Expert Review of Clinical Immunology

ISSN: 1744-666X (Print) 1744-8409 (Online) Journal homepage: http://www.tandfonline.com/loi/ierm20

Overcoming challenges in the diagnosis and

treatment of myasthenia gravis

Amelia Evoli, Raffaele Iorio & Emanuela Bartoccioni

To cite this article: Amelia Evoli, Raffaele Iorio & Emanuela Bartoccioni (2016) Overcoming
challenges in the diagnosis and treatment of myasthenia gravis, Expert Review of Clinical
Immunology, 12:2, 157-168, DOI: 10.1586/1744666X.2016.1110487

To link to this article: http://dx.doi.org/10.1586/1744666X.2016.1110487

Published online: 16 Dec 2015.

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 EXPERT REVIEW OF CLINICAL IMMUNOLOGY, 2016
VOL. 12, NO. 2, 157–168
http://dx.doi.org/10.1586/1744666X.2016.1110487

REVIEW

Overcoming challenges in the diagnosis and treatment of myasthenia gravis

Amelia Evolia, Raffaele Iorioa,b and Emanuela Bartoccionic
a
Institute of Neurology, Catholic University, Roma, Italy; bDon Gnocchi ONLUS Foundation, Milan, Italy; cInstitute of General Pathology,
Catholic University, Roma, Italy

ABSTRACT ARTICLE HISTORY

In recent years, the discovery of new autoantigens and the use of sensitive assays have expanded Received 31 August 2015
the clinical spectrum of myasthenia gravis (MG). In particular, antibodies binding to clustered Accepted 16 October 2015
acetylcholine receptors and to the low-density lipoprotein receptor-related protein 4 have not KEYWORDS
only bridged a significant gap in diagnosis but also have relevant clinical implications. MG AChR; clustered-AChR;
management includes different therapeutic options, from symptomatic agents as the only immunosuppressants; Lrp4;
therapy in mildly affected cases to combined long-term immunosuppression and thymectomy myasthenia gravis; MuSK;
Downloaded by [University of New England] at 09:05 11 November 2017

in patients with severe disabling disease. MG biological diversity can influence the response to thymectomy
therapies and should be taken into account when planning treatment. Biologic agents are
promising, though their use is currently limited to patients with refractory disease.

Introduction with thymoma, a tumor of thymic epithelial cells, that is

Myasthenia gravis (MG) is the most common disorder of
neuromuscular junction (NMJ), with a prevalence
approaching 200/million. It affects all races and can
occur at any age, from the first year to the nineties. In
Western countries, MG shows a peak age of onset in the
third decade in women and a larger peak in the sixth
and seventh decades in men, while in Asian countries
there is a high prevalence of limited forms of the dis-
ease with childhood onset.[1] MG is increasingly recog-
nized as a heterogeneous disease including distinct
clinical entities.
MG is due to autoantibodies (Abs) that, with differ-
ent mechanisms, impair neuromuscular transmission
(NMT) and cause fatigable weakness of voluntary mus-
cles. The great majority of patients have serum Abs to
the acetylcholine receptor (AChR). These Abs are mainly
of IgG1 and IgG3 isotypes, induce severe alterations of
the postsynaptic membrane through complement acti-
vation and increased AChR degradation, and, to a lesser
extent, interfere with the neurotransmitter binding site.
[2] MG with AChR Abs (AChR-MG) is frequently asso-
ciated with thymus alterations that are thought to play
a critical role in the disease pathogenesis. Thymus folli-
cular hyperplasia (TFH), typically found in female
patients with early onset MG, is considered the site
where the autoimmune response to the AChR is
initiated and maintained. Conversely, defects of T cell
selection appears to be responsible for MG association
present in 10–15% of patients, more commonly in the
fifth and sixth decades of life.[3]
Serum Abs to the muscle-specific tyrosine kinase
receptor (MuSK) are detected in 5–8% of MG patients.
MuSK is the core of a multiprotein complex, that is
critical for AChR clustering and postsynaptic configura-
tion.[4] As experimental models have shown, MuSK Abs
interfere with the protein function[5]; although being
mostly IgG4, they cannot activate complement and are
rather inefficient in antigen cross-linking.[2] In addition,
the low-density lipoprotein receptor-related protein 4
(Lrp4), a receptor for agrin that mediates MuSK activa-
tion,[6] has been identified as autoantigen in MG (see
Figure 1). The frequency of Lrp4 Abs in patients with
neither AChR- nor MuSK-Abs shows marked variability in
different studies [7]; the Ab effects and clinical pheno-
types are not fully characterized. While the search for
novel antigens in MG is actively pursued, there are still
some patients without detectable Abs. The frequency of
these so-called ‘triple-negative’ cases is unknown.
In MG, pathogenic heterogeneity is associated with
marked variability in weakness extension and severity,
from purely ocular symptoms to severe life-threatening
disease. A correct diagnosis is crucial in order to offer
patients appropriate treatment.
MG management has steadily improved over the
years. However, there are still uncertainties and contro-
versies both in the diagnostic workup and therapeutic
strategy. This review will discuss how recent advances

CONTACT Amelia Evoli a.evoli@rm.unicatt.it

© 2015 Taylor & Francis
 158 A. EVOLI ET AL.
Downloaded by [University of New England] at 09:05 11 November 2017

