Department of Clinical and Experimental Medicine, Neurology Unit, University of Pisa
Refractory Myasthenia Gravis:
clinical and serological features of our Pisan
cohort
PhD Program Director Prof. Stefano Del Prato
Tutor:
Prof. Gabriele Siciliano
Co-tutor:
Dott. Michelangelo Maestri Tassoni
PhD Candidate:
Dott.ssa Anna De Rosa
Table of contents
Abstract ... 6
CHAPTER 1: Myasthenia Gravis (MG) ... 9
1.1 Clinical features ... 9
1.2 Epidemiology ... 14
1.3 Serological features ... 14
1.3.1 MG with anti-AChR antibodies ... 14
1.3.2 MG with anti-MuSK antibodies ... 16
1.3.3 "Seronegative" MG ... 18
1.4 Diagnosis ... 20
1.5 Medical therapy ... 22
CHAPTER 2: The thymus... 26
2.1 Role of the thymus in MG ... 26
2.2 Thymic epitelial tumors ... 26
2.2.1 Epidemiology ... 26
2.2.2 Histology ... 26
2.3 Thymoma-associated Myasthenia Gravis (TAMG) ... 28
2.3.1 Clinical features of TAMG ... 29
2.3.2 Serological features of TAMG ... 30
2.3.3 Genetics and Epigenetics of TAMG... 32
2.3.4 Recurrent Thymoma associated with MG ... 39
2.4 Thymic hyperplasia in MG ... 39
2.5 Thymectomy ... 42
3.1 Clinical, serological features and Thymic abnormalities in Refractory MG... 46
CHAPTER 4: Aim of the study ... 47
CHAPTER 5: Refractory MG with anti-MuSK antibodies: clinical and serological
features ... 48
5.1 Methods ... 48
5.2 Results... 48
5.3 Potential novel tratment for anti-MuSK-MG ... 51
CHAPTER 6: Refractory MG associated with Thymoma ... 56
6.1 Methods ... 56
6.2 Results... 58
6.2.1 Variables associated with AChR-Ab levels ... 60
6.2.2 Variables associated with worsening of MG ... 62
6.2.3 Variables associated with the risk of recurrence ... 64
6.2.4 Variables associated with the postintervention status ... 66
6.2.5 Serological features and relashionship among Neuromyotonia, MG and Thymoma ... 67
6.2.6 Neuromyotonia as clinical predictor of of Thymoma recurrences... 72
CHAPTER 7: Conclusions ... 73
References ... 77
List of Abbreviations and acronyms ... 89
List of Publications ... 91
Abstract
1. Riassunto
Background: although there are a large number of treatment options, approximately 10% of patients with generalized MG do not respond to conventional treatments. This subgroup of subjects falls under the definition of “Refractory Myasthenia Gravis patients”. In literature Authors agree that, compared to those with non-refractory myasthenia, these patients have an earlier onset, are predominantly female, have a thymoma and a form of generalized myasthenia gravis associated with anti-MuSK antibodies (anti-MuSK Abs). In order to improve the picture of patients with refractory MG it is of utmost importance defining clinically and serologically the different subtypes of MG.
Aim: to analyze the clinical, serological and histopathological characteristics of patients with Refractory Myasthenia associated with anti-MuSK antibodies and with thymoma afferent at Myasthenia Clinic of Azienda Ospedaliera Universitaria Pisana (AOUP). Further aims were: to determine if inhibitor of the tyrosine phosphatase Shp2 (NSC-87877) could represent a new treatment strategy for MuSK-MG, to identify clinical and serological features capable of predicting the risk of relapse in thymoma patients and to define the percentage of both MuSK and thymoma patients who achieve MG remission.
Methods and Materials: A total number of 268 patients with thymomatous MG and 71 patients with MuSK-MG were studied retrospectively. Of both cohorts we assessed: age of MG onset, MG clinical status according to MGFA (Myasthenia Gravis Foundation of America), treatment and post-intervention status. For patients with thymoma we also evaluated: epoch of thymectomy, oncological features and surgical approach. Sera of MuSK-MG patients were sent in Oxford and anti-MuSK Abs were detected both with radioimmunoassay (RIA) and cell-based assay (CBA). Moreover, we tested NSC-87877 for its ability to increase MuSK phosphorylation and to reverse or prevent the effects of MuSK-Abs in vitro models.
In thymoma patients, AChR-Ab dosages were measured both before and after thymectomy and those with symptoms of spontaneous muscle overactivity were selected for autoantibody testing using immunohistology for neuronal cell-surface proteins and cell-based assays for contactin-associated protein 2 (CASPR2), leucine-rich glioma inactivated 1 (LGI1), glycine receptor and Netrin-1 receptor antibodies.
Results: pharmacological/complete stable remission (PR/CSR) was reached in 26/69 (37.7%) of Musk patients. There were no differences in rate of remission between different MuSK-MG phenotypes (p>0.05). 80% of our MuSK-MG cohort positive by RIA responded poorly to cholinesterase inhibitors or with cholinergic side effects. Our in-vitro studies demonstrated the beneficial effect of SHP2 inhibition in reversing the effects of MuSK-Abs on AChR clusters. In our cohort of patients with thymoma, PR/CSR was attained in 120 (56.6%) patients and CSR was achieved in a higher percentage of patients with a mild MG status before thymectomy than others. There were no differences in rate of remission between thymoma recurrences and monophasic thymoma (p=0.08). AChR-Ab titres decreased after first thymectomy (p < 0.001) and this reduction was more pronounced in female patients (p = 0.05), in patients diagnosed with MG at an older age (p = 0.003), and in those with a milder MG stage before surgery (p = 0.02) or higher Masaoka-Koga stage (p=0.005) than others. The risk of relapse was closely linked with the age of the patient, the Masaoka-Koga stage, the surgical approach and the presence of Neuromyotonia (p < 0.001).
Conclusions: The reduction of AChR-Ab titres after thymectomy confirms an immunological role of thymoma in the pathogenesis of MG. Our observations on thymoma recurrences provide a pragmatic risk stratification for tumour vigilance in patients with thymomatous MG. A relevant percentage of thymoma and anti-MuSK patients can achieve a pharmacological/complete stable remission. Our study shows that the knowledge of the molecular mechanisms that occur in the development and progression of MG is essential to develop a more precise targeting treatment for patients with Refractory Myasthenia Gravis.
Chapter 1
Myasthenia Gravis
Myasthenia Gravis (MG) is an autoimmune neuromuscular disease due to autoantibodies that interfere with the Neuromuscular Junction (NMJ). In most cases, MG is caused by circulating antibodies directed against the acetylcholine receptor (AChR), located on the post-synaptic membrane of the muscle junction, which determine a marked reduction in the number of AChR and an unfolding of the post-synaptic membrane with disappearance of the junctional folds (1).These morphological alterations are the basis of the clinical and electrophysiological characteristics of the disease.
1.1 Clinical features
The pathognomonic symptom of MG is the abnormal muscle fatigue due to a block of neuromuscular transmission.
All skeletal muscles can be affected, while smooth and cardiac muscles are spared. Characteristically, muscle atrophy is not observed and osteo-tendon reflexes are preserved. The muscles most frequently affected are the extrinsic ocular muscles (EOM) whose deficits cause diplopia and ptosis. Although ocular symptoms are very frequent at presentation, only in 15-20% of patients MG remains confined to EOM, as generally within two years from onset, weakness spreads to other muscle groups (2).
