Research

JAMA Neurology | Original Investigation

Durability of the Rituximab Response in Acetylcholine

Receptor AutoantibodyPositive Myasthenia Gravis
Kimberly R. Robeson, MD; Aditya Kumar, MD; Benison Keung, MD; Daniel B. DiCapua, MD; Emily Grodinsky, MD; Huned S. Patwa, MD;
Panos A. Stathopoulos, MD, PhD; Jonathan M. Goldstein, MD; Kevin C. OConnor, PhD; Richard J. Nowak, MD, MS

Editorial
IMPORTANCE Myasthenia gravis (MG), an autoimmune disorder of neuromuscular Supplemental content
transmission, is treated by an array of immunotherapeutics, many of which are nonspecific.
Even with current therapies, a subset of patients has medically refractory MG. The benefits of
B-celltargeted therapy with rituximab have been observed in MG; however, the duration of
these benefits after treatment is unclear.

OBJECTIVE To evaluate the durability of response to rituximab in the treatment of

acetylcholine receptor autoantibodypositive (AChR+) generalized MG.

DESIGN, SETTING AND PARTICIPANTS This retrospective case series study included 16 patients
with AChR+ MG referred to an MG clinic from January 1, 2007, to December 31, 2015. The
patients were treated with rituximab and followed up for 18 to 84 months after treatment.

MAIN OUTCOMES AND MEASURES Assessment of long-term clinical response, durability of

response and/or relapse rate, AChR autoantibody levels, adverse effects, and inflammatory
markers.

RESULTS In the 16 patients (6 men and 10 women; median age, 42 [range, 18-69] years),
clinical improvement was observed in parallel with complete withdrawal or reduction of other
immunotherapies, with all patients achieving complete stable remission, pharmacologic
remission, or minimal manifestations based on the Myasthenia Gravis Foundation of America
postintervention status criteria. Nine patients (56%) had a relapse during a mean follow-up of
36 (range, 24-47) months. Seven patients (44%) remained relapse free with a mean
follow-up of 47 (range, 18-81) months since the last rituximab treatment. All values were
normalized to a pretreatment anti-AChR antibody level of 100% and the mean levels after
each rituximab cycle were calculated. A 33% decrease was seen after cycle 1 of rituximab
treatment (100% vs 67%; P = .004); 20% after cycle 2 (compared with cycle 1) (67% vs
47%; P = .008); and 17% after cycle 3 (compared with cycle 2) (47% vs 30%; P = .02).
However, the serum cytokine levels measured were found to be unchanged.

CONCLUSIONS AND RELEVANCE Rituximab therapy appears to be an effective option in

patients with refractory AChR+ MG, who were observed to have a durable response after Author Affiliations: Program in
treatment. Identification of markers of disease relapse and sustained remission are critical Clinical and Translational
next steps in the development of pathophysiology-relevant, evidence-based practice Neuromuscular Research, Division of
Neuromuscular Medicine,
parameters for rituximab in the treatment of MG.
Department of Neurology, Yale
University School of Medicine,
New Haven, Connecticut (Robeson,
Kumar, Keung, DiCapua, Patwa,
Stathopoulos, OConnor, Nowak);
Department of Neurology, Hospital
for Special Surgery, New York,
New York (Grodinsky, Goldstein).
Corresponding Author: Richard J.
Nowak, MD, MS, Program in Clinical
and Translational Neuromuscular
Research, Division of Neuromuscular
Medicine, Department of Neurology,
Yale University School of Medicine,
JAMA Neurol. doi:10.1001/jamaneurol.2016.4190 PO Box 208018, New Haven, CT
Published online November 21, 2016. 06520 (richard.nowak@yale.edu).

