle specific tyrosine kinase antibody (MuSK Ab)-positive myasthenia gravis (MG) vary depending on the muscles tested. We analyzed RNS responses of limb and facial muscles in MuSK MG and double-seronegative MG patients. We retrospectively compared RNS responses between 45 MuSK Ab-positive and acetylcholine receptor antibody (AChR Ab)-negative MG (MuSK MG) and 29 MuSK Ab-negative and AChR Ab-negative (double-seronegative, DSN) MG patients. RNS of the abductor digiti minimi, flexor carpi ulnaris, trapezius, orbicularis oculi, and nasalis muscles was performed. Abnormal RNS responses in limb muscles were observed in 22.2% of MuSK MG and 58.6% of DSN MG. Abnormal facial responses were observed in 77.8% of MuSK MG and 65.5% of DSN MG. Overall, abnormal responses in any of the five muscles were observed in 86.7% of MuSK MG and 72.4% of DSN MG. Abnormal RNS responses in the abductor digiti minimi or flexor carpi ulnaris were less frequent in MuSK MG (8.9% and 15.6%) than in the DSN MG (37.9%



and 55.2%), whereas that in other muscles were similar between groups. Abnormal facial responses but normal limb responses were independently associated with MuSK MG (odds ratio 5.224; 95% confidence interval 1.3 – 20.99). Abnormal RNS responses primarily in facial muscles without involvement of limb muscles were more pronounced in MuSK MG than in DSN MG. RNS of both face and limb muscles in AChR Ab-negative MG can increase test sensitivity and aid in early suspicion of MuSK MG.

87. Early Fast-acting Treatment Strategy Against Generalized Myasthenia Gravis Kimiaki Utsugisawa, MD1, Yuriko Nagane, MD1, Tetsuya Akaishi, MD2, Yasushi Suzuki, MD3, Tomihiro Imai, MD4, Emiko Tsuda, MD4, Naoya Minami, MD5, Akiyuki Uzawa, MD6, Naoki Kawaguchi, MD7, Masayuki Masuda, MD8, Shingo Konno, MD9, Hidekazu Suzuki, MD10, Hiroyuki Murai, MD11, and Masashi Aoki, MD2 1 Hanamaki General Hospital, Hanamaki, Japan; 2 Tohoku University Graduate School of Medicine, Sendai, Japan; 3 Sendai Medical Center, Sendai, Japan; 4 Sapporo Medical University Hospital, Sapporo, Japan; 5 Hokkaido Medical Center, Sapporo, Japan; 6 Chiba University School of Medicine, Chiba, Japan; 7 Chiba Neurology Clinic, Chiba, Japan; 8 Tokyo Medical University, Tokyo, Japan; 9 Toho University Oh-hashi Medical Center, Tokyo, Japan; 10 Kinki University School of Medicine, Osaka, Japan; 11 Kyushu University Graduate School of Medicine, Fukuoka, Japan Long-term full remission is rare in myasthenia gravis (MG) patients. Minimal manifestations (MM) or better status with prednisolone at ≤ 5 mg/day (MM-or-better-5 mg) was identified as a practical treatment target, as the health-related quality of life of patients with this status is as good as that of complete stable remission. However, the percentage of patients who achieve MM-or-better-5 mg is not high, this indicates that changes in treatments are needed to further increase treatment success. Therapeutic strategies that aggressively use non-oral fast-acting immunotherapies such as plasmapheresis, intravenous methylprednisolone, and intravenous immunoglobulin from early stages of treatment (early fast-acting treatment, EFT) are reported. We clarified the effects of EFT strategies on the time course for achieving the treatment target in generalized MG. This retrospective study of 923 consecutive MG patients analyzed 688 generalized MG patients who had received immunotherapy during the disease course. The time course to first achieve MM-or-better-5 mg for ≥6 months up to 120 months after starting immunotherapies was analyzed by the Kaplan-Meier method, and was compared between 2 patient groups undergoing different treatment strategies of the EFT and non-EFT. Achievement of MM-or-better-5 mg for ≥ 6 months was more frequent and earlier in the EFT group (P = 0.0004, Wilcoxon test; P = 0.0001, log-rank test). Multivariate Cox regression analysis calculated a hazard ratio of 1.98 (P < 0.0001) for utilization of EFT. Dosing regimens of oral steroids in EFT produced no differences in the time course. EFT strategies are advantageous for early achievement of MM-or-better-5mg.