Less commonly, the facial and bulbar districts are affected at the onset, but their involvement is very frequent during the course of the disease (80% of cases). Typical symptoms are the weakness of lower facial muscles (“vertical” smile) which characterizes the myasthenic facies, dysarthria with rhinolalia, difficulty in chewing and dysphagia. Weakness of neck extensors may cause a “dropped head”. As for the limbs, proximal muscles are prevalently involved,
especially in the upper limbs; among distal muscles, finger extensors are more commonly affected, while weakness of foot extensors is not unusual. Involvement of the respiratory muscles leads to respiratory failure (myasthenic crisis) that occurs in 15-20% of patients, more frequently in the first two years from onset (3).Ventilation failure is caused by weakness of the diaphragm and intercostal muscles together with upper airway obstruction by bronchial secretions and saliva aspiration.
The natural history of MG is characterized, especially in the initial stages, by a fluctuating course, with periods of worsening alternating with phases of clinical improvement or remission. Several factors can favor the onset or myasthenic crises: physical and psychological trauma, surgery, the puerperium, infectious diseases, drugs interfering with NMJ (especially some antibiotics, anticholinergics and anesthetics, see table 1) (4), overmedication of cholinesterase inhibitors, initiation of steroid treatment (3).
Over the years, different clinical classification systems of MG have been used. One of the first classifications, which has been universally applied for many years, is the one proposed by Osserman (5)(table 2).
Table 2. Osserman Classification.
Since 2000, the MG Foundation of America (MGFA) scale has been mostly used in clinical practice (6)(Table 3).
The outcome of MG following treatment is typically assessed using the postintervention status scale (PIS; Table 4).
Table 4. Post intervention status scale
1.2 Epidemiology
The incidence of MG is between 4 and 11/1,000,000 inhabitants, while the prevalence is approaching 150/million, with an increase in recent decades, probably due to the refinement of diagnostic techniques, the prolongation of life and a real increase in the frequency of the disease (7). The disease can begin at any age, but there are basically two incidence peaks: the first in the second-third decade with a clear prevalence for women, the second in the sixth-seventh decade with greater frequency in males.
1.3 Serological features
1.3.1 Myasthenia Gravis with anti-AChR antibodies
MG with antibodies to acetylcholine receptor (AChR-Ab) is the most common form of MG; in fact, about 50% of patients with ocular MG and 85% of those with generalized MG have got these antibodies.
AChR is a transmembrane glycoprotein of p.m. 275kD, consisting of 4 subunits (αβγδ in embryonic muscle and αβεδ in adult muscle), which form a pentameric structure α2βγδ / α2βεδ
Post Intervention status
Complete stable remission (CSR) No symptoms/signs for at least 1 yr. Isolated weakness of eyelid closure accepted Pharmacologic remission (PR) As per CSR but on MG treatment. Excludes cholinesterase inhibitors
Minimal manifestations (MM) Asymptomatic non-functionally limiting weakness, detectable only by careful examination Improved (I) Significant improvement in pre-treatment clinical status or reduction of MG treatment Unchanged (U) No significant change in clinical stats or reduction of MG medications
Worse (W) Substantial worsening of clinical status or increase in MG medications
surrounding the ion channel (8). Each α subunit contains a binding site for ACh; therefore, a receptor binds two neurotransmitter molecules (see figure 1).
Fig. 1: Pentameric structure of AChR.
When both α subunits are engaged in binding ACh, a modification of the conformation of the AChR takes place; this allows the opening of the channel and the entry into the muscle fiber of ions (mainly Na +), responsible for the depolarization of the post-synaptic membrane and the genesis of the action potential.
The receptor gene has been cloned and it is possible to synthesize the subunits that constitute it starting from the cDNA. Each subunit consists of a large extracellular N-terminal region, 4 transmembrane hydrophobic regions (M1-M4), a cytoplasmic loop and an extracellular C-terminal portion (9).
The extracellular portion of the α subunit contains, in addition to the binding site for ACh, the so-called "main immunogenic region" (MIR), which includes the sequence 66-76, against which most of the antibodies present in patients are directed (10).
The AChR-Ab are IgG and mainly belong to the IgG1 and IgG3 subclasses. The mechanisms by which anti-AChR antibodies cause the post-synaptic membrane alterations typical of MG are essentially three (11):
1) complement-mediated lysis of the post-synaptic membrane. It is able to explain the morphological alterations, such as the “simplification” of the membrane with the disappearance of the junctional folds;
2) antigenic modulation, responsible for an increase in internalization and degradation of AChRs, by cross-linking of receptor molecules by divalent IgG;
3) "block" of the link with the neurotransmitter.
On the basis of a clinical-pathogenetic classification (12),we can distinguish three forms of MG with AChR-Ab:
1) Early onset form (EOMG, ≤ 50), more frequent in females (with a female: male ratio of 3: 1), suggesting a role of sex hormones. It is associated with a high AChR-Ab titre, follicular thymic hyperplasia and HLA A1 B8 DR3 DQ2. Patients with EOMG respond to thymectomy and frequently present with other autoimmune diseases (OAIDs) (5% –30%), among which thyroid disease predominates (13) (14) (15) (Rotondo Dottore G. et al, Eur Thyroid J, accepted, 2020).
2) Late onset form (LOMG, > 50 years), more frequent in males, weakly associated with the HLA B7 DR2 haplotype.
3) Form associated with thymoma (TAMG), more common in the fifth-seventh decade, with no significant gender prevalence or HLA associations (12) (16).
1.3.2 Myasthenia Gravis with anti-MuSK antibodies (MuSK-MG)
Around 5-8% of patients have antibodies directed against the extracellular region of the muscle-specific receptor tyrosine kinase
(MuSK). MuSK is activated by agrin, secreted by the motor nerve terminal, through its co-receptor, the lipoprotein-related receptor 4 (LRP4). MuSK activation triggers an intracellular pathway leading to AChR clustering (6). The main effect of anti-MuSK Abs is a block of MuSK-LRP4 binding (17).
The role of MusK in AChR grouping is demonstrated by the absence of AChR clusters in MuSK - / - mice (18).Unlike anti-acetylcholine receptor antibodies, most of anti-MuSK Abs belong to
the IgG4 immunoglobulin subclass (17), do not activate complement and their role is still poorly understood (19)(20)(21).Passive transfer of the IgG4 component of MuSK-MG serum is especially effective in inducing the experimental disease (22) and IgG4-Abs behave as if they are functionally monovalent (23). Human IgG4 is an unusually dynamic antibody, with half molecule exchange (“Fab-arm exchange”) resulting in asymmetrical, bispecific antibodies with two different antigen binding sites, which contributes to its anti-inflammatory activity in vivo and in vitro (21). Anti-MuSK Abs of the IgG1 subclass are also present in most MuSK-MG patients, but at much lower levels than the IgG4 Abs (19). The role the IgG1 Abs play in MuSK-MG is still unknown.
Another key protein in MuSK activation is Dok7 (down-stream-of-tyrosine-kinase-7), which binds to a tyrosine-phosphorylated site in the juxta-membranous domain of MuSK (NPXY553). The first stage of MuSK activation seems to be the autophosphorylation of Y553 in the NPXY553 site that recruits Dok7, creating a tetramer in which a dimer of Dok7 binds two monomers of MuSK (24).
MuSK-MG is characterized by involvement of bulbar and axial muscles, with dysarthria, dysphagia, weakness of facial and neck muscles and frequent respiratory crises; ocular symptoms are mild and often transient; limb muscles can be totally spared (25). Three main categories of MuSK-MG have been suggested: 1) severe facio-pharyngeal weakness; 2) predominant neck and respiratory weakness; 3) clinical features indistinguishable from non-MuSK-Ab MG (non-MuSK-MG) (25) (26).
MuSK-MG shows a strong prevalence in women and a peak onset age in the fourth decade and, in contrast to other forms of MG, MuSK-MG has a strong association with HLA, particularly with HLA-DQ5 (27).