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 Research Original Investigation
M
yasthenia gravis (MG) is an autoimmune disorder af-
fecting neuromuscular transmission with an esti-
mated annual incidence of about 1.7 to 21.3 cases per
1 million person-years and prevalence as high as 15 to 179 per
1 million persons. 1 Control of symptoms can be initially
achieved with acetylcholinesterase inhibitors; however, most
patients require immunotherapy such as corticosteroids, aza-
thioprine, cyclosporine, mycophenolate mofetil, plasma ex-
change, and intravenous immunoglobulin at some point in their
disease course to achieve sustained clinical remission.2-4 Thy-
mectomy, regardless of the presence of a thymoma, is also con-
sidered a therapeutic option based on prior clinical experi-
ence; however, results and conclusions from the recently
completed placebo-controlled thymectomy trial in nonthy-
momatous MG are pending at this time.5 Despite these thera-
peutic options, a subset of patients has medically refractory
MG or intolerable adverse effects from medication.6
Autoreactive B cells have a clear pathogenic role in the de-
velopment of MG, and B-celldirected therapy has emerged as
a highly effective tool in managing other autoimmune dis-
eases such as rheumatoid arthritis and neuromyelitis optica.7,8
Several groups9-18 have also observed the benefits of ritux-
imab, a chimeric anti-CD20 monoclonal antibody, in patients
with MG. In addition to significant clinical improvement,
rituximab also allowed for tapering and subsequent discon-
tinuation of other immunotherapies in patients with acetyl-
choline receptor autoantibodypositive (AChR+) and muscle-
specific kinase autoantibodypositive MG.9 However, the
duration of clinical remission among patients treated with ri-
tuximab while not receiving other immunotherapy, espe-
cially considering the prior refractory nature of their disease,
is unclear. The number of rituximab treatment cycles neces-
sary to achieve long-term remission also remains unknown.
Herein, we report our experience with the long-term effects
of rituximab in 16 patients with refractory AChR+ MG who were
followed up for 18 to 84 months, representing to our knowl-
edge the longest follow-up of a single MG cohort to date.
Methods
Patients
This retrospective study included patients with generalized MG
referred to the Yale Myasthenia Gravis Clinic, New Haven, Con-
necticut, from January 1, 2007, to December 31, 2015. Sixteen
patients with AChR+ refractory generalized disease and a mini-
mum of 12 months of follow-up after completion of the initial
set of rituximab treatment cycles were identified (Table). This
study was approved by the institutional review board of Yale
University as part of an observational study examining the
treatment and disease course of MG. All patients provided writ-
ten informed consent.
Disease was defined as refractory when the immuno-
therapy dosage could not be lowered without clinical re-
lapse, inadequate clinical control of the disease was achieved
during the immunotherapy regimen, or severe adverse ef-
fects due to current immunosuppressive therapy were present.
Pretreatment and posttreatment immunotherapy regimens,
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Durability of the Rituximab Response in Myasthenia Gravis
Key Points
Question Is the rituximab response in treatment-refractory
acetylcholine receptor autoantibodypositive myasthenia gravis
(AChR+ MG) durable?
Findings In this case series study of 16 patients with AChR+ MG
who were treated with rituximab and followed up for 18 to 84
months, all patients were observed to have clinical improvement.
Nine patients had a relapse within a mean of 36 months after the
last treatment cycle; the remaining 7 continued to maintain clinical
benefit during a mean follow-up of 47 months.
Meaning Rituximab appears to be an effective option with
sustained long-term benefit after treatment in patients with
refractory AChR+ MG.
clinical symptoms, and examination findings were evalu-
ated. The Myasthenia Gravis Foundation of America (MGFA)
clinical classification criteria19 and postintervention status were
used to assign clinical state a minimum of 12 months after
completion of the initial set of rituximab cycles. The number
of administered rituximab treatment cycles, time since the last
treatment cycle, and times to relapse and postrelapse treat-
ments were reviewed. In addition, anti-AChR antibody lev-
els, measured by conventional radioimmunoassays (refer-
ence value, 0.02 nmol/L; Mayo Medical Laboratories), were
assessed in patients before initiation of rituximab therapy, at
the end of each cycle, at the time of clinical relapse, and at the
last follow-up. Statistical analysis was performed using Graph-
Pad Prism (GraphPad Software), and P values were calculated
using nonparametric Friedman and Wilcoxon matched-pairs
signed rank tests.
Rituximab
Because no established infusion protocol for rituximab use in
MG currently exists, we used a standard protocol adopted from
the nonHodgkin lymphoma regimen of 4 weekly infusions of
375 mg/m2. One cycle is defined as 1 infusion per week for 4
consecutive weeks. The interval between cycles was 6 months.
Infusions were completed per protocol in the outpatient in-
fusion center at our institution. Our patients were treated with
an initial 2- to 4-cycle regimen. The number of cycles was
mainly based on reaching a symptom-free state and patient tol-
eration of tapering or withdrawal of other immunotherapies
(ie, corticosteroids). The number of rituximab treatment cycles
or interval between cycles was not dictated by B-cell counts.
Conventional Immunotherapy
All prior immunotherapies were reviewed. These included
prednisone, which is the standard first-line agent; plasma ex-
change; azathioprine; mycophenolate mofetil; and intrave-
nous immunoglobulin.
Safety and Adverse Effects
In addition to clinical follow-up, we reviewed the infusion cen-
ter notes and monitored complete blood cell counts and liver
function test profiles to evaluate the safety profile. Periodic
jamaneurology.com
 Table. Patient Characteristics and Response to Rituximab Treatment
Time From Follow-up
Patient No./ MGFA Class Thymectomy No. of Time From Since Initial MGFA PIS
Sex/Age at Symptoms Therapy to Rtx Thymus Initial Diagnosis Rtx Regimen After Time to Treatment MGFA
Start of Rtx Refractory Before at Start of Treatment, Pathologic Rtx to First Rtx Completed, Initial Rtx Relapse, After Current PIS Current
Therapy, y Criteriaa Rtx Therapy Rtx Regimen mo Finding Cycles Cycle, mo mo Regimen mo Relapse Therapy Status

