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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 CME Quiz at
jamanetworkcme.com
B-cell–targeted therapy with rituximab have been observed in MG; however, the duration of
these benefits after treatment is unclear.
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.
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.
60 (Reprinted) jamaneurology.com
M
yasthenia gravis (MG) is an autoimmune disorder af-
fecting neuromuscular transmission with an esti- Key Points
mated annual incidence of about 1.7 to 21.3 cases per
Question Is the rituximab response in treatment-refractory
1 million person-years and prevalence as high as 15 to 179 per acetylcholine receptor autoantibody–positive myasthenia gravis
1 million persons. 1 Control of symptoms can be initially (AChR+ MG) durable?
achieved with acetylcholinesterase inhibitors; however, most
Findings In this case series study of 16 patients with AChR+ MG
patients require immunotherapy such as corticosteroids, aza-
who were treated with rituximab and followed up for 18 to 84
thioprine, cyclosporine, mycophenolate mofetil, plasma ex- months, all patients were observed to have clinical improvement.
change, and intravenous immunoglobulin at some point in their Nine patients had a relapse within a mean of 36 months after the
disease course to achieve sustained clinical remission.2-4 Thy- last treatment cycle; the remaining 7 continued to maintain clinical
mectomy, regardless of the presence of a thymoma, is also con- benefit during a mean follow-up of 47 months.
sidered a therapeutic option based on prior clinical experi- Meaning Rituximab appears to be an effective option with
ence; however, results and conclusions from the recently sustained long-term benefit after treatment in patients with
completed placebo-controlled thymectomy trial in nonthy- refractory AChR+ MG.
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 symptoms, and examination findings were evaluated. The My-
Autoreactive B cells have a clear pathogenic role in the de- asthenia Gravis Foundation of America (MGFA) clinical classi-
velopment of MG, and B-cell–directed therapy has emerged as fication criteria19 and postintervention status were used to as-
a highly effective tool in managing other autoimmune dis- sign clinical state a minimum of 12 months after completion
eases such as rheumatoid arthritis and neuromyelitis optica.7,8 of the initial set of rituximab cycles. The number of adminis-
Several groups9-18 have also observed the benefits of ritux- tered rituximab treatment cycles, time since the last treat-
imab, a chimeric anti-CD20 monoclonal antibody, in patients ment cycle, and times to relapse and postrelapse treatments
with MG. In addition to significant clinical improvement, ritux- were reviewed. In addition, anti-AChR antibody levels, mea-
imab also allowed for tapering and subsequent discontinua- sured by conventional radioimmunoassays (reference value,
tion of other immunotherapies in patients with acetylcholine ≤0.02 nmol/L; Mayo Medical Laboratories), were assessed in
receptor autoantibody–positive (AChR+) and muscle-specific ki- patients before initiation of rituximab therapy, at the end of each
nase autoantibody–positive MG.9 However, the duration of clini- cycle, at the time of clinical relapse, and at the last follow-up.
cal remission among patients treated with rituximab while not Statistical analysis was performed using GraphPad Prism
receiving other immunotherapy, especially considering the prior (GraphPad Software), and P values were calculated using non-
refractory nature of their disease, is unclear. The number of ri- parametric Friedman and Wilcoxon matched-pairs signed rank
tuximab treatment cycles necessary to achieve long-term re- tests.
mission also remains unknown. Herein, we report our experi-
ence with the long-term effects of rituximab in 16 patients with Rituximab
refractory AChR+ MG who were followed up for 18 to 84 months, Because no established infusion protocol for rituximab use in
representing to our knowledge the longest follow-up of a single MG currently exists, we used a standard protocol adopted from
MG cohort to date. the non–Hodgkin 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
Methods infusion center at our institution. Our patients were treated
Patients with an initial 2- to 4-cycle regimen. The number of cycles
This retrospective study included patients with generalized MG was mainly based on reaching a symptom-free state and
referred to the Yale Myasthenia Gravis Clinic, New Haven, Con- patient toleration of tapering or withdrawal of other immu-
necticut, from January 1, 2007, to December 31, 2015. Sixteen notherapies (ie, corticosteroids). The number of rituximab
patients with AChR+ refractory generalized disease and a mini- treatment cycles or interval between cycles was not dictated
mum of 12 months of follow-up after completion of the initial by B-cell counts.
set of rituximab treatment cycles were identified (Table). This
study was approved by the institutional review board of Yale Conventional Immunotherapy
University as part of an observational study examining the All prior immunotherapies were reviewed. These included
treatment and disease course of MG. All patients provided writ- prednisone, which is the standard first-line agent; plasma ex-
ten informed consent. change; azathioprine; mycophenolate mofetil; and intrave-
Disease was defined as refractory when the immuno- nous immunoglobulin.