In patients with positive anti-MuSK MG, thymic tissue appears substantially normal (28)(29) so there is no response to thymectomy (see section 2.5). Many of the standard treatments of AChR-MG are of limited effectiveness in MuSK-MG, including cholinesterase inhibitors.
Therefore, current treatment involves immunosuppression, primarily by corticosteroids or B cell depletion agents.
1.3.3 "Seronegative" Myasthenia Gravis
The term “seronegative myasthenia” (SNMG) was traditionally applied to those patients who tested negative for AChR-Ab and MuSK on radioimmnoassays (RIAs). However, numerous lines of evidence suggest that MG without antibodies detectable with the usual serological techniques is not a completely different disease from the others; a) the clinical features do not differ from seropositive MG; b) plasma-exchange (P-E) therapy results in clinical improvement, c) mother-to-fetus transfer has been reported and d) passive transfer of seronegative sera in animals caused MG (30)(31). These observations suggested the presence of antibodies in seronegative MG that were not detectable with the traditional RIAs available. Subsequently, cell-based assays (CBAs), using human embryonic cells transfected with recombinant AChR subunits and rhapsin, allowed the detection of low affinity AChR-Abs - and, therefore, not detectable with the common radioimmunoassay - in 66% of SNMG patients and in 50% of patients with previously seronegative ocular MG (32)(33); such patients have clinical features similar to the classic form of MG (32).
Furthermore, within the "seronegative" MG there are forms associated with the presence of anti-LRP4 antibodies, whose frequency varies from 3-54% (34)(35)(36)(37) and whose role in the grouping of AChR at the postsynaptic membrane was described above. The thymus of these patients is atrophic and normal for age, although some cases with hyperplastic thymus have been described (38)(39).
Recently, new postsynaptic membrane proteins have been discovered such as agrin, titin and ryanodine, sometimes in association with known antibodies (40). Antibodies directed against agrin have also been found in some myasthenic patients with anti-AChR, MuSK, or LRP4
antibodies (41).Patients with anti-LRP4 Abs are mostly affected by mild generalized or ocular MG (37).
Antibodies directed against titin, a filamentous muscle protein involved in the maintenance of cellular flexibility, have been reported in approximately 40% of patients with AChR-Ab. The presence of these antibodies is related to age, since they prevail in 6% of EOMG forms and in 50-60% of non-thymomatous LOMG forms (42).
On the other hand, antibodies against the ryanodine receptor have been found in late-onset myasthenic patients with thymoma and with significant respiratory and neck muscle
involvement (43).
Fig. 2: Normal junction. Normal neuromuscular junction function, with major components implicated in MG. The action potential at the level of the presynaptic nerve terminal causes the opening of voltage-gated calcium channels, causing the release of acetylcholine and agrin into the synaptic space. The binding of acetylcholine to acetylcholine receptors (AChRs) determines the opening of the sodium channels, thus promoting muscle contraction. Agrin binds to the complex formed by low-density lipoprotein associated with protein 4 (LRP4) and MuSK, causing the AChR grouping which is required for the stabilization of the postsynaptic structures of the neuromuscular junction (N. E. Gilhus, et al, 2016 (44))
Fig. 3: Junction in MG. The main pathogenetic mechanisms caused by anti-AChR antibodies in MG include the activation of complement at the NMJ level, which causes the formation of membrane complexes (MACs) on the muscle membranes and the destruction of the typical folds in the sarcolemma (1); antigenic modulation that results in the internalization and degradation of (2); and the binding of anti-AChR antibodies at the level of the binding site of (3) which would directly block the bond with acetylcholine and, consequently, the opening of sodium channels. Anti-MuSK and anti-LRP4 antibodies block the intermolecular interactions of MuSK or LRP4 respectively and could therefore inhibit the normal mechanisms for maintaining the organization of NMJ (4). Antibodies that play a pathogenic role in MG are highlighted in red (N. E. Gilhus, et al, 2016 (44)).
1.4 Diagnosis
Diagnosis of MG is based on anamnestic (history of strength deficit with a fluctuating course) and clinical data (deficit and exhaustion in "typical" muscle areas in the absence of signs of lesion of the central and peripheral nervous system). The clinical diagnosis of MG is confirmed by electromyography (EMG) studies, pharmacologic testing, serum Ab assay (4).
Electromyography consists of:
a) significant decrease (> 10%) of the compound muscle action potential (cMAP) during repetitive nerve stimulation (RNS) at low frequency (3-5 Hz);
b) increased jitter and signs of blocking of single fiber electromyography (SF-EMG);
Pharmacologic testing consists of a clear clinical improvement following the administration of cholinesterase inhibitors (ChE-Is), such as prostigmine (neostigmine) i.m. and edrophonium (Tensilon) i.v.
Immunological tests demonstrate the presence of specific serum antibodies. This test is usually performed with RIAs. Recently other more sensitive techniques for detecting the presence of antibodies have been described, such as the CBA (see above) (45). Among the diagnostic tests, the antibody dosage is certainly the most reliable, being endowed with almost absolute specificity. In patients with neither AChR or MuSK Abs on standard RIA, EMG confirmation is crucial (4).
The Tensilon / Prostigmine test, while of great diagnostic value due to its high sensitivity and specificity, can give rise to false positive responses in diseases such as Lambert-Eaton myasthenic syndrome, amyotrophic lateral sclerosis, diabetic neuropathy and brain tumors (46)(47).
Among the electrophysiological tests, the RNS appears to have high specificity and moderate sensitivity, since, if different districts are examined, it is positive in about 70% of patients with mild forms and up to 90% of cases with severe forms of the disease.
In comparison, SF-EMG, although more sensitive, appears to have less specificity (48) and may also be positive in other pathologies, for example in Miller Fischer Syndrome and in Guillain-Barrè axonal variants.
In patients with anti-MuSK antibodies, diagnosis can be difficult because the response to ChE-Is may be ambiguous or absent and electrophysiological tests may be negative (49). Given the particular distribution of deficits, RNS in the limbs frequently does not allow to confirm the diagnosis. SF-EMG, considered the most sensitive diagnostic test, however, shows a lower percentage of positivity than typical MG (50). However, the assay of anti-MuSK antibodies, being of high specificity, allows in these cases to confirm the diagnosis (11).
After having ascertained the diagnosis of MG, each patient must undergo a radiological study of the thymus to rule out the presence of a thymoma (51).
It is also useful to investigate the presence of other autoimmune pathologies, which are frequently associated with MG and to carry out a screening of any internal pathologies that can complicate the course of the disease and interfere with therapies.
1.5 Medical therapy
Current therapeutic measures have significantly improved the expectation and quality of life of myasthenic subjects. There is no ideal treatment plan for all patients, as everyone needs an individual treatment plan. Therapeutic options consist of: cholinesterase inhibitors (ChE-Is), short and long-term immunosuppressive therapies (IS), thymectomy (see section 2.5).
The goal of treating MG should be to achieve satisfactory symptom control with an improvement in muscle strength minimizing adverse effects (AEs) (52).
Recently, numerous international guidelines for the treatment of myasthenia have been published, which review the different therapeutic options of the disease (53)(54)(52)(4). Symptomatic treatment is based on ChE-Is that still represent the treatment of first choice in patients with MG (53). ChE-Is can be administered orally, more commonly, but also intramuscularly or intravenously, especially in emergency situations or in the immediate post-surgical phase. It is known that in most patients with anti-MuSK antibodies, the response to pyridostigmine is unsatisfactory and very often causes severe cholinergic effects even with very low doses.
Immunosuppressive therapy is indicated in all patients with disabling symptoms, not sufficiently compensated by ChE-Is.
Corticosteroids (mainly prednisone and prednisolone), used since 1970 as a treatment for MG (55), are the first choice in patients with severe symptoms and, at low dosages, they represent the most effective treatment in ocular myasthenia.