jamaneurology.com
1/F/51 1, 2 IIIb: diplopia, P, 60 mg 7 Normal 3 41 84 CSR 44 IVIg, MM Rtxb MM-1: diplopia,
dysarthria, dysphagia, AZA, 150 mg dysarthria
limb weakness
2/F/53 1, 2, 3 IIIb: diplopia, ptosis, P, 60 mg 42 Unavailable; no 3 60 81 CSR NA NA None CSR
dysarthria, dysphagia, thymoma as per
limb weakness medical record
3/F/36 1, 2 IIb: ptosis, limb P, 60 mg 6 Thymoma type B3 2 9 68 CSR 39 PPX, IVIg Rtx every PR
weakness 6 mo
4/M/30 1, 2 IIa: limb weakness P, 60 mg 9 Thymic follicular 3 13 61 CSR 29 P, Rtx Rtx every PR
AZA, 150 mg hyperplasia 6 mo
5/F/38 1, 2, 3 IIa: ptosis, dysarthria, P, 60 mg 14 Unavailable; 3 13 60 CSR 38 Rtx Rtx every PR
dysphagia, limb AZA, 150 mg thymoma as per 6 mo
weakness chest CT
Durability of the Rituximab Response in Myasthenia Gravis

6/M/62 1, 2, 3 I: diplopia P, 50 mg NA NA 2 59 68 CSR 39 IVIg AZA, 100 mg PR

AZA, 150 mg
7/F/22 1, 2, 3 IIIa: ptosis, dysarthria, IVIg, 35 g every 38 Unavailable; no 3 51 77 CSR NA NA None CSR
dysphagia, limb other week thymoma as per
weakness medical record
8/M/26 1, 2, 3 IIIa: ptosis, diplopia, P, 60 mg 2 Normal 3 10 55 MM-0: ptosis, NA NA None MM-0: diplopia,
limb weakness Pyr prn diplopia, limb limb weakness

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weakness
9/M/63 1, 2 IIIb: diplopia, ptosis, P, 60 mg 32 Thymoma type B2 2 19 64 MM-0: 29 IVIg, Rtx IVIg every PR
dysarthria, dysphagia, AZA, 150 mg dysarthria 4 wk
10/F/36 1, 2, 3 I: diplopia, ptosis P, 10 mg 7 Invasive thymoma 3 11 53 PR 31 IVIg AZA, 100 mg PR
AZA, 100 mg type B3
c
11/F/24 1, 3 IIa: ptosis, limb P, 70 mg NA NA 2 32 51 PR NA NA P, 25 mg PR
weakness
12/F/28 1, 2, 3 IIa: ptosis, diplopia, P, 40 mg 22 Normal 4 61 66 MM-0: ptosis, 47 IVIg P, 20 mg MM-1: ptosis,
limb weakness MMF, 2 g diplopia AZA, 150 mg diplopia
13/F/65 2, 3 IIIa: ptosis, limb P, 20 mg 51 Thymoma class B2 2 57 43 PR 24 IVIg, Rtx P, 10 mg PR
weakness MMF, 1 g
14/F/33 1 IIa: diplopia, limb P, 60 mg 6 Thymic 2 22 24 CSR NA NA None CSR
weakness hyperplasia; no

Copyright 2016 American Medical Association. All rights reserved.