Figure 1. Schematic representation of the acetylcholine receptor, the muscle-specific tyrosine kinase receptor and low-density
lipoprotein receptor-related protein 4 complex at the neuromuscular junction.
AChE: acetylcholinesterase; AChR: acetylcholine receptor; Lrp4: Low-density lipoprotein receptor-related protein 4; MuSK: muscle-
specific tyrosine kinase receptor.Modified and reprinted with permission from Evoli and Iorio, Clinical Experimental
Neuroimmunology, 2015; license number: 3696030334051.

in clinical and basic research may help clinicians in the
care of these patients.
Establishing the diagnosis of MG
MG is suspected in patients with history and signs of
fluctuating weakness of voluntary muscles, worsening
on exertion and improving at rest. Diagnosis confirma-
tion is then achieved through: a) detection of serum Abs;
b) electromyography (EMG) studies showing compound
muscle action potential decrement on low-rate repetitive
nerve stimulation (RNS) or increased jitter on single-fiber
(SF) EMG; c) clinical response to acetylcholinesterase inhi-
bitors (AChE-Is). Positive results on b) and c) confirm a
postsynaptic defect of NMT, while detection of specific
Abs establishes the diagnosis of MG.
Routine serological testing includes AChR and MuSK
Ab assays. Given to their high prevalence in MG, AChR
Abs are the first to be tested when MG is suspected on
clinical grounds; patients with negative results on this
assay should be tested for MuSK Abs. The coexistence
of these Abs is very rare.[8] AChR and MuSK Abs are
considered very specific for MG and, in practice, their
detection in patients with consistent symptoms con-
firms the diagnosis, with no real need for additional
testing. On the other hand, when Ab assays are nega-
tive, EMG confirmation is crucial.
Electrophysiological examination should include
conventional needle EMG and neurographic studies
to rule out diseases that can mimic MG. RNS is the
routine technique in the investigation of NMT disor-
ders; it is considered fairly specific, though a decre-
mental response can also be found in motor neuron
disease and radiculopathy.[9] The diagnostic sensitiv-
ity of RNS is related to weakness pattern. The rate of
positive results is around 70% in patients with general-
ized disease when both distal and proximal muscles
are tested, while it is less than 50% in ocular myasthe-
nia and in patients with focal cranial symptoms.[10]
SF-EMG is regarded as the most sensitive diagnostic
test in MG, as positive results can be recorded in >90%
of patients, including those with limited weakness,
when appropriate muscles are examined. However,
increased jitter is far from specific and can be found
in several neuropathic and myopathic conditions.[9] In
addition, SF-EMG technique is time-consuming and
requires patient’s cooperation. In clinical practice, the
choice between RNS and SF-EMG depends on their
relative sensitivity in relation to weakness extension:
SF-EMG has a stronger indication in subjects with
purely ocular or focal symptoms and in those with
mild weakness; RNS is generally adequate in patients
with more relevant generalized symptoms.
A clear-cut clinical improvement on administration
of short-acting AChE-Is, as edrophonium chloride iv. or