In subjects who need high maintenance doses, it is advisable to combine a second immunosuppressant that allows a significant reduction of the steroid. Immunosuppressants are
mostly used as “steroid-sparing agents”, can replace steroids in long-term treatment and can be used in monotherapy in patients with mild non-progressive MG (4). The drugs currently in use are cytostatics (azathioprine and, more recently, mycophenolate mofetil) and calcineurin inhibitors (cyclosporine and tacrolimus) (56). In isolated cases, unresponsive to conventional treatments, Rituximab (anti-CD20 monoclonal antibodies) can be used (57).
Azathioprine is the drug of first choice among non-steroidal immunosuppressants and is also effective in monotherapy; however, due to the prolonged latency of action, it is usually administered together with steroids, at least in the initial phases (58).
Cyclosporin A is usually used in association with corticosteroids with good therapeutic results (59), but is still considered a second-choice immunosuppressant, as an alternative to azathioprine, due to its high nephrotoxic power.
Good therapeutic results have been reported with the use of mycophenolate mofetil and tacrolimus (60), but the use of these drugs is currently limited to patients who do not respond to conventional treatment (61).
Several studies have reported a good response to rituximab, a CD20 B-cell depleting monoclonal Ab. The results of the ongoing trial in AChR-MG [ClinicalTrials.gov Identifier: NCT02110706] have not yet been published. A very recent meta-analysis has identified MuSK-myasthenia gravis, younger age and mild-to-moderate disease as predictors of a favorable response to rituximab (62). In a number of cases, treatment led to eventual elimination of the need for other immune-directed treatments, e.g., steroids, and without the necessity for repeated rituximab infusions (63). Recent guidelines have supported earlier use of this agent when an initial standard treatment does not induce rapid remission (52). Eculizumab, which inhibits C5 and prevents membrane attack complex formation, was evaluated in a phase III study in Refractory Generalized without finding significant improvements, probably because of statistic method limitations (64)
"Short-term" immunosuppressive treatments are plasma exchange (P-E) and intravenous immunoglobulins (IVIG). The indication for the use of these therapeutic procedures is mainly represented by the worsening phases of the disease, such as myasthenic crises, by refractory to drug treatment and in preparation for thymectomy surgery (65)(66). P-E and IVIG have comparable efficacy in the treatment of severe exacerbations of MG but also in patients with moderate to severe variable disease (67)(68).
Guidelines suggest that the choice between the two treatments should depend on individual patient’s factors, including comorbidities (eg. P-E is contraindicated in sepsis and immunoglobulins are contraindicated in renal insufficiency or hypercoagulable states). P-E present a greater rapidity of clinical response but may involve a greater risk of side effects. On the other hand, the English guidelines suggest the use of IVIG as first treatment after steroids for the management of myasthenia gravis in intensive care units (53).
In recent years, therefore, the use of IVIG has met an increasing approval, in particular in the treatment of the critical phases of MG, also due to the simplicity of the therapeutic procedure and the low percentage of side effects. Recently, thanks to the Myasthenia Clinic of Pisa, the Italian Medicines Agency (AIFA) has included IVIG in the list of dispensable medicines at full charge of National health service for the treatment of MG (Determina n. 7385/2020) (20A00644) (GU Serie Generale n.26 del 01-02-2020) with the following therapeutic indications:
-myasthenic crisis; -exacerbations of MG;
-in the early stages of MG, waiting for the effect of cortisone and/or immunosuppressive therapy;
-as preparation for thymectomy
-in patients with MG non-responsive to steroid and / or immunosuppressive drug therapies or having contraindications to their use.
Chapter 2
The Thymus
2.1 Role of the Thymus in MG
For a long time, the high frequency of thymic changes (follicular hyperplasia and thymoma are found in approximately 50-55% and 10-20% of myasthenic patients, respectively) and the clinical benefit induced by thymectomy have suggested a pathogenetic role of the thymus in MG (69). The Thymus is the organ responsible for the maturation and differentiation of T lymphocytes and an alteration of this process is involved in the pathogenesis of MG.
Thymic follicular hyperplasia is typical of juvenile onset MG, characterized by the high frequency of association with the HLA A1 B8 DR3 DQ2 haplotype; on the contrary, thymoma is more frequent in the fifth and sixth decade and is not associated with a particular HLA haplotype (12)(16).
2.2 Thymic epithelial tumors
2.2.1 Epidemiology
Thymomas have an incidence of 1.7 per million people per year in Europe and 0.13 per 100,000 people per year in the US. The incidence increases with age: 0.4 per million / year in young people (<25 years), 1.9 in patients between 25 and 64 years and 4.2 in patients over 65 years (70).
2.2.2 Histology
Thymoma is a rare mediastinal tumor of the thymus epithelial cells containing non-neoplastic lymphocytes. In 1999, the World Health Organization (WHO) proposed a histological
classification based on the morphology and lymphocyte / epithelial cell ratio (69). Subsequently, this classification was modified and updated in 2014 by A. Marx (71). The most widely used WHO classification identifies 6 types of TET (thymic epithelial tumors):
- type A, also called medullary or spindle-cell thymoma, without atypia or neoplastic lymphocytes;
- type AB, also called mixed thymoma, as it is similar to type A but has foci of neoplastic lymphocytes;
- type B, subdivided on the basis of the proportional increase of thymocytes respect to lymphocytes and cellular atypia in:
- type B1, or lymphocytic thymoma - type B2, or cortical thymoma
- type B3, also called epithelial thymoma or well differentiated thymic carcinoma - type C, thymic carcinomas.
Type A and type AB have a benign clinical course, while types B1-B3 are considered low to moderate malignancy. Thymic carcinomas classified as type C thymomas follow a malignant course and can metastasize and are not associated with MG (72). The staging of thymomas was instead proposed by Masaoka and colleagues in 1981 (73), it is usually referred to as the “Masaoka system” and was modified in 1994 by Koga (74). This classification describes the size of the tumor and its degree of invasion:
- stage I: capsulated tumor
- stage IIa: microscopic transcapsular invasion
- stage IIb: macroscopic invasion of the perithymic fat that does not infiltrate the mediastinal pleura or pericardium
- stage III: macroscopic invasion of surrounding organs such as the pericardium, large vessels or the lung
-stage IVa: pleural or pericardial metastases - stage IVb: lymphatic or blood metastases
2.3 Thymoma-associated Myasthenia Gravis (TAMG)
The role of thymoma in the pathogenesis of MG has not yet been fully understood. First, the AChR, the target antigen of the autoimmune response, is not fully expressed in the neoplastic thymus; furthermore, lymphocytes extracted from thymoma do not produce autoantibodies (75). Thymoma can instead generate CD4 + AChR-specific (76) or T cells sensitized against muscle antigens (commonly expressed in thymoma); these lymphocytes would be exported to the periphery where they could give rise to tissue damage with release of antigens and production of antibodies in the lymphnodes (77). Neoplastic thymic epithelial cells (TECs) are able to support the maturation of T lymphocytes (78); in particular they are able to induce the maturation of lymphocyte precursors up to the phenotypic stage DP (double-positive T cells: mature T cells that truly express both CD4 and CD8) as occurs in the normal thymus; however, unlike the latter, the subsequent stages of maturation would occur in an incomplete or pathological manner, as evidenced by the presence of an abnormal percentage of immature CD4+ cells within the thymoma. The ability to induce T lymphocyte maturation differs between different isotypes; the existence of this thymopoietic activity in thymomas is correlated to the presence of paraneoplastic pathologies, including MG (79).