germinal centers
15/M/65 3 I: diplopia P, 40 mg NA NA 2 23 24 CSR NA NA None CSR
16/M/16 1, 2, 3 IIIa: ptosis, dysathria, P, 25 mg 40 Normal 2 90 18 CSR NA NA None CSR
limb weakness IVIg, every 3
wk
Abbreviations: AZA, azathioprine; CSR, complete stable remission (no symptoms or signs of MG for at least 1 year inhibitors] but no signs or symptoms for at least 1 year); prn, as needed; Pyr, pyridostigmine; Rtx, rituximab.
and no therapy for MG during that time); CT, computed tomography; IVIg, intravenous immunoglobulin; a
Refractory criteria include the following: (1) inability to lower immunotherapy without clinical relapse, (2) not
MGFA, Myasthenia Gravis Foundation of America; MM, minimal manifestations (no symptoms of functional clinically controlled with the immunotherapy regimen, and (3) severe adverse effects due to immunotherapy.
limitation from MG but some weakness on examination of some muscles); MMF, mycophenolate mofetil; b
Receiving Rtx for treatment of rheumatoid arthritis.
MM-0, MM no therapy for MG for at least 1 year; MM-1, MM with some form of immunotherapy for MG (excluding
c
cholinesterase inhibitors); NA, not applicable; P, prednisone; PIS, postintervention status; PLEX, plasma The patient had an unplanned pregnancy during the second cycle of Rtx treatment. Treatment was
exchange; PR, pharmacologic remission (some form of immunotherapy for MG [excluding cholinesterase discontinued. She had an uncomplicated pregnancy and delivery.
Original Investigation Research