neostigmine im., strongly supports the diagnosis of MG
and a positive response to this ‘pharmacological’ test
has been reported in approximately 90% of MG
patients.[10] However, the response rate is much
lower (50–70%) in MuSK-MG [11]; in addition, a positive
reaction to AChE-I is not specific for MG, as it is usually
present in congenital myasthenic syndromes and can
be found in Lambert–Eaton myasthenic syndrome,
amyotrophic lateral sclerosis (ALS), Guillain–Barrè syn-
drome, and, even, in patients with intracranial
tumors.[1]
In patients with negative AChR and MuSK Ab assays,
MG must first be differentiated from other primary dis-
orders of NMT, while, in selected cases, ALS, Miller
Fisher syndrome, cranial neuropathy and mitochondrial
myopathy must be considered in the differential diag-
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nosis. Among NMT disorders, Lambert–Eaton myasthe-
nic syndrome and botulism show characteristic clinical
and EMG findings that can assist in establishing the
correct diagnosis, while MG differentiation from a con-
genital myasthenic syndrome may be far more challen-
ging. Patients with purely ocular symptoms, and
negative or equivocal results of EMG (including SF-
EMG) tests should undergo brain MRI, and MG diagno-
sis should be reconsidered after the exclusion of other
conditions that may cause ptosis and/or diplopia.
New Abs have recently added to laboratory testing
in MG. Herein, we discuss the contribution of standard
and newly described Ab assays to the clinical diagnosis.
Diagnostic yield of AChR and MuSK Abs
AChR- and MuSK Abs are routinely measured by a
radioimmunoprecipitation assay (RIPA) that is sensitive,
quantitative and widely available. AChR Ab testing by
enzyme-linked immunoassay (ELISA), that offers the
obvious advantage of not requiring radioactive iso-
topes, proved less sensitive, especially in patients with
border-line Ab titers.[12]
By RIPA, Abs to AChR are detected as IgG binding to
125
I-α-bungarotoxin-labeled solubilized AChR (binding
Abs). Modulating and blocking Abs, which can be mea-
sured with distinct assays,[13] increase to a limited
extent the sensitivity of the standard RIPA and have
not entered routine practice. Binding AChR Abs are
detected in 80–90% of patients with generalized MG,
in roughly 50% of those with symptoms restricted to
ocular muscles, and in virtually all cases with thymoma-
associated MG.[8,14] Diagnostic sensitivity is increased
by testing nonimmunosuppressed patients, while
AChR-negative patients with recent-onset disease may
turn positive when retested after 6–12 months (sero-
conversion).[8] On the other hand, AChR Ab positivity
EXPERT REVIEW OF CLINICAL IMMUNOLOGY 159
rate is lower in childhood-onset MG,[15] and in milder
forms of the disease.[13]
MuSK Abs are detected by the standard RIPA in
30–40% of AChR-negative patients, with variable fre-
quency across populations.[8] These Abs are very rare
in ocular MG and are most commonly associated with a
clinical pattern of predominant weakness in cranial,
neck and respiratory muscles. MuSK-MG is uncommon
in childhood, with a mean age of onset in the fourth
decade.[11]
The diagnostic accuracy of the standard RIPA for
AChR Ab ranges 97–99%,[10] as positive results may
be rarely found in other autoimmune conditions and
in thymoma without MG, while MuSK Abs have never
been reported in non-MG patients.[8] On the whole,
standard RIPA for AChR- and MuSK Abs can confirm
the diagnosis of MG in around 90% patients, with a
higher rate of positive results in adults with generalized
disease.
MG with neither AChR nor MuSK Abs on the stan-
dard assay (so-called double seronegative myasthenia
gravis (dSN-MG) has variable frequency in different
patient cohorts (in part, depending on the relative
rate of MuSK-MG), wide clinical spectrum and broad
range of age-of-onset. In recent years, development of
sensitive cell-based assays (CBAs) has led to detection
of new Abs in this heterogeneous population.
In CBA, antigens are expressed in cell lines, usually
human embryonic kidney cells, transfected with the
appropriate cDNAs and the Ab binding is detected by
indirect immunofluorescence. This sensitive nonra-
dioactive method could ideally be used in patients’
first screening. However, in comparison with RIPA,
CBA cannot provide an exact quantification of Ab
titer, and its use is currently limited to selected labora-
tories with specific facilities and expertise.[16,17]
Clustered-AChR Abs
A significant improvement in the serological diagnosis
of MG was the demonstration of serum Abs to clus-
tered AChR. Earlier reports had shown that a propor-
tion of dSN patients had a clinical picture
undistinguishable from AChR-MG [18,19] and often
harbored thymus hyperplastic changes, that are defi-
nitely uncommon in MuSK-MG.[20–22] These observa-
tions suggested the possible presence of Abs unable
to attach to AChR in solution, but able to bind to
densely packed AChRs, as they are in vivo.[23] To test
this hypothesis, the neuroscience group in Oxford
developed a CBA, using human embryonic kidney
293 cells transfected with cDNA from AChR subunits
and from rapsyn, in order to have, on cell surface,
 160 A. EVOLI ET AL.