Fig. 4: Possible consequences of reduced generation of regulatory T cells in the pathogenesis of MG. In the normal thymus, there would be a critical balance between the generation of self-reactive and regulatory T cells. Under normal conditions, the generation and elimination of regulatory T cells may be sufficient to control the few self-reactive T cells present in the blood even of healthy subjects. However, in thymomas, the balance is altered by an increased number of self-reactive T cells and a reduction in regulatory ones. This imbalance could be a contributing factor in the breakdown of peripheral tolerance and therefore in the pathogenesis of autoimmunity (Ströbel P. et al., 2003 (80)).
2.3.1 Clinical features of TAMG
The high frequency of autoimmune manifestations associated with thymoma (> 50%) assumes that it is not a mere coincidence, considering that the thymus is the site of induction of central self-tolerance (81).
The most frequent autoimmune disease associated with thymoma is certainly MG; in fact, approximately 10–20% of myasthenic patients have a thymoma and approximately 30% of patients with thymoma have a thymoma associated MG (TAMG) (82).
TAMG occurs more frequently after the age of 50, is not associated with sex or a strong HLA association. Thymoma and Myasthenia Gravis are usually diagnosed at the same time or the thymoma is diagnosed later although it is not uncommon for patients with thymoma to
cells, in which the disturbance of either component may lead to autoimmunity (Fig. 4). Our findings may explain why paraneoplastic MG often deteriorates after thymoma resection (Somnier, 1994; Beeson et al., 1998), since
removal of the tumor also implies removal of the small “thymoma-specific” regT cell pool. In MG patients, these regT cells are obviously not sufficient to control autoimmunity, but the clinical course of MG after
FIGURE 4 Potential consequences of reduced generation of regulatory T cells in the pathogenesis of MG. In the normal thymus, there may be a critical balance between generation of autoreactive and regulatory T cells. Under normal conditions, thymic generation and export of regulatory T cells may suffice to control the few autoreactive T cells present in the blood also of healthy subjects. In thymomas, however, this balance is disturbed by increased numbers of autoreactive T cells and decreased numbers of regulatory T cells. This imbalance may be a contributing factor in breaking peripheral tolerance and in the pathogenesis of autoimmunity.
FIGURE 3 Generation of mature T cells with a regulatory (CD4þCD25þ) phenotype is highly reduced in thymomas. FACS analysis of 9 MG(þ) and 13 MG(2 ) thymomas as well as 13 non-neoplastic control thymuses showing a statistically significant reduction of CD4þCD25þ regulatory T cells in both MG(þ) and MG(2) thymomas, although reduction of this T cell subset was more pronounced in MG(2) tumors.
subsequently develop MG (75). In most studies, the incidence of MG in patients with thymoma is higher in histological type B2 (WHO classification) (83)(84).
In the past, MG was considered an unfavorable prognostic factor in the case of thymoma due to the high surgical risk and higher perioperative mortality (5)(85). On the contrary, recent studies have shown that myasthenic symptoms are a favorable prognostic factor in patients with thymoma (86)(87)(88). This change of course is the consequence of several factors: the improvements in medical therapy for MG, the earlier diagnosis of thymoma and the hypothetical beneficial effect of steroids, widely used in the treatment of both diseases (87). In fact, it has been suggested that steroid therapy causes an apoptotic effect both on the lymphocyte component of tumors and on the neoplastic TECs, thus reducing the tumor mass (89). It is for this reason that corticosteroids are also used in the case of thymoma, particularly in the presence of metastatic and advanced thymomas (90).
2.3.2 Serological features of TAMG
Patients with TAMG typically present with generalized myasthenia and anti-AChR antibodies; rare exceptions have been described in seronegative or anti-MusK patients while no associations with anti-LRP4 antibodies have been reported (91)(92)(37). A part from MG, many other autoimmune neurological diseases also co-occur in patients with tumors of the thymus (Table 5), which is normally the classic site of central self-tolerance induction (81). In a subgroup of TAMG patients is also possible to find the presence of autoantibodies directed against voltage gated potassium channels (VGKCs) which cause the so-called “Acquired Neuromyotonia” (NMT). NMT, also known as Isaac's Syndrome, is a paraneoplastic syndrome characterized by spontaneous discharges on electromyography due to hyperexcitability of peripheral nerve fibers (93).Isaac in fact described it as a “syndrome of continuous muscular activity at rest” (CMFAS) (94). Clinically it is characterized by continuous motor twitching and myochimias, fasciculations, cramps and hyperhidrosis. In 20% of cases, an association with
signs of CNS involvement such as behavioral changes, confusion or insomnia is also described. Indeed, anti-VGKCs antibodies, in particular those directed against leucin-rich glioma-inactived protein 1 (LGI-1) and contactin-associated protein-like 2 (CASPR 2), are not only present in the peripheral nervous system but also in the central nervous system, where they play an important role in neuronal excitability.
The pathogenesis of NMT is probably multifactorial, with autoimmune and paraneoplastic mechanisms involved.
The association of this syndrome with myasthenia and thymoma is frequently reported in the literature: thymoma is in fact present in about 20% of patients with Isaac's syndrome and most of them have antibodies directed against Caspr2 (95). Isaac's syndrome is the second most frequent autoimmune disease after MG in patients with thymoma and thymectomy, in association with immunosuppressive and immunomodulatory therapy, improves hyperexcitability symptoms (96)(97).
CASPR 2 antibodies can also cause Morvan Syndrome (MoS), an autoimmune encephalitis caused by the presence of anti-VGKC antibodies directed against the nervous system, both peripheral (PNS) and central (CNS) causing a set of symptoms including acquired NMT, neuropsychiatric disorders, dysautonomia, disorders sleep, especially insomnia, and neuropathic pain. CASPR2 antibodies are found in 79% of cases with MoS and are often associated with thymoma (98).
Patients with MoS have a poor response to immunosuppressive therapy and generally require second or third line immunosuppressants in higher doses than MG (98). The prognosis of patients with MoS associated with thymoma is worse than with TAMG or relapsing thymomas (99). Recently, new surface antibodies, called antibodies directed against Netrin-1 receptors, have been detected in patients with thymoma and NMT (100).
Table 5. Autoimmune neurological diseases associated with thymomas (adapted from Marx A. et al., 2010 (81))
Notes: In many “historic” reports, histological diversity of thymomas was not appreciated. AChR, acetylcholine receptor; CRMP (1–5), collapsing response mediator protein (1–5); GAD, glutamic acid decarboxylase; NEC, neuroendocrine carcinoma; NNA-1 (¼Hu), neuronal nuclear antigens; RyR, ryanodine receptor; StrA, striational antigens, including titin; VGKC, voltage-gated potassium channel; NA, information not available.
2.3.3 Genetics and epigenetics of TAMG
Like isolated MG, epigenetic factors play an important role in the association between Thymoma and MG.
The molecular events that characterize thymic neoplasms, including point mutations, chromosomal aberrations, and epigenetic modifications, such as changes in DNA methylation, have been described in the last few years (101)(102).
In particular, high DNA methylation levels of genes involved in one-carbon metabolism (MTHFR) and DNA methylation (DNMT1, DNMT3A, and DNMT3B) seem to increase the risk to develop TAMG (103)(102).
In 2013 Coppedè et al. investigated the presence of a common polymorphism (-579G>T: rs1569686) in the promoter of the DNMT3B gene coding for the DNA methyltransferase 3B in
324 AChR+ MG patients and 735 healthy matched unrelated controls afferent at the Myasthenia Clinic of Azienda Ospedaliera Universitaria Pisana (AOUP), at the Institute of General Pathology of the Catholic University of Rome, and at the Department of Translational Research and New Technologies in Medicine and Surgery of the University of Pisa (103).