(Reprinted) JAMA Neurology Published online November 21, 2016

E3
 Research Original Investigation
Figure 1. Durability of Response to Rituximab
Patient 1
Patient 2
Patient 3
Patient 4
Patient 5
Patient 6
Patient 7
Patient 8
Patient 9
Patient 10
Patient 11
Patient 12
Patient 13
Patient 14
Patient 15
Patient 16
90 80 70 60 50 40 30 20 10 0 10 20
Time, mo
measurement of B-cell counts was performed per best medi-
cal practice after completion of the initial set of cycles from a
safety perspective (ie, safety of vaccinations, etc). However,
owing to the retrospective nature of this work, we did not have
complete longitudinal data on B-cell counts for analysis.
Serum Affinity Proteomics
Preinfusion and postinfusion serial blood samples were col-
lected from 4 patients and 10 healthy control individuals af-
ter obtaining informed consent. Serum was obtained by cen-
trifugation of whole-blood samples and was cryopreserved at
80C. A fluorescent multiplexed magnetic beadbased screen-
ing assay (R&D Systems, Inc) was performed in accordance with
the manufacturers protocol for the following 10 cytokines:
interleukin 4 (IL-4), IL-5, IL-6, IL-10, IL-17A, IL-17F, tumor ne-
crosis factor, interferon , vascular endothelial growth fac-
tor, and resistin. The samples were analyzed in duplicate and
diluted 2-fold. Results were expressed as a mean value in pic-
tograms per milliliter. We used the Mann-Whitney test to
determine statistical significance (P < .05).
Results
Of the 16 patients in the study (6 men and 10 women; median
age, 42 [range, 18-69] years), 15 were receiving prednisone be-
fore initiating rituximab therapy (Table). Eight patients were
also receiving a corticosteroid-sparing agent (azathioprine in
6 and mycophenolate mofetil in 2). Thirteen had undergone
thymectomy (thymoma [5 patients], thymic hyperplasia [2 pa-
tients], and normal thymus [6 patients]). Eight patients were
treated with 2 cycles; 7 patients, with 3 cycles; and 1 patient,
with 4 cycles. A change in clinical status to improved was ob-
served in parallel with complete withdrawal or reduction of
other immunotherapies with all patients achieving complete
stable remission, pharmacologic remission, or minimal mani-
festations (MM) based on the MGFA postintervention status
criteria. After completing the initial set of rituximab treat-
ment cycles, 10 patients (63%) achieved complete stable re-
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Durability of the Rituximab Response in Myasthenia Gravis
Time of diagnosis
Time of relapse
Last follow-up Time of diagnosis, last follow-up visit,
and time of clinical relapse are
30 40 50 60 70 80 90 displayed. The black vertical line
crossing the x-axis at 0 indicates
initiation of treatment with rituximab.
mission; 3 patients (19%), pharmacologic remission (with aza-
thioprine in patient 10 and with prednisone in patients 11 and
13). The remaining 3 patients (19%) achieved MM-0 (minimal
manifestations but no therapy for MG). The 13 patients (with
complete stable remission and MM-0) who were able to taper
and discontinue all other immunotherapies were able to do so
in a mean of 8.3 (range, 1-15) months since the last infusion.
No infusion reactions were seen. One patient developed
leukopenia (white blood cell count, 2700/L [to convert to 109
per liter, multiply by 0.001]) after the second cycle, but this
resolved without intervention. Treatment had to be stopped
in 1 patient owing to an unplanned pregnancy during the sec-
ond cycle. She went on to have an uncomplicated pregnancy
and delivery.
Nine of 16 patients (56%) experienced a relapse in a mean
of 36 (range, 24-47) months after the last rituximab treat-
ment cycle (Figure 1). The 4 patients who had received 2 cycles
had a relapse within a mean follow-up of 33 months. The 4 pa-
tients who had received 3 cycles had a relapse within a mean
follow-up of 36 (range, 29-44) months. One patient received
4 cycles and had a relapse at 47 months. All of these patients
improved again after further immunosuppression therapy (in-
travenous immunoglobulin or plasma exchange in 7; high-
dose prednisone in 1; and an additional cycle of rituximab in
4; some received more than 1 treatment for relapse). The MGFA
postintervention status at their most recent follow-up was
pharmacologic remission (n = 7) or MM-1 (MM with some form
of immunotherapy for MG) (n = 2).
Seven of 16 patients (44%) remained clinically stable with
follow-up ranging from 18 to 81 months (mean follow-up, 47
months) (Figure 1). The MGFA postintervention status at their
most recent follow-up was complete stable remission (n = 5),
pharmacologic remission (n = 1), or MM-0 (n = 1).
Because we observed a mean time to relapse of 36 months,
we compared durability of benefit in patients followed up for
more than 48 months (12 patients) and 48 months or less (4 pa-
tients) after completion of the initial treatment regimen. Of those
patients with follow-up of more than 48 months, 8 of 12 pa-
tients had a relapse in a mean follow-up of 37 (range, 29-47)
jamaneurology.com
 Durability of the Rituximab Response in Myasthenia Gravis
months. The remaining 4 patients did not have a relapse, with
a mean follow-up period of 66 (range, 51-81) months. Of those
patients with a follow-up of 48 months or less, 1 of 4 had a re-
lapse at 24 months. The remaining 3 patients did not have a re-
lapse with a mean follow-up period of 22 (range, 18-24) months.
We also reviewed the time from diagnosis to initiation of first
treatment with rituximab in our cohort, which was a mean of 36
(range, 9-90) months. The mean duration of disease before treat-
ment in the relapse group was 31 (range, 9-61) months; in the non-
relapse group, 41 (range, 10-90) months (Figure 1). We observed
no difference in the time from diagnosis to initiation of rituximab
therapy and response durability based on these data.
A total of 13 patients in our cohort had a thymectomy.
Among the 6 patients who underwent thymectomy less than
12 months before starting treatment, 4 had a relapse in a mean
time of 36 (range, 29-44) months. Among the 7 patients who
underwent thymectomy more than 12 months before starting
treatment, 4 had a relapse in a mean time of 35 (range, 24-47)
months. The relapse rate was 67% in the group with thymec-
tomy less than 12 months before rituximab treatment (mean,
6.2 months) and 57% in the group with thymectomy more than
12 months before rituximab treatment (mean, 34 months). The
relapse rates appear similar between these 2 groups. Only 1 of
3 patients who did not undergo thymectomy had a relapse at
39 months. However, no firm conclusions can be drawn from
such a small number of patients.
Anti-AChR Antibody Levels
The following statistically significant decreases in anti-AChR
antibody levels was observed after treatment with the initial
set of rituximab cycles (Figure 2): 33% after cycle 1 (100% vs
67%; P = .004); 20% after cycle 2 (compared with cycle 1) (67%
vs 47%; P = .008); and 17% after cycle 3 (compared with cycle
2) (47% vs 30%; P = .02). In the patients who did not have a
relapse (eFigure 1A in the Supplement), a sustained decrease
in anti-AChR antibody level was noted until their last
follow-up (mean antibody level before rituximab treatment,
24.05 nmol/L; after last cycle, 10.8 nmol/L; and at last follow-
up, 15.65 nmol/L; P = .01). In the patients who had a relapse
after initial cycles of rituximab treatment (eFigure 1B in the
Supplement), no significant difference was noted in the anti-
AChR antibody levels at relapse (mean antibody level after last
cycle, 2.72 nmol/L; at the time of relapse, 2.87 nmol/L; P = .22).
However, 1 patient had a dramatic rise in anti-AChR antibody
level at the time of a second relapse that occurred in the set-
ting of a new diagnosis of stage IV adenocarcinoma with an
unknown primary site of origin.
Serum Cytokine Levels
Levels of 8 of the 10 cytokines measured (IL-4, IL-5, IL-6, IL-
10, IL-17A, IL-17F, tumor necrosis factor, and interferon ) were
below the level of detection for the assay, implying that their
levels in serum were not elevated. No statistically significant
differences in serum levels of resistin and vascular endothe-
lial growth factor were observed between healthy donor and
patient serum samples before the initiation of rituximab treat-
ment (eFigure 2 in the Supplement). We also found no appre-
ciable change in the concentrations of resistin and vascular
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Original Investigation Research
Figure 2. Levels of Acetylcholine Receptor (Anti-AChR) Autoantibodies
Before and After Treatment
a b c,d
125
100
Anti-AChR Antibody Level, %
75
50
25
0
Pretreatment Cycle 1 Cycle 2 Cycle 3
Anti-AChR antibody levels (normalized to a pretreatment value of 100%) before
and after each treatment cycle with rituximab. The orange bars indicate the
median values. Eight patients were treated with 2 cycles of rituximab; 7, with 3
cycles. One patient not shown because data were unavailable had 4 cycles of
treatment. Data points indicate individual patients.
a
Compared with pretreatment levels, P = .005 in anti-AChR antibody levels
after cycle 1.
b
Compared with cycle 1, P = .009 in anti-AChR antibody levels after cycle 2.
c
Compared with cycle 2, P = .01 in anti-AChR antibody levels after cycle 3.
d
Compared with pretreatment levels, P < .001 in anti-AChR antibody levels
after all 3 cycles.
endothelial growth factor with rituximab treatment or after
achieving a state of clinical remission, during longitudinal
follow-up. One of the 4 patients studied experienced a re-
lapse, but there was no significant difference observed in the
levels of these 2 cytokines before and after relapse.
Discussion
The need for additional treatments, particularly for patients
who do not respond to or have intolerable adverse effects from
existing immunosuppressive therapy, has led to an interest in
targeted immunotherapies. Rituximab is an appealing choice
owing to its B-celltargeting mechanism of action and the pre-
cedence for its use in the treatment of other autoimmune
diseases, such as rheumatoid arthritis.7,20,21
In this retrospective analysis of 16 patients with refrac-
tory AChR+ MG, rituximab appears to have a durable re-
sponse. Our results support the hypothesis that rituximab can
be helpful in managing refractory MG. These findings are in
agreement with previous reports of its benefit.9,10,22,23
The number of rituximab treatment cycles necessary to
achieve disease remission has been unclear. A minimum of 2
cycles appear to be needed, because most of the patients in our
cohort required approximately 1 year to taper other thera-
pies. As observed in previous studies,10 some patients may
need additional cycles. Although patients treated with more
cycles tended to have a longer-lasting response, our sample size
in each group is too small to draw any firm conclusions. To date,
(Reprinted) JAMA Neurology Published online November 21, 2016 E5
 Research Original Investigation Durability of the Rituximab Response in Myasthenia Gravis