therapy dosage could not be lowered without clinical relapse,
inadequate clinical control of the disease was achieved during Safety and Adverse Effects
the immunotherapy regimen, or severe adverse effects due to In addition to clinical follow-up, we reviewed the infusion cen-
current immunosuppressive therapy were present. Pretreat- ter notes and monitored complete blood cell counts and liver
ment and posttreatment immunotherapy regimens, clinical function test profiles to evaluate the safety profile. Periodic
jamaneurology.com
Durability of the Rituximab Response in Myasthenia Gravis
Durability of the Rituximab Response in Myasthenia Gravis Original Investigation Research
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 Time of diagnosis
Time of relapse
Patient 15
Last follow-up Time of diagnosis, last follow-up visit,
Patient 16
and time of clinical relapse are
−90 −80 −70 −60 −50 −40 −30 −20 −10 0 10 20 30 40 50 60 70 80 90 displayed. The black vertical line
Time, mo crossing the x-axis at 0 indicates
initiation of treatment with rituximab.
measurement of B-cell counts was performed per best medi- mission; 3 patients (19%), pharmacologic remission (with aza-
cal practice after completion of the initial set of cycles from a thioprine in patient 10 and with prednisone in patients 11 and
safety perspective (ie, safety of vaccinations, etc). However, 13). The remaining 3 patients (19%) achieved MM-0 (minimal
owing to the retrospective nature of this work, we did not have manifestations but no therapy for MG). The 13 patients (with
complete longitudinal data on B-cell counts for analysis. complete stable remission and MM-0) who were able to taper
and discontinue all other immunotherapies were able to do so
Serum Affinity Proteomics in a mean of 8.3 (range, 1-15) months since the last infusion.
Preinfusion and postinfusion serial blood samples were col- No infusion reactions were seen. One patient developed
lected from 4 patients and 10 healthy control individuals af- leukopenia (white blood cell count, 2700/μL [to convert to ×109
ter obtaining informed consent. Serum was obtained by cen- per liter, multiply by 0.001]) after the second cycle, but this
trifugation of whole-blood samples and was cryopreserved at resolved without intervention. Treatment had to be stopped
−80°C. A fluorescent multiplexed magnetic bead–based screen- in 1 patient owing to an unplanned pregnancy during the sec-
ing assay (R&D Systems, Inc) was performed in accordance with ond cycle. She went on to have an uncomplicated pregnancy
the manufacturer’s protocol for the following 10 cytokines: in- and delivery.
terleukin 4 (IL-4), IL-5, IL-6, IL-10, IL-17A, IL-17F, tumor ne- Nine of 16 patients (56%) experienced a relapse in a mean
crosis factor, interferon γ, vascular endothelial growth fac- of 36 (range, 24-47) months after the last rituximab treat-
tor, and resistin. The samples were analyzed in duplicate and ment cycle (Figure 1). The 4 patients who had received 2 cycles
diluted 2-fold. Results were expressed as a mean value in pic- had a relapse within a mean follow-up of 33 months. The 4 pa-
tograms per milliliter. We used the Mann-Whitney test to de- tients who had received 3 cycles had a relapse within a mean
termine statistical significance (P < .05). 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-
Results dose prednisone in 1; and an additional cycle of rituximab in
Of the 16 patients in the study (6 men and 10 women; median 4; some received more than 1 treatment for relapse). The MGFA
age, 42 [range, 18-69] years), 15 were receiving prednisone be- postintervention status at their most recent follow-up was
fore initiating rituximab therapy (Table). Eight patients were pharmacologic remission (n = 7) or MM-1 (MM with some form
also receiving a corticosteroid-sparing agent (azathioprine in of immunotherapy for MG) (n = 2).
6 and mycophenolate mofetil in 2). Thirteen had undergone Seven of 16 patients (44%) remained clinically stable with
thymectomy (thymoma [5 patients], thymic hyperplasia [2 pa- follow-up ranging from 18 to 81 months (mean follow-up, 47
tients], and normal thymus [6 patients]). Eight patients were months) (Figure 1). The MGFA postintervention status at their
treated with 2 cycles; 7 patients, with 3 cycles; and 1 patient, most recent follow-up was complete stable remission (n = 5),
with 4 cycles. A change in clinical status to improved was ob- pharmacologic remission (n = 1), or MM-0 (n = 1).