Disease diagnosis was based on characteristic signs and symptoms of MG coupled with an anti-AChR antibody positive test. The main clinical characteristics of MG patients are listed in Table 6. The mean age (±S.D.) of the patients was 56.0 (± 16.5) years. According to disease onset age, patients were divided into early onset (≤50 years) and late onset (>50 years). According to the Osserman classification, they could be divided into pure ocular (class I) and generalized MG (class IIA, IIB, III, IV). MG was associated with different autoimmune disorders (AID) in 51 (15.7 %) out of 324 patients (see Table 6 for details). All patients had computed tomography (CT) scans of the chest and thymectomy was performed in 179 out of 324 patients according to the CT scan findings. Overall 94 patients (29.0%) had a thymoma.
The study was performed in accordance to the Declaration of Helsinki, and was approved by the Ethics Committees of the Pisa University Hospital and of the Catholic University of Rome.
Our study suggests that the presence of the DNMT3B -579T allele might represent a risk factor for the development of MG-associated thymomas, particularly in carriers of the homozygous TT genotype.
Therefore, the analysis of this polymorphism could help to identify those AChR+ MG individuals at increased risk to develop a thymoma (103).
In 2016 we investigated the methylation levels of genes involved in DNA methylation reactions (DNMTs), such as DNMT1, DNMT3A, and DNMT3B, and in one-carbon metabolism, such as methylenetetrahydrofolate reductase (MTHFR), in blood and tumor tissue DNA from 69 thymoma-associated myasthenia gravis patients; for 44 of them we also had thymic epithelial tissue adjacent to the tumor (102).
TAMG patients were always recruited at the Myasthenia Clinic of AOUP. The diagnosis of MG was made on the clinical symptoms together with the detection of positivity of AChR antibodies. Clinical stages of MG were assessed according to the Osserman and MGFA classifications. 97% of the patients were taking steroids at the time of surgical specimen collection. In addition, 10% of them were following immunosuppressant and/or immunomodulatory therapy, 6% followed a cisplatin-based chemotherapy prior to surgery and 4% underwent radiotherapy. Demographic characteristics of the population are shown in Table 7. DNA samples were obtained from both the surgically resected tumor tissue and from the adjacent normal tissue available from 44 of them (Table 8). An aliquot of blood in EDTA tubes was also collected from each patient and stored at -20 C until assayed. Each patient gave informed written consent for genotype analysis before blood drawing. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of the Pisa University Hospital (Protocol number 21302/2015).
Table 7. Demographic and clinico-pathological characteristics of 369 the studied population (adapted from Lopomo A. et al., 2016 (102))
Abbreviations: TAMG: thymoma-associated myasthenia gravis; MG: myasthenia gravis; F: females; M: males; N = number; NS: not specified; y: years. MGFA: MG Foundation of America, classification at thymectomy. *Therapy: Almost all the patients (97%) were taking steroids prior to and/or at the time of surgical specimen collection. Immuno:
immunomodulatory/immunosuppressant drugs (azathioprine, methotrexate, intravenous immunoglobulins, cyclosporine). Chemo: cisplatin-based chemotherapy. Radio: radiotherapy.
Table 8. Methylation levels (%) of the studied genes in blood and tumor tissue of total thymoma-associated MG (TAMG) patients (n = 69). Blood and tumor tissues were taken from the same donors, and the p-value refers to a t-test for paired samples (Lopomo A. et al., 2016 (102))
Figure 5 shows the mean methylation levels of the studied genes in blood, tumor tissue, and thymic epithelial tissue adjacent to the cancer, in the 44 patients for whom adjacent tissue was available. MTHFR shows higher methylation levels in tumor tissue with respect to blood (p = 0.0053) and to adjacent tissue (p < 0.001). DNMT3A shows higher methylation levels in tumor tissue and adjacent thymic tissue with respect to blood that results in them being completely demethylated (p < 0.001), but no significant difference was observed between thymomas and adjacent thymic tissue. The other two genes, DNMT3B and DNMT1, show very low methylation levels, no more than 2%, and no statistically significant difference was observed among the studied tissues. Age, gender, and clinico-pathological features had no effect on the mean methylation levels of the studied genes.
Fig. 5. Methylation levels (%) of the studied genes ((A) MTHFR; (B) DNMT3A; (C) DNMT3B; and (D) DNMT1) in blood, tumor tissue and adjacent thymic tissue of thymoma-associated MG (TAMG) patients for whom adjacent tissue was available (n = 44). The significant p-values (in red) refer to t-tests for paired samples (Lopomo A. et al., 2016 (102))
We observed that MTHFR and DNMT3A promoters show different methylation levels between blood and tumor tissue; this data was observed in the total population composed of 69 patients as well as in the smaller subgroup of 44 patients for whom healthy thymic specimens were available. In the subgroup of 44 patients, different MTHFR methylation levels between the adjacent thymic tissue and the tumor tissue were noticed; in particular, tumor tissue showed on average higher methylation levels with respect to adjacent healthy tissue. For DNMT3A, higher methylation levels in the healthy tissue adjacent to a tumor were observed with respect to blood. Comparing the methylation levels of the studied genes in different tissues, statistically significant correlations of MTHFR promoter methylation levels between blood and tumor tissue, adjacent thymic tissue and blood, and tumor tissue and adjacent tissue were observed. In conclusion, the study revealed increased MTHFR promoter methylation in thymomas and some degrees of methylation of the DNMT3A gene in thymic tissue with respect to blood (102). A recent whole-genome methylation study has suggested that thymomas associated to MG are epigenetically distinct from thymomas that develop in people without MG (104). The
methylation levels of the DNA repair and tumor suppressor genes MLH1, MGMT, CDKN2A and RASSF1A have been frequently investigated in thymic epithelial tumors (TETs), including thymomas (105)(106).
Recently, in the same cohort of 69 TAMG patients described before (102) we investigated the methylation levels of the DNA repair and tumor suppressor genes MLH1, MGMT, CDKN2A and RASSF1A in blood and tumor tissue DNA, as well as in the healthy thymic epithelial tissue adjacent to the tumor available from 44 patients. The study revealed that the promoter methylation levels of MLH1, MGMT, CDKN2A and RASSF1A genes are not increased in thymomas with respect to the healthy tissues of MG patients, and do not correlate with histological or pathological features (107).
Fig. 6 Methylation levels of the studied genes in blood (red), healthy thymic tissue (blue), and tumor tissue (green). A) MLH1. B) MGMT. C) CDKN2A. D) RASSF1A. All the investigated genes resulted largely hypo-methylated in the studied tissues and no significant difference among them was observed. Data are shown as means ± SEM (mean standard error), and for each gene we compared 69 blood samples, 69 thymomas and 44 healthy thymuses. The p-value reflects the Bonferroni’s corrected p-value of the comparison of mean methylation levels among the three groups (Coppedè F. et al., 2020 (107))
2.3.4 Recurrent Thymoma associated with Myasthenia Gravis
The potential malignancy of thymoma is due to its risk of recurrence after removal (108). In 2010, the International Thymic Malignancy Interest Group (ITMIG) defined that: (1) the term “recurrence” is appropriate if the tumor has been eradicated; (2) relapses are classified as local (anterior mediastinum), regional (intrathoracic not contiguous with the thymus), and distant (intrapulmonary and extrathoracic) (109).
Recurrences of thymoma are rare (110) and disease-free time varies from case to case.
Recurrence of thymoma is usually insidious as most patients are asymptomatic (111). Some patients may occasionally present with symptoms of chest tightness such as chest pain and dyspnea, similar to thymoma symptoms (108). Chest Computed Tomography (CT) scan is the routine exam to identify the presence of a thymoma, but it is also the most targeted screening exam to highlight a thymic recurrence (112). Literature data report that thymomas can recur even years after the first intervention (113)(114); long-term surveillance, extended to at least 20 years, could therefore be essential for early identification of relapse (112).