guidelines on when to discontinue or repeat rituximab treat-
ment are yet to be established. As a practical matter, patients
with evidence of clinical disease relapse and a minimum of 6
months since the last cycle should be considered for retreat-
ment in the absence of medical contraindications.
The relapse rate in our cohort was 56%, typically occur-
ring about 3 years after the last rituximab treatment cycle. This
relapse rate is similar to those of previously reported cohorts,10
but after a longer duration of disease stability. After an induc-
tion regimen, a mean time to relapse of 17 (ranging, 6-34)
months was observed in an independent study.10 The pa-
tients with relapse in our cohort were able to achieve clinical
improvement again after treatment with further immunosup-
pression. Thymectomy is certainly a possible confounder. Ac-
knowledging the limitation of our sample size, thymectomy
status, timing of thymectomy or pathologic findings in the thy-
mus did not seem to influence disease relapse or durability of
response in our cohort.
Biomarkers would be very helpful in guiding clinicians as
to whom to offer additional cycles. Anti-AChR antibody lev-
els can be helpful in assessing the response to treatment be-
cause these levels were noted to decrease after the adminis-
tration of rituximab. However, their role in predicting relapse
is less clear because we did not note any significant increase
in the levels at the time of relapse, although our small sample
size limits definitive conclusion.
Our analysis of 10 cytokines that have been associated with
the immunopathogenesis of MG24,25 did not reveal any appre-
ciable changes with B-cell depletion, clinical remission, or re-
lapse. Acknowledging the limitation of sample size, further
studies are needed to attribute value to these cytokines as bio-
markers and to identify other indicators of disease activity and
response to therapy.
The effects of rituximab on B cells as well as putative T-cell
mediated immune dysregulation in MG need further investiga-
tion. We plan to apply recently developed assays26,27 to the pro-
spective clinical trial of rituximab in MG currently under way.28
All patients followed up in this study tolerated rituximab
with no severe hematologic derangements or infusion reac-
tions. Although the most common adverse effect is an infu-
sion reaction, progressive multifocal leukoencephalopathy is
also of concern after rituximab therapy29,30; however, the rela-
tive risk is thought to be low.31 A recent case report32 has de-
scribed the occurrence of progressive multifocal leukoen-
cephalopathy in a patient with seronegative MG, having been
treated with rituximab in addition to prednisone, azathio-
prine, mycophenolate mofetil, intravenous immunoglobu-
lin, and plasma exchange at different times during the course
of disease. What role aggressive, long-term immunosuppres-
sion therapy had in this case is unclear. Nevertheless, clinical
monitoring per best medical practice standards and minimiz-
ing combination immunosuppressive regimens is required and
strongly advised when considering the initiation of ritux-
imab therapy.
Conclusions
We found B-cell depletion therapy to be an effective option with
sustained long-term benefit after treatment in patients with
refractory AChR+ MG. This study represents, to our knowl-
edge, one of the largest single-center studies with extended
long-term follow-up. A prospective, placebo-controlled clini-
cal trial is currently under way that will further help to evalu-
ate the efficacy, safety, and pharmacodynamics of rituximab
in MG.28 Identification of markers of disease activity, respon-
siveness to therapy, clinical relapse, and remission are criti-
cal next steps in the development of evidence-based practice
parameters for rituximab in the treatment of MG as well as other
potential target therapeutics.