88. Czech National Registry of Myasthenia Gravis Stanislav Vohanka, PhD1, Magda Horakova, MD1, and Jana Strenkova, MSc2 1 Department of Neurology, University Hospital and Medical Faculty Masaryk University Brno, Czechia; 2 Institute of Biostatistics and Analyses, Masaryk University, Brno, Czechia So far there was no national registry of myasthenia gravis in Czechia. The new registry has been designed and supervised by Czech Neuromuscular Society. Technical solution and hosting has been provided by Masaryk University in Brno. Data of the registry are stored in a database system based on a modified version of TrialDB and fully customized to the structure of the myasthenia project. The on-line application is accessible to users via the internet browser. The security of individual records within the registry is



guaranteed via de-identified data collection. Each patient's identity is replaced with a number (ID) which does not allow any backward identification of that person. The unequivocal identification of patient is only known to the authorized health care professional. An encryption protocol is used for data transfer. For this reason, any communication between the client and server is realized via the secure protocol HTTPS, using the SSL (Secure Socket Layer) encryption. The registry has four principal parts: enrolment, follow-up, therapy, and pregnancy. All claims for personal data protection were met. Laws and regulations in Czechia require having an informed consent from all patients whose data are used in the registry. The registry was launched in spring 2016: 7 from 9 Czech neuromuscular centers were connected during one year and 171 patient´s files were included (approx. 10% of the estimated number). The first year with myasthenia registry demonstrated that it is the reliable and convenient tool for gathering and management of the data of patients with myasthenia gravis.

89. A Cohort of 50 Patients with Lambert-Eaton Myasthenic Syndrome: Long Term Follow-up of

Survival State Yiqi Liu, Kai Qiao, MD, PhD, Jie Lin, MD, PhD, Dongyue Yue, MD, Chongbo Zhao, MD, PhD, Jiahong Lu, MD, PhD, and Jianying Xi, MD, PhD Department of Neurology, Huashan Hospital, Fudan University, Shanghai, China In order to summarize the clinical characteristics and obtain the survival state of patients with Lambert-Eaton myasthenic syndrome (LEMS), we retrospectively reviewed the clinical information of 50 patients electrophysiologically confirmed LEMS in Huashan Hospital, Fudan University between 1997 to 2016 and also followed up with them in outpatient or by telephone. Fifty patients were divided in to three groups: (1) SCLC-LEMS group: 17 patients with confirmed SCLC; (2) NT-LEMS group: 19 patients who were ruled out tumor and followed up at least 2 years; (3) unclassified group: 8 patients with lung or mediastinum tumor (pathology was not available), 1 with colonial cancer and 5 patients who was followed up less than 2 years. Onset age of SCLC-LEMS group (59.6 ± 8.7y) was later than NT-LEMS group (48.5 ± 15.9y, p = 0.02) while no difference of weakness distribution, occurrence of fluctuation, autonomic dysfunction, ataxia was seen between two groups (p > 0.0.5). Among all patients, 92.5% (37/40) showed >100% increment at high-rate stimulation and 97.0% (32/33) showed >100% increment of CMAP after exercise, and 80% (12/15) patents showed positive P/Q VGCC antibody. Among 25 patients with tumors, 14 died, 9 survived and 2 lost to follow up. We obtained the survival curve of 16 patients in SCLC-LEMS group and median survival time of 7 deceased patients was 17 months (three patients not receiving any treatment for cancer). One patient in NT-LEMS group with symptoms of ataxia died of fungal infection after immunosuppression therapy. In conclusion, our data firstly provided the clinical characteristics and survival state of Chinese patients with LEMS.

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