AChR clusters similar to those at the NMJ. With this
assay, they were able to detect AChR Abs in a high
proportion of dSN patients, including some cases with
purely ocular disease.[23–25] These Abs were found
to be mostly complement-activating IgG1,[23] and
proved pathogenic in a passive transfer model.[25]
Recently, the same authors reported clustered-AChR
Abs in 38% dSN-MG patients most frequently in
patients with prepubertal age of onset and purely
ocular symptoms.[26] These results, if confirmed,
would strengthen the diagnostic value of clustered-
AChR Abs, given their high positivity rate in patient
subgroups, as ocular myasthenia and pediatric cases,
in whom standard Ab assays have a lower diagnostic
yield and diagnostic confirmation may be trying. Two
recent reports [27,28] came to somewhat different
Downloaded by [University of New England] at 09:05 11 November 2017
conclusions. In a French study, clustered-AChR Abs
were detected in 16% dSN-MG, mostly affected with
mild disease.[27] In a Chinese study, patients with Abs
only to clustered AChR accounted for 45.8% dSN
cases, with a 27% rate of ocular myasthenia and a
strong association with thymoma.[28] These discre-
pancies may be due to technical differences, possible
biases in patients’ selection or referral, and ethnic
factors, as already noted for MuSK Abs.
CBA may improve MuSK Ab detection in RIPA-nega-
tive patients.[24,29] In a multicenter study, MuSK Abs
were detected in 13% of ‘triple-negative’ patients, in
1.9% of controls and in 5.1% of patients with other
neurologic autoimmune diseases; interestingly, subjects
with MuSK Abs only on CBA had a milder phenotype
that those positive on standard RIPA.[29]
Low-density lipoprotein receptor-related protein 4-
Abs
Of late, clinical research has made significant progress
in the search for new pathogenic Abs in MG. Lrp4
represented an ideal candidate antigen since its identi-
fication with the long-sought coreceptor for agrin, in
2008.[30] In the subsequent years, while the crucial role
of Lrp4 at the NMJ was further clarified in experimental
studies,[6,31] several groups reported serum IgG to
Lrp4 in MG patients.
Lrp4 Abs mainly belong to IgG1 subclass and were
shown to interfere with Lrp4-agrin binding [32] and
with agrin-induced AChR clusters.[33] Mice immunized
with Lrp4 ectodomain developed MG-like weakness
associated with electrophysiological signs of NMT
impairment.[34] Similar alterations were induced by
injection of Abs from Lrp4-immunized rabbits.[34]
Passive-transfer studies by patients’ IgG injection have
not been reported so far.
The proportion of Lrp4-positive patients among dSN
cases varied from 0 to 50% in different reports.
[7,24,32,33,35,36] This variability could be due to the use
of different assays, such as luciferase-reporter immunopre-
cipitation,[32] ELISA [33] and CBA.[7,24,35,36]
Furthermore, a multicenter study, in which Lrp4 Abs
were detected in 18.7% of 635 dSN patients, showed
differences in Ab frequency (from 7 to 37%) in samples
from different countries,[7] suggesting a possible role of
ethnic factors in disease susceptibility. In the same study,
more than 20% of Lrp4-positive patients had ocular
myasthenia, and most of those with generalized disease
had mild to moderate symptoms. In some patients, Lrp4
Abs were found to be associated, with AChR or MuSK Abs,
more commonly with the latter [7,32,35,36]; these double
positive cases tended to have more severe symptoms.[7]
Recently, Lrp4 Abs were detected in serum and cer-
ebrospinal fluid samples from patients with ALS. In this
study, Lrp4 positivity rate was much higher in ALS
(23%) than in MG (1–2%).[37] These findings are worry-
ing, as they cut down Lrp4-Ab specificity for MG, and
may even complicate clinical diagnosis. ALS with bulbar
onset is sometimes mistaken for MG, and, in these
cases, a slight reaction to AChE-Is may be seen and a
decremental response on RNS and increased jitter on
SF-EMG can be present.[9]
Ab-associated MG clinical patterns are summarized
in Table 1.
In the immunological diagnosis of MG, current knowl-
edge confirms RIPA as first-line assay in the detection of
AChR and MuSK Abs and in Ab status monitoring. The
diagnostic yield of these Abs can be increased by the use
of sensitive CBAs. Clustered-AChR Ab assay proved an
effective and specific tool in MG diagnosis; larger size
studies including samples from different countries could
be helpful in clarifying its epidemiological and clinical
association. Lrp4 confirms the third most frequent Ab in
MG patients; standardization of assay techniques, further
evaluation of Ab specificity and Ab-phenotype correla-
tion will better define its clinical relevance.
Other Abs
Abs to agrin, ColQ and cortactin have recently been
reported in MG patients. Their role in the disease patho-
genesis has not been proved and they are not currently
used in diagnostic assays.
Neuronal agrin is a large proteoglycan secreted by
motor neurons and enriched at the basal lamina. Agrin-
Lrp4 binding promotes the formation of a tetrameric
complex that binds to and activates MuSK, triggering
intracellular signaling leading to AChR clustering.[4] Abs
to agrin were detected by ELISA and CBA, in a total of