As reported in the literature, thymoma recurrences are closely related to the histological grade (WHO and Masaoka-Koga classification) (115)(108); in fact, relapsing thymomas are more frequently of type B2 and B3 according to the WHO classification and belong to the more aggressive Masaoka-Koga stages, such as stage IIb, III, IV-III / IV. Authors have shown that an early relapse (<40 months after surgery) is a negative prognostic factor (116), and a localized and single recurrence implies a better prognosis (115). The most frequent relapses are pleural or intrathoracic ones, while distant metastases are rare (117).
2.4 Thymic hyperplasia in Myasthenia Gravis
Up to 80% of the patients with early disease onset (EOMG, age of onset ≤50 years) have follicular hyperplasia, in which there are ectopic germinal centers (GCs) containing a large number of B cells producing anti-AChR antibodies (38)(118). It was suggested that after a
triggering event, such as viral infection, in a genetically favorable background, thymic epithelial cells overproduce inflammatory cytokines, which ultimately contribute to the attraction of B cells from the periphery. Estrogens promote the proliferation of B cells, which could explain why follicles are primarily found in females, and B cells produce anti-AChR antibodies (38). Indeed, it was observed that MG patients with a hyperplastic gland have a significantly greater chance of achieving reduced disease severity or remission after thymectomy, compared to other histological subtypes (119).
A part from MG, other autoimmune diseases have been associated with thymus enlargement, especially thymus hyperplasia, including autoimmune thyroid diseases, especially Graves’ disease (GD).
With the aim of investigating the role of the thymus in humoral thyroid autoimmunity in humans, we performed a prospective study in patients with MG (Rotondo Dottore G. et al, Eur Thyroid J, accepted, 2020). The primary outcome of the study was the behavior of serum anti-thyroglobulin (TgAbs) and anti-thyroperoxidase (TPOAbs) autoantibodies in relation to thymectomy over a 48-week follow-up period, with visits at 24 and 48 weeks. The secondary outcome was the relationship between TgAbs and TPOAbs and the features of MG, namely the Osserman class, the presence of thymus involvement, as well as serum AchR-Abs and MuSK Abs.
As shown in Fig. 7, the prevalence of detectable serum TgAbs and/or TPOAbs decreased significantly over the follow-up period (p=0.003) in the 12 patients with detectable autoantibodies at baseline that underwent thymectomy for either a thymoma (8 patients), or a thymus hyperplasia (4 patients). At 48 weeks, only ~40% of these patients still had detectable serum TgAbs and/or TPOAbs, but the reduction of the autoantibody prevalence was already evident at 24 weeks. In contrast, the prevalence of detectable serum TgAbs and/or TPOAbs did not diminish significantly in the 10 patients with detectable autoantibodies at baseline that did
not undergo thymectomy, with a significant difference between thymectomized and non-thymectomized patients (p<0.001).
In the study we provided evidence for a central role of the thymus in humoral thyroid autoimmunity. This observation is supported by the disappearance of anti-thyroid autoantibodies from the bloodstream over a 48-month follow-up period in a relevant proportion (~60%) of patients following thymectomy, which was not instead observed in patients who did not undergo thymectomy (Rotondo Dottore G. et al, Eur Thyroid J, accepted, 2020).
Fig. 7: Prevalence of serum anti-thyroglobulin (TgAbs) and anti-thyroperoxidase (TPOAbs) over time in patients with detectable concentrations at baseline, selected out of 107 consecutive patients with Myasthenia Gravis, in relation to thymectomy. a) Prevalence of serum TgAbs and/or TPOAbs; b) prevalence of TgAbs; c) prevalence of TPOAbs. P values were obtained by Fisher Exact test (Rotondo Dottore G. et al, accepted, 2020).
2.5 Thymectomy
Thymectomy in MG is now a universally recommended treatment in forms of myasthenia with positive antibodies to acetylcholinic receptor (82) in order to improve myasthenic symptoms and also allow, in many cases, to achieve complete remission of the disease (120). The indication for thymectomy is based on the hypothesis that the thymus is the site of sensitization against AChR (121), on the evidence that it is a source of production of anti-AChR antibodies (122) and on clinical data showing how the removal of the thymus, in this category of patients, is associated with a significant increase in the frequency of clinical remissions (123)(124). The importance of thymectomy in the treatment of MG is based on several lines of evidence that support the central role of the thymus in the pathogenesis of this disease. The multicenter, randomized MGTX (Multinational Thymectomy Trial in Non-Thymomatous Myasthenia Gravis) trial, the results of which were published in August 2016, was instrumental in validating the therapeutic role of radical thymectomy in patients with non-thymomatous MG (125). The current indications for thymectomy surgery in MG concern patients with thymoma and cases of generalized myasthenia with EOMG and anti-AChR antibodies in which the thymus usually presents changes attributable to follicular hyperplasia. Furthermore, this type of treatment is considered more effective when it is carried out earlier than at the onset of symptoms. In non-thymomatous seropositive forms with late onset there are no absolute contraindications to the operation but it must be evaluated on a case by case basis (126). Numerous techniques have been described over the years to remove the thymus in MG. The MGFA classification of thymectomy divides four distinct approaches (Table 9).
Tab. 9. MGFA classification of the different surgical approaches
Currently, the extensive removal of the thymus and perithymic fat is recognized as the best technique in patients with MG as the only one capable of ensuring stable remission or good control of the disease (6)(125).
Fig. 8: Anatomy of the thymus. Black portion: thymus; gray portion: perithymic fat (Jaretzki A. et al, 2000 (6))
possible. In addition, multiple resectional techniques should not be reported as a single cohort.
The Thymectomy Classification (Table 5) is based on published reports. The techniques are grouped according to the primary approach (transcervical, videoscopic, trans-sternal, or combinations) and are described briefly. Refer-enced reports are recommended for details. Because, within each category, there may be variations in the extent of the resection from surgeon to surgeon, the extent of the
resec-tion for each patient cohort must be recorded. In all pro-spective studies it is recommended that detailed descrip-tions of the operative technique be supplied, accompanied by drawings and photographs of typical specimens. Ideally, a video of the technique should also be available.
At this time, two types of transcervical thymectomy are performed. The “Basic” resection employs an intracapsular extraction of the mediastinal thymus via a cervical incision and is limited to the removal of the central cervical– mediastinal lobes (Figure 1, A and B). No other tissue is removed in either the neck or the mediastinum [25, 26]. The original “Extended” resection employs a special manubrial retractor for improved exposure of the mediastinum. The mediastinal dissection is extracapsular and includes re-section of the visible mediastinal thymus and fat. Sharp dissection may or may not be performed on the pericar-dium. The neck exploration and dissection varies in extent and may or may not be limited to exploration and removal of the cervical–mediastinal extensions [27, 28]. Variations include the addition of a partial median ster-notomy [29] and the associated use of mediastinoscopy [30].
A number of variations in videoscopic technique are Fig 1. Anatomy of the thymus. This illus-tration represents what is now generally accepted as the surgical anatomy of the thymus [41]. The frequencies (percent oc-currence) of the variations are noted. Black ! thymus; gray ! fat that may contain islands of thymus and micro-scopic thymus. A-P window ! aorto-pul-monary window. Source: Neurology 1997;48(suppl 5):S52–S63.
Table 5. Thymectomy Classification
T-1 Transcervical Thymectomy (a)-Basic (b)-Extended T-2 Videoscopic Thymectomy (a)-“Classic” (b)-“VATET” T-3 Transsternal Thymectomy (a)-Standard (b)-Extended
T-4 Transcervical & Transsternal Thymectomy
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In patients with positive anti-MuSK MG, thymic tissue appears substantially normal (28)(29). The absence of histological changes is associated, in these subjects, with a poor clinical efficacy of thymectomy (49)(31) without a significant reduction in the antibody titre (127). In seronegative patients there are no definitive data on the role of thymectomy and therefore no indication is currently being given to the intervention.