served in parallel with complete withdrawal or reduction of Because we observed a mean time to relapse of 36 months,
other immunotherapies with all patients achieving complete we compared durability of benefit in patients followed up for
stable remission, pharmacologic remission, or minimal mani- more than 48 months (12 patients) and 48 months or less (4 pa-
festations (MM) based on the MGFA postintervention status tients) after completion of the initial treatment regimen. Of those
criteria. After completing the initial set of rituximab treat- patients with follow-up of more than 48 months, 8 of 12 pa-
ment cycles, 10 patients (63%) achieved complete stable re- tients had a relapse in a mean follow-up of 37 (range, 29-47)
guidelines on when to discontinue or repeat rituximab treat- tion. We plan to apply recently developed assays26,27 to the pro-
ment are yet to be established. As a practical matter, patients spective clinical trial of rituximab in MG currently under way.28
with evidence of clinical disease relapse and a minimum of 6 All patients followed up in this study tolerated rituximab
months since the last cycle should be considered for retreat- with no severe hematologic derangements or infusion reac-
ment in the absence of medical contraindications. tions. Although the most common adverse effect is an infu-
The relapse rate in our cohort was 56%, typically occur- sion reaction, progressive multifocal leukoencephalopathy is
ring about 3 years after the last rituximab treatment cycle. This also of concern after rituximab therapy29,30; however, the rela-
relapse rate is similar to those of previously reported cohorts,10 tive risk is thought to be low.31 A recent case report32 has de-
but after a longer duration of disease stability. After an induc- scribed the occurrence of progressive multifocal leukoen-
tion regimen, a mean time to relapse of 17 (ranging, 6-34) cephalopathy in a patient with seronegative MG, having been
months was observed in an independent study.10 The pa- treated with rituximab in addition to prednisone, azathio-
tients with relapse in our cohort were able to achieve clinical prine, mycophenolate mofetil, intravenous immunoglobu-
improvement again after treatment with further immunosup- lin, and plasma exchange at different times during the course
pression. Thymectomy is certainly a possible confounder. Ac- of disease. What role aggressive, long-term immunosuppres-
knowledging the limitation of our sample size, thymectomy sion therapy had in this case is unclear. Nevertheless, clinical
status, timing of thymectomy or pathologic findings in the thy- monitoring per best medical practice standards and minimiz-
mus did not seem to influence disease relapse or durability of ing combination immunosuppressive regimens is required and
response in our cohort. strongly advised when considering the initiation of ritux-
Biomarkers would be very helpful in guiding clinicians as imab therapy.
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
Conclusions
is less clear because we did not note any significant increase We found B-cell depletion therapy to be an effective option with
in the levels at the time of relapse, although our small sample sustained long-term benefit after treatment in patients with
size limits definitive conclusion. refractory AChR+ MG. This study represents, to our knowl-
Our analysis of 10 cytokines that have been associated with edge, one of the largest single-center studies with extended
the immunopathogenesis of MG24,25 did not reveal any appre- long-term follow-up. A prospective, placebo-controlled clini-
ciable changes with B-cell depletion, clinical remission, or re- cal trial is currently under way that will further help to evalu-
lapse. Acknowledging the limitation of sample size, further ate the efficacy, safety, and pharmacodynamics of rituximab
studies are needed to attribute value to these cytokines as bio- in MG.28 Identification of markers of disease activity, respon-
markers and to identify other indicators of disease activity and siveness to therapy, clinical relapse, and remission are criti-
response to therapy. cal next steps in the development of evidence-based practice
The effects of rituximab on B cells as well as putative T-cell– parameters for rituximab in the treatment of MG as well as other
mediated immune dysregulation in MG need further investiga- potential target therapeutics.
ARTICLE INFORMATION with drug and placebo provided by Genentech 2. Gold R, Schneider-Gold C. Current and future
Accepted for Publication: August 26, 2016. through an investigator-initiated trial agreement, standards in treatment of myasthenia gravis.
which is separate from the research in this article. Neurotherapeutics. 2008;5(4):535-541.
Published Online: November 21, 2016. Dr O’Connor reports receiving honoraria (speaking
doi:10.1001/jamaneurol.2016.4190 3. Sathasivam S. Steroids and immunosuppressant
fees) from Genentech. No other disclosures were drugs in myasthenia gravis. Nat Clin Pract Neurol.
Author Contributions: Drs Robeson and Kumar reported. 2008;4(6):317-327.
contributed equally to this study and are co–first Funding/Support: Dr Nowak was supported in
authors. Dr Nowak had full access to all the data in 4. Conti-Fine BM, Milani M, Kaminski HJ.
part, by award U01NS084495-01A1 from the Myasthenia gravis: past, present, and future. J Clin
the study and takes full responsibility for the NINDS of the NIH. This study was supported, in
integrity of the data and the accuracy of the data Invest. 2006;116(11):2843-2854.
part, by grant R01AI114780 from the National
analysis. Institute of Allergy and Infectious Diseases of the 5. clinicaltrials.gov. A Multi-Center, Single-Blind,
Study concept and design: Robeson, Kumar, Keung, NIH (Dr O’Connor). Randomized Study Comparing Thymectomy to No
Goldstein, O’Connor, Nowak. Thymectomy in Non-Thymomatous Myasthenia
Acquisition, analysis, or interpretation of data: All Role of the Funder/Sponsor: The funding sources Gravis (MG) Patients Receiving Prednisone.
authors. had no role in the design and conduct of the study; NCT00294658. https://clinicaltrials.gov/ct2/show
Drafting of the manuscript: Robeson, Kumar, collection, management, analysis, and /NCT00294658. Accessed June 13, 2016.
Nowak. interpretation of the data; and preparation, review,
or approval of the manuscript; and decision to 6. Suh J, Goldstein JM, Nowak RJ. Clinical
Critical revision of the manuscript for important characteristics of refractory myasthenia gravis
intellectual content: All authors. submit the manuscript for publication.
patients. Yale J Biol Med. 2013;86(2):255-260.
Statistical analysis: Robeson, Kumar, Nowak.
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