Table 1. Clinical characteristics of MG subtypes.
AChR-MG (85% patients)†
Early-onset Thymoma
Age of onset <50 years 40–60 years
(mostly)
Males:Females 1:4 1:1
Ocular myasthenia 10–15% 2–3%
Weakness pattern Any Any
Thymus changes (prevalent) Thymic follicular AB and B‡
hyperplasia thymomas
†
An additional 3–4% of MG patients should have clustered-AChR Abs.
‡
According to WHO classification.
AChR: acetylcholine receptor; Lrp4: low-density lipoprotein receptor-related protein 4;
MG: myasthenia gravis; MuSK: muscle-specific tyrosine kinase receptor.
34 MG patients in different studies, often in association
with other disease-specific Abs, mostly AChR Abs.[38–
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41] Sera from agrin-positive patients inhibited agrin-
induced MuSK phosphorylation and AChR clustering in
myotubes.[38]
At the NMJ, AChE is anchored at the basal lamina by
its triple collagen tail (ColQ). Abs to ColQ were detected
in 12/415 (2.9%) MG sera and in 1/43 (2.3%) control
samples. All ColQ-positive patients were women and
were prevalently affected with generalized disease; 7/
12 of these cases were also positive for AChR-Abs or
MuSK-Abs.[42]
Cortactin is a tyrosine kinase substrate and a regu-
lator of F-actin assembly that acts downstream of agrin/
MuSK in promoting AChR clustering.[43] Abs to cortac-
tin were reported in 19.7% of dSN patients, but also in
4.8% of AChR- or MuSK-positive patients, 12.5% of sub-
jects with other autoimmune diseases and 5.2% of
healthy controls.[44]
Additional testing
Patients with AChR-MG may have serum Abs to titin
and the ryanodine receptor (striatonal Abs). These Abs
are not diagnostic of MG, but are strongly associated
with thymoma (titin Abs are positive in 95% and ryano-
dine receptor Abs in 70% of thymoma patients) and to
a lesser extent with late-onset nonthymoma MG.[8,44]
Striatonal Abs are markers of thymoma, at least in
young MG patients,[45] though binding intracellular
antigens and their pathogenicity remain uncertain.
Kv1.4 Abs that target the muscle voltage-gated potas-
sium channel were reported in 12–15% of Japanese MG
patients, often in association with severe MG and myo-
carditis with arrhythmias.[46] These findings were not
confirmed in Caucasian patients.[47]
Upon MG diagnosis, all patients should undergo a
radiological study of the mediastinum by computed
tomography or MRI. As the presence of a thymoma is
EXPERT REVIEW OF CLINICAL IMMUNOLOGY 161
Late-onset MuSK-MG (5–8%) Lrp4-MG (~2%)
≥50 years Any Any
1.7:1 1:4.5 1:2
20–25% Very rare >20%
Any Prevalently bulbar Any
Atrophy with rare Normal-for-age Hyperplastic changes in
germinal centers some cases
uncommon in ocular myasthenia, and very rare in chil-
dren and in patients with MuSK or Lrp4 Abs, its exclu-
sion should be especially careful in patients at higher
risk for such an association, that is, adult subjects with
generalized AChR-MG.
Treating MG
There is general agreement that MG is one of the more
treatable neuromuscular diseases. Current treatment,
although largely unspecific, has dramatically reduced
mortality and can restore satisfactory life conditions in
the great majority of patients. However, there are still
relevant drawbacks and controversies that can compli-
cate management even in specialized centers: MG is a
chronic condition and many patients need long-term
treatment with exposure to serious side effects; few
therapeutic modalities have been evaluated in rando-
mized controlled trials (RCTs) and current guidelines
mainly rely on expert opinions and retrospective stu-
dies; the response to treatment can differ among
patients depending on MG biological diversity.
Most patients present with isolated eyelid ptosis and
diplopia, but only in 15–20% of these cases,[48] symp-
toms remain confined to extrinsic ocular muscles; in the
other patients, generally within 2 years from the onset,
weakness spreads to other muscle groups. The outcome
of generalized MG is largely unpredictable, as some
patients may suffer from rapidly progressive symptoms
with early respiratory crises, while others have mild
weakness for the whole course of their disease.
Validated biomarkers that may predict disease severity
and response to therapy have not yet been identified.
Clinical management is based on the use, usually in
combination, of different therapeutic options. Intensity
of care is determined by weakness extension and sever-
ity, while treatment strategy should also take into
account the disease pathophysiology (associated Abs
 162 A. EVOLI ET AL.
and thymus pathology), and should be tailored, as far
as possible, on individual patients.
Symptomatic treatment
Symptomatic agents relieve MG symptoms by enhan-
cing NMT. They do not affect Ab production and patho-
genic effects, and do not influence the natural history
of the disease.
Oral AChE-Is (pyridostigmine is the agent most com-
monly used) are the first-line treatment in MG, although
normal muscle strength can be restored only in sub-
jects with mild symptoms.
Most AChR-MG patients respond more or less to
pyridostigmine that is well tolerated at standard dosage
ranging 180–360 mg/day in adults. Side effects are
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generally mild and subside with dose reduction. AChE-
I overmedication can lead to depolarization blockade at
NMJ with increased muscle weakness.[48]
On the other hand, MuSK-MG patients, for the most
part, do not improve on AChE-Is, and standard pyridos-
tigmine doses frequently induce nicotinic side effects
and may even worsen weakness up to a cholinergic
crisis.[11] A relative deficiency of AChE at motor end-
plate due to Abs interference with MuSK-ColQ binding
could account for the cholinergic hypersensitivity in
MuSK-MG.[49] In recent passive transfer studies, pyri-
dostigmine injection exacerbated morphological and
functional end-plate alterations, while 3,4-diaminopyr-
idine, which increases quantal release without prolong-
ing ACh half-life at the synaptic cleft, enhanced NMT
without causing postsynaptic alterations.[50] In addi-
tion, treatment with the β2-adrenergic agent, albuterol
reduced weakness in mice injected with IgG from
MuSK-MG patients.[51] There are very few data on the
effects of these agents in the human disease. To date, a
partial effect of 3,4-dyaminopiridine in two children,[52]
and response to both ephedrine and albuterol in a
patient with severe MuSK-MG,[53] have been reported.
Long-term immunomodulation: thymectomy
Thymectomy is indicated in all MG patients with evi-
dence of thymoma. In these cases, surgery is the main-
stay of oncologic treatment (that includes radio- and
chemotherapy for invasive tumors), while it does not
appear to improve significantly MG course.
In patients without thymoma, thymectomy is aimed
at the treatment of MG (‘therapeutic thymectomy’), its
rationale resting on the pathogenic role of the thymus
that has been convincingly shown in AChR-MG.[3,54,55]
The efficacy of therapeutic thymectomy has never been
evaluated in RCTs, and confounding factors, as
interstudy variability in surgical approach, patients’
selection, associated therapies and results’ evaluation,
make the comparison between retrospective reports
difficult.[56–58] Currently, thymectomy is recom-
mended, in nonthymomatous patients with generalized
MG, as an option to increase the chance of improve-
ment and remission.[56] Recent studies support this
view, reporting a better outcome in thymectomized
than in unthymectomized patients, in particular in sub-
jects with early onset MG associated with TFH [58,59]
and AChR Abs.[22,60] The results of a multicenter, sin-
gle-blinded randomized trial in nonthymomatous
AChR-MG patients are awaited soon.[61]
The indication to thymectomy should be extended
to generalized MG with clustered-AChR Abs, as in these
patients, the disease pathogenesis is very likely to be
the same as in ‘typical’ AChR-MG and both TFH and
thymoma have been reported.[23,27,28] On the other
hand, the role of thymectomy in patients with other
Abs is highly controversial.
In MuSK-MG, thymus hyperplastic changes are
uncommon [3,20,21] and clinical studies failed to
show a definite benefit from thymectomy, as most
thymectomized patients remained dependent on
immunosuppressive therapy and few achieved remis-
sion.[62,63] In Lrp4-MG, TFH appears to be more rare
than in AChR-MG [8]; the response to thymectomy has
not been specifically addressed.
The presence of a thymoma has been reported so far
in three MuSK-MG patients [11,64] and in a single case
with Lrp4 Abs.[36] This association, rare as it may be,
justifies a radiological study of the mediastinum in
these patients.
Short-term immunomodulation: plasma-exchange
and intravenous immunoglobulin
Plasma-exchange (PLEX), that removes Abs from circu-
lation, and intravenous immunoglobulin (IVIg) that
interferes with Ab activity, have a rapid albeit short-
lived effect in MG, and additional immunosuppression
is usually needed. PLEX standard protocol consists of
five exchanges of one plasma volume on alternate days;
IVIg are usually administered at 2 g/kg over 2–5 days.
Their main indication is the treatment of disease exacer-
bations in patients with severe symptoms or respiratory
crises. Additional indications include prevention of MG
deterioration at the start of steroid therapy, preparation
for thymectomy and as periodic treatment in selected
cases unresponsive to conventional immunosuppres-
sion.[54,55]
Some years ago, PLEX and IVIg were shown to have
comparable efficacy in the treatment of severe MG