Thymectomy, even in the presence of thymoma, is an intervention of choice that requires adequate preparation of the patient, who must have achieved good compensation for myasthenic symptoms. This compensation takes place thanks to a dedicated specialist team based on the close collaboration between the three specialists most involved in the care of the patient with MG: neurologist, thoracic surgeon and anesthetist. This multidisciplinary collaboration represents a fundamental element not only to reduce intra and post-operative complications, but also and above all to guarantee the patient a greater possibility of achieving remission or stable clinical improvement (88).
Minimally invasive robotic thymectomy is a safe alternative to sternotomic thymectomy in forms of Myasthenia Gravis associated with thymic hyperplasia or low-grade, non-invasive and small thymomas (128)(129). The robotic technique therefore represents a valid surgical option especially in young patients, with clinically well compensated myasthenia and in whom minimally invasive and faster recovery can represent an additional factor for an optimal therapeutic result (126).
Chapter 3
Refractory Myasthenia Gravis
Although there are a large number of treatment options, approximately 10% of patients with generalized MG do not respond to conventional treatments. This subgroup of myasthenic patients are defined “Refractory Myasthenia Gravis patients” (130)(131).
There is no univocal definition of "refractory myasthenia" but several are reported in the literature that share the concept of variability of the response to therapy over time (132)(133):
• loss of response to conventional treatments (both in terms of doses and duration of treatment);
• presence of serious adverse events with conventional treatments; • request for excessive doses of potentially harmful agents;
• Comorbid conditions that restrict the use of conventional therapies (131); • request for repeated short-term "rescue" treatments (IVIG or PLEX); • presence of frequent myasthenic crises (130)(134).
Current international guidelines define refractory myasthenia as follows:
Post-operative status is unchanged or worsened after corticosteroids and at least 2 other immunosuppressive agents, used in adequate doses for an adequate length of time, with persistent symptoms or side effects limiting their use, as defined by the patient and physician (52).
In order to improve the picture of patients with refractory MG it is of utmost importance defining clinically and serologically the different subtypes of MG.
3.1 Clinical, serological features and thymic abnormalities in Refractory myasthenia
Several authors have studied the characteristics associated with refractory myasthenia. A single-center retrospective study (n = 128) identified 19 patients with treatment-refractory MG (135). Compared to patients with non-refractory myasthenia these had an earlier onset, were predominantly female, had a thymoma and a form of generalized myasthenia gravis associated with anti-MuSK antibodies. Furthermore, a significantly greater proportion of patients with refractory MG had undergone thymectomy, suggesting underlying biological differences between patients with refractory and non-refractory MG (135).
These results were confirmed by another retrospective study involving seven centers (n = 512), in which 76 patients (14.8%) were considered refractory (134).
In this study characteristics associated with patients with refractory MG were similar to previous papers and included MuSK antibody positivity, female gender, having undergone thymectomy, presence of thymoma, and younger age of onset. Patients with refractory MG also had a higher prevalence and poor control of type 2 diabetes and dyslipidemia, due to increased steroid therapy (134).
Patients can remain refractory for more than 10 years (136) and are often considered refractory until they respond to therapy or become asymptomatic without therapeutic intervention. These patients experience periodically acute exacerbations of symptoms, including life-threatening symptoms, such as difficulty in swallowing and breathing, leading to myasthenic crisis (for the definition of “myasthenic crisis” see paragraph 1.1).
In addition to the risk of myasthenic crisis, patients with refractory MG have a severely impaired quality of life due to generalized muscle weakness and / or ocular symptoms with difficulty driving and performing common daily activities (131)(136). There is a need not only for more evidence-based therapies, but also for other measures to improve the quality of life in patients with MG (137).
Chapter 4
Aim of the study
The aim of the present study was to analyze the clinical and serological characteristics of patients with Refractory Myasthenia associated with anti-MuSK antibodies and with thymoma afferent at Myasthenia Clinic of Azienda Ospedaliera Universitaria Pisana (AOUP).
Thanks to the large number of the Pisan cohort and the close collaboration with oncology specialists, thoracic surgeons and colleagues of Oxford and Pavia, we were able to investigate these subgroups of patients.
Further aims were:
-to determine if inhibitor of the tyrosine phosphatase Shp2 (NSC-87877) could represent a new treatment strategy for MuSK-MG
-to identify clinical and serological features capable of predicting the risk of relapse in thymoma patients
Chapter 5
Refractory MG with anti-MuSK antibodies: clinical and serological features
Clinical features of MuSK MG have already been explained in Chapter 1.
The first aim of this chapter was to analyze the serological characteristics of these patients. The second aim was to compare the clinical features of MuSK-RIA and MuSK-CBA positive sera (45).
5.1 Methods
Sera of 71 MuSK-MG patients were sent to Dr. Saif Huda in Oxford. The MG diagnosis was established on the basis of characteristic clinical features, detection of MuSK-Abs (determined by RIA), and a beneficial response to immunotherapy. The MuSK-Ab status of the patients was first re-checked by MuSK-RIA with sera diluted 1:10 samples. MuSK-MG sera were subsequently tested by CBAs.
5.2 Results
From the serological point of view, MuSK-Abs were detected both with RIA and CBA assays in 69/71 (97.2%).
The clinical features of MuSK-RIA patients are summarized in Table 9. Patients were predominantly female [(M:F; 1:9; median age: 40 (1-79) yrs]; only 7/71 (10%) were male and they were similar in demographic and clinical features to female patients. The median time from symptom onset to diagnosis was 1 (0-10) year. Oropharyngeal involvement was the most prominent symptom in 69/71 (97.2%) and, at first review, 49/67 (73.1%) had an MGFA>2. Four patients presented with respiratory failure requiring immediate intubation and ventilation.
Ocular symptoms were present in 58/71 (81.7%) of patients and 16/71 (22.5%) had an involvement of limb muscles. A family history of AChR-MG was noted in 4/71 (5.6%) patients and concurrent autoimmune disease in 12/71 (16.9%) of patients (most commonly thyroid disease). A ‘Guptill phenotype’ (26) was available in 70 patients of which 28/70 (40%) had features that were indistinguishable from non-MuSK-MG, 27/70 (38.6%) had predominant faciopharyngeal involvement, and 15/70 (21.4%) patients had predominant neck extensor and respiratory weakness. The individual clinical data of all the MuSK-MG positive patients are summarized in Table 10.
SF-EMG was positive in 9/20 patients whereas for 51 patients this data was not available. RNS-EMG was positive in 31/48 patients, in 2 patients was borderline whereas for 21 patients this data was not available.
Immunotherapy, of which the mainstay was prednisone, was given in 69/71 (97.2%) of patients. Ten patients received azathioprine as a ‘steroid-sparing’ agent. IVIG were required in 10/71 (14%) patients. Two patients also received P-E at regular intervals. Immunotherapy was beneficial in 67/69 (97.1%) patients. At follow-up 11/70 (15.7%) of patients had an MGFA>2 compared to 49/67 (71.3%) at onset (p<0.0001), (median follow-up- 9 (2-34) yrs).
Accordingly, a PIS of minimal manifestations was attained in 40/67 (59.7%) and
pharmacological/complete stable remission in 26/69 (37.7%) of patients. There were no differences in rate of remission between different MuSK-MG phenotypes (p>0.05). Thymectomy was performed in 6/71 (9%) of patients. Histological features were normal in 3/6 and suggestive of thymic hyperplasia in the remaining three samples. Post-operative improvement was not noted in any of these patients.