ARTICLE INFORMATION
Accepted for Publication: August 26, 2016.
Published Online: November 21, 2016.
doi:10.1001/jamaneurol.2016.4190
Author Contributions: Drs Robeson and Kumar
contributed equally to this study and are cofirst
authors. Dr Nowak had full access to all the data in
the study and takes full responsibility for the
integrity of the data and the accuracy of the data
analysis.
Study concept and design: Robeson, Kumar, Keung,
Goldstein, OConnor, Nowak.
Acquisition, analysis, or interpretation of data: All
authors.
Drafting of the manuscript: Robeson, Kumar,
Nowak.
Critical revision of the manuscript for important
intellectual content: All authors.
Statistical analysis: Robeson, Kumar, Nowak.
Study supervision: Nowak.
Conflict of Interest Disclosures: Dr Nowak reports
receiving grant U01NS084495-01A1 from the
National Institute of Neurological Disorders and
Stroke (NINDS), National Institutes of Health (NIH),
to conduct the currently underway prospective
phase 2 trial of rituximab in myasthenia gravis along
with drug and placebo provided by Genentech
through an investigator-initiated trial agreement,
which is separate from the research in this article.
Dr OConnor reports receiving honoraria (speaking
fees) from Genentech. No other disclosures were
reported.
Funding/Support: Dr Nowak was supported in
part, by award U01NS084495-01A1 from the
NINDS of the NIH. This study was supported, in
part, by grant R01AI114780 from the National
Institute of Allergy and Infectious Diseases of the
NIH (Dr OConnor).
Role of the Funder/Sponsor: The funding sources
had no role in the design and conduct of the study;
collection, management, analysis, and
interpretation of the data; and preparation, review,
or approval of the manuscript; and decision to
submit the manuscript for publication.
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