exacerbations.[65] A recent RCT has reached the same
conclusion in patients with moderate to severe disease,
showing that the two treatments were similar for extent
of clinical improvement, response rate, duration of
effect and tolerance.[66] The choice between PLEX
and IVIg is based on patient’s comorbidities, proce-
dure’s potential risks and medical facilities. However,
in patients with life-threatening symptoms, PLEX is pre-
ferred because of its more rapid effect, while for out-
patients IVIg is more suitable.[67] Both AChR-MG and
MuSK-MG respond very well to PLEX, while IVIg effec-
tiveness appears to be less consistent in patients with
MuSK Abs.[11]
Adverse effects can be reduced by screening patient’s
comorbidities before treatment. IVIg may induce anaphy-
laxis in IgA-deficient patients and, at the dosage used in
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MG, is contraindicated in subjects with congestive heart
disease and renal function impairment. Otherwise, side
effects observed with IVIg are generally mild (nausea,
headache, chills and fever) though, more rarely hemoly-
tic anemia and thromboembolic complications can
occur. PLEX is contraindicated in patients with sepsis,
epilepsy, severe arterial hypotension and heart failure.
Most adverse events are associated with vascular access
such as infection, thrombosis; hemodynamic instability
related to volume shifts requires careful monitoring dur-
ing the procedure.[55]
Different techniques of immunoadsorption (IA) have
been successfully used in MG (mostly in AChR-MG), with
the same indications as PLEX. This semiselective IA, that
removes circulating IgG leaving other plasma compo-
nents unaltered, can represent a valuable alternative in
patients requiring intensive PLEX protocols.[68] An even
more attractive option would be the selective depletion
of pathogenic Abs. The efforts to develop antigen-spe-
cific IA have achieved encouraging results in experi-
mental studies.[69]
Subcutaneous IgG has recently been evaluated for
maintenance therapy in inflammatory neuromuscular
diseases.[70] Because of ease of administration and
tolerability, it appears to be a sensible alternative to
IVIg. An open label study of subcutaneous IgG in MG
patients is under way.
Immunosuppressive therapy
Immunosuppression is the mainstay treatment for MG.
It is performed in patients with disabling symptoms, not
adequately controlled with symptomatic agents, in
association or as an alternative to thymectomy.
Treatment approach is determined by weakness sever-
ity and rate of progression, and by patient’s character-
istics (age, lifestyle and associated conditions). Once
EXPERT REVIEW OF CLINICAL IMMUNOLOGY 163
sustained improvement is achieved, medications are
gradually reduced to the minimum effective dose in
order to minimize adverse effects.[48]
Corticosteroids have been in use for many years, and
treatment drawbacks and complications (including the
risk of MG deterioration at the start of treatment) are
well known.[71] Nonetheless, these agents still repre-
sent the first option in MG whereby immunosuppres-
sion is required on account of their efficacy (70–80% of
response rate) and rapid-onset effect, that is advanta-
geous in severely affected cases.[72] Prednisone and
prednisolone are the preparations most commonly
used, starting with high doses or with a progressively
increasing regimen,[71] up to 1 mg/kg/day in patients
with generalized MG; an alternate-day schedule is gen-
erally preferred in chronic administration.[54] High-dose
steroids, in association with PLEX, are the first-choice
treatment for respiratory crises, while lower doses can
be sufficient in patients with mild or purely ocular
symptoms.[73]
Different classes of immunosuppressants, including
cytostatic drugs (azathioprine, mycophenolate mofetil
and methotrexate), the alkylating agent cyclophospha-
mide and calcineurin inhibitors (cyclosporine A and tacro-
limus), are used in MG treatment. These agents can be
used in monotherapy, but because of delayed-onset
effect, they are frequently administered in association
with prednisone (to reduce its dosage and treatment
duration) and can replace steroids in long-term treatment.
Positive clinical evidence, though from studies based on
small sample size is available for azathioprine (as steroid-
sparing agent), cyclosporine A and iv. pulse cyclopho-
sphamide.[55] On the other hand, three RCTs, two on
mycophenolate mofetil [74,75] and one on tacrolimus
[76] failed to demonstrate significant efficacy (in these
trials, due to the study design, the investigational drug
effect was probably masked by the concomitant steroid
treatment). Clinical benefit of mycophenolate [77] and
tacrolimus [78] was reported in retrospective, open label
studies and small trials, and, also in view of their favorable
side-effect profile, these agents are used as second- or
third-line therapy.
In many countries, azathioprine (2–3 mg/kg body
weight (b.w.)/day as starting dose, 1 mg/kg as mainte-
nance dose) is the first-choice immunosuppressant in
MG patients. As patients with thiopurine methyl trans-
ferase deficiency may develop severe bone-marrow
toxicity, thiopurine methyl transferase activity should
be measured before treatment. Mycophenolate mofetil
(at a standard dose of 2–2.5 g/daily) is generally used as
second-choice; cyclosporine A (at an initial dose of
4–6 mg/kg b.w. and a maintenance dose <3–4 mg/kg/
day) and tacrolimus (3 mg/day or 0.1 mg/kg b.w./day)
 164 A. EVOLI ET AL.
are third-choice agents, though the use of cyclosporine
A is limited by nephrotoxicity.[48,54,55,72] In a recent
single-blinded study, methotrexate was found to be
useful as steroid-sparing agent, with similar efficacy
and tolerability to azathioprine [79]; a phase-II trial of
methotrexate is ongoing. The use of cyclophosphamide
on account of significant toxicity is mostly reserved to
patients unresponsive to other agents.[55]
Immunosuppressive therapy, based on corticoster-
oids and afore-mentioned drugs (also called ‘conven-
tional immunosuppression’) is currently performed in
>80% patients with generalized AChR-MG and in nearly
all MuSK-MG cases, with an overall response rate
around 90% and an outcome of minimal manifestations
or better [80] in 66% of patients.[72] However, disease
relapses are frequent on dose tapering or after treat-
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ment withdrawal, and most patients need long-term
therapy. Patient candidates to immunosuppression
should be screened for chronic infections like viral
hepatitis and tuberculosis. Bone mineral density assess-
ment and calcium, vitamin D and bisphosphonate sup-
plement should be started concurrently with
corticosteroids to prevent osteoporosis, and other
acute and long-term steroid complications should be
promptly diagnosed and treated. In patients taking
immunosuppressants, side-effect monitoring through
specific and periodic blood tests is necessary. As
patients’ cooperation is of the utmost importance,
patients and relatives must be fully informed of poten-
tial risks and expected benefit.
Refractory MG
MG is defined as refractory to conventional treatment,
when patients complain of disabling weakness or
severe disease relapses in spite of adequate immuno-
suppressive treatment, and/or they require too high
doses of steroids or other immunosuppressive agents
with serious side effects.[81] The frequency of refractory
MG was found to be around 10%, with the highest
prevalence rate among patients with thymoma-asso-
ciated AChR-MG and MuSK-MG.[82] These findings are
not surprising, considering that these patient groups
are generally affected with severe disease and tend to
remain dependent on immunosuppressive therapy.[72]
The reasons why some patients, at some point of
their disease, become resistant to treatment and exactly
when the autoimmune response goes out of control are
largely unknown. Multiple factors are likely to be
involved, including failure of regulatory signals, dys-
function of B cell maturation and survival, generation
of long-lived autoreactive plasma cells, changes in Ab
subclass and epitope specificity.[83–86]
Refractory MG can been managed with intermittent
PLEX/IA or IVIg treatments, in association with immu-
nosuppressive therapy.[55] This option provides short-
term benefit, has a significant impact on patient’s life-
style, is costly and not exempt from side effects.
Intravenous cyclophosphamide, both on high-doses
(200 mg/kg in 4 consecutive days) [81] and as pulse
treatment (at 0.75 mg/m2 every 4 weeks for 6 cycles)
followed by conventional immunotherapy,[87] resulted
in significant improvement in all forms of MG. However,
its use is limited by relevant toxicity.
The use of biologics as monoclonal Abs (-mAbs) and
therapeutic fusion proteins (-cepts) is rapidly expanding
in the treatment of autoimmune diseases. So far, B cell
depletion and complement inhibition have proved ben-
eficial in patients with refractory MG.
Rituximab, a chimeric anti-CD20 mAb, causes a pro-
found depletion of circulating pre-B and B lymphocytes
without reducing total IgG levels. To date, 170 MG
patients treated with rituximab have been reported in
the English literature. The results of a recent meta-ana-
lysis showed a higher response rate in MuSK-MG (88.8%)
than in AChR-MG (80.4%),[88] thus confirming previous
reports,[89,90] together with a trend toward an inverse
correlation between disease duration and response to
rituximab; treatment was generally well tolerated.[88]
Rituximab efficacy and tolerability in MG have been
evaluated, so far, in single-case reports and small open-
label studies, characterized by great variability in treat-
ment schedule, result evaluation criteria and follow-up
duration. A phase-II RCT is currently under way. Decrease
in specific Ab levels [89] and reconstitution of T and B
regulatory cells have been proposed as biomarkers of
response to rituximab.[90,91]
Eculizumab, a humanized mAb, inhibits the
Complement component 5 preventing the formation
of the terminal C cascade. It proved effective and well
tolerated in a small Phase II RCT in AChR-MG patients.
[92] A phase III RCT is ongoing.
Cyclophosphamide and rituximab, as well as conven-
tional immunosuppressants, do not deplete long-lived
plasma cells, that, having entered the immunological
niche, can persistently secrete Abs. Different agents tar-
geting plasma cells and niche survival factors, like chemo-
kines and their receptors, cytokines such as interleukin-6,
B-cell activating factor (BAFF) and a proliferation-inducing
ligand (APRIL), are being evaluated in the treatment of
autoimmune diseases.[84] Currently, belimumab, a human
anti-BAFF mAb, and the proteasome inhibitor bortezomib
that eliminates both short-lived and long-lived plasma
cells are the object of RCTs in MG.
Improving our understanding of mechanisms
involved in treatment resistance and the availability of
 EXPERT REVIEW OF CLINICAL IMMUNOLOGY 165

biomarkers predictive of MG flares could optimize the
use of disease-modifying medications.
Expert commentary and five-year view
Recently, the effects of MuSK Abs have been defined in
experimental studies and new Ab targets have been
identified in MG patients. These developments have
expanded our understanding of the pathophysiology of
the disease and improved its management. However,
new Ab assays require specific facilities and expertise,
and are currently performed in few laboratories. In clin-
ical practice, the diagnosis of MG can be challenging in
patients with negative results on AChR and MuSK Ab
standard assays. In these cases, diagnostic confirmation
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MG. Possible candidates, as subsets of regulatory cells,
cytokines and microRNAs are currently under investiga-
tion.[66,93] Epigenetics and exosomes are promising
research fields for biomarker discovery.[94,95] In particular,
the proteomic and microRNA profiling of thymus- or mus-
cle-derived exosomes may provide useful insights for the
monitoring of MG course.
Epitope mapping can elucidate the pathogenic
effects of Abs [86] and, together with changes in IgG
subclasses, may correlate more closely with clinical
changes that total Ab titer.
Possible candidate targets for new therapies are T
follicular helper cells, a subset of T lymphocytes that
plays a key role in B cell maturation. Compelling evi-
dence exists in mice and in humans that aberrant gen-
eration and/or activation of T follicular helper cells may

requires the exclusion of conditions that can mimic MG

and should rely more on the clinical context than on
positive results on a single test.
Current treatment is mostly based on unspecific
immunosuppression and has considerable limitations.
RCTs are difficult in a rare disease like MG, but recently
completed trials have provided evidence for therapeutic
approaches that had long been in clinical use.
Complement inhibition and B-cell targeting therapies
seem to be promising approaches as rescue treatment
for refractory disease. A better understanding of the
mechanisms leading to nonresponsiveness to immuno-
suppressants may justify a more extensive and earlier
use of biologic agents. However, growing expenditures
on extensive use of off-label treatments is already a
cause for concern in payers and patients, and a major
challenge in the next future will be how to grant effec-
tive and safe therapies at sustainable costs.
Biomarkers that can predict the disease course and
response to treatment have long been sought after in
contribute to the pathogenesis of autoimmune dis-
eases.[85] MAbs targeting molecules expressed by this
lymphocytes subset (e.g. ICOS) are currently evaluated
in the treatment of systemic lupus erythematosus.[96]
Different forms of antigen-specific immunotherapy
[54,97] have been successfully developed in animal mod-
els, and, the next years will see more efforts to fill the
gap between experimental studies and human disease.
Financial & competing interests disclosure
The authors have no relevant affiliations or financial invol-
vement with any organization or entity with a financial
interest in or financial conflict with the subject matter or
materials discussed in the manuscript. This includes
employment, consultancies, honoraria, stock ownership
or options, expert testimony, grants or patents received
or pending, or royalties.

Key issues
● Acetylcholine receptor (AChR) antibodies (Abs) are the first to be tested when myasthenia gravis (MG) is
suspected.
● Muscle-specific tyrosine kinase receptor (MuSK) Abs should be tested in all AChR negative patients. MuSK-
MG is more likely in young adult women with predominant weakness of bulbar and neck muscles.
● Clustered-AChR Abs seem to be more common in young patients (including children) with ocular or mild
generalized disease.
● Further studies are needed to assess the specificity of low-density lipoprotein receptor-related protein 4 Abs
for MG. These Abs should be assayed in AChR and MuSK negative patients. The associated phenotype
appears to be similar to AChR-MG.
● The frequency of triple-negative (AChR/MuSK/low-density lipoprotein receptor-related protein 4 negative)
MG could be estimated at 3–4%.
● Most MuSK-MG patients are unresponsive to or intolerant of acetylcholinesterase inhibitors. 3,4-diamino-
pyridine and albuterol might be beneficial in these cases.
● In uncontrolled studies, rituximab proved effective in MG, especially in MuSK-MG.
 166 A. EVOLI ET AL.

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