A retrospective multicenter study on clinical and serological parameters in patients with MuSK myasthenia gravis with and without general immunosuppression.

Introduction: Muscle-specific kinase (MuSK)- myasthenia gravis (MG) is caused by pathogenic autoantibodies against MuSK that correlate with disease severity and are predominantly of the IgG4 subclass. The first-line treatment for MuSK-MG is general immunosuppression with corticosteroids, but the effect of treatment on IgG4 and MuSK IgG4 levels has not been studied. Methods: We analyzed the clinical data and sera from 52 MuSK-MG patients (45 female, 7 male, median age 49 (range 17-79) years) from Italy, the Netherlands, Greece and Belgium, and 43 AChR-MG patients (22 female, 21 male, median age 63 (range 2-82) years) from Italy, receiving different types of immunosuppression, and sera from 46 age- and sex-matched non-disease controls (with no diagnosed diseases, 38 female, 8 male, median age 51.5 (range 20-68) years) from the Netherlands. We analyzed the disease severity (assessed by MGFA or QMG score), and measured concentrations of MuSK IgG4, MuSK IgG, total IgG4 and total IgG in the sera by ELISA, RIA and nephelometry. Results: We observed that MuSK-MG patients showed a robust clinical improvement and reduction of MuSK IgG after therapy, and that MuSK IgG4 concentrations, but not total IgG4 concentrations, correlated with clinical severity. MuSK IgG and MuSK IgG4 concentrations were reduced after immunosuppression in 4/5 individuals with before-after data, but data from non-linked patient samples showed no difference. Total serum IgG4 levels were within the normal range, with IgG4 levels above threshold (1.35g/L) in 1/52 MuSK-MG, 2/43 AChR-MG patients and 1/45 non-disease controls. MuSK-MG patients improved within the first four years after disease onset, but no further clinical improvement or reduction of MuSK IgG4 were observed four years later, and only 14/52 (26.92%) patients in total, of which 13 (93.3%) received general immunosuppression, reached clinical remission. Discussion: We conclude that MuSK-MG patients improve clinically with general immunosuppression but may require further treatment to reach remission. Longitudinal testing of individual patients may be clinically more useful than single measurements of MuSK IgG4. No significant differences in the serum IgG4 concentrations and IgG4/IgG ratio between AChR- and MuSK-MG patients were found during follow-up. Further studies with larger patient and control cohorts are necessary to validate the findings.

Outcomes after robotic thymectomy in nonthymomatous versus thymomatous patients with acetylcholine-receptor-antibody-associated myasthenia gravis.

The aim of this study was to investigate the surgical and long-term neurological outcomes of patients with acetylcholine-receptor-antibody-associated myasthenia gravis (AChR-MG) who underwent robotic thymectomy (RATS). We retrospectively analyzed the clinical-pathological data of all patients with AChR-MG who underwent RATS using the DaVinci® Robotic System at the MUMC+ between April 2004 and December 2018. Follow-up data were collected from 60 referring Dutch hospitals. In total, 230 myasthenic patients including 76 patients with a thymoma (33.0%) were enrolled in this study. Mean follow-up time, procedure time and hospitalization were, respectively 65.7 ± 43.1 months, 111±52.5 min and 3.3 ± 2.2 days. Thymomatous patients had significantly more frequently and more severe complications than nonthymomatous patients (18.4% vs. 3.9%, p<0.001). Follow up data was available in 71.7% of the included patients. The Myasthenia Gravis Foundation of America postintervention score showed any kind of improvement of MG-symptoms after RATS in 82.4% of the patients. Complete stable remission (CSR) or pharmacological remission (PR) of MG was observed in 8.4% and 39.4% of the patients, respectively. Mean time till CSR/PR remission after thymectomy was 26.2 ± 29.2 months. No statistical difference was found in remission or improvement in MGFA scale between thymomatous and nonthymomatous patients. RATS is safe and feasible in patients with MG. The majority of the patients (82.4%) improved after thymectomy. CSR and PR were observed in 8.4% and 39.4% of the patients, respectively, with a mean of 26.2 months after thymectomy. Thymomatous patients had more frequently and more severe complications compared to nonthymomatous patients.

Novel treatment strategies for acetylcholine receptor antibody-positive myasthenia gravis and related disorders.

The presence of autoantibodies directed against the muscle nicotinic acetylcholine receptor (AChR) is the most common cause of myasthenia gravis (MG). These antibodies damage the postsynaptic membrane of the neuromuscular junction and cause muscle weakness by depleting AChRs and thus impairing synaptic transmission. As one of the best-characterized antibody-mediated autoimmune diseases, AChR-MG has often served as a reference model for other autoimmune disorders. Classical pharmacological treatments, including broad-spectrum immunosuppressive drugs, are effective in many patients. However, complete remission cannot be achieved in all patients, and 10% of patients do not respond to currently used therapies. This may be attributed to production of autoantibodies by long-lived plasma cells which are resistant to conventional immunosuppressive drugs. Hence, novel therapies specifically targeting plasma cells might be a suitable therapeutic approach for selected patients. Additionally, in order to reduce side effects of broad-spectrum immunosuppression, targeted immunotherapies and symptomatic treatments will be required. This review presents established therapies as well as novel therapeutic approaches for MG and related conditions, with a focus on AChR-MG.

Robotic Thymectomy for Thymomas: A Retrospective Follow-up Study in the Netherlands.

BACKGROUND: The Maastricht University Medical Center+ is a Dutch center of expertise appointed by the Netherlands Federation of University Medical Centers for the treatment of thymomas. The aim of this study was to investigate the long-term oncologic, surgical, and neurologic outcomes of all patients who underwent a robotic thymectomy for a thymoma at Maastricht University Medical Center+. METHODS: We retrospectively analyzed the clinical-pathologic data of all consecutive patients with a thymoma who underwent robotic thymectomy using the DaVinci robotic system at Maastricht University Medical Center+ between April 2004 and December 2018. Follow-up data were collected from 60 referring Dutch hospitals. RESULTS: In total, 398 robotic thymectomies were performed, and 130 thymomas (32.7%) were found. Median follow-up time was 46 months; median procedure time, 116 minutes; and median hospitalization time, 3 days. In 8.4% of patients, a conversion was performed, and in 20.8%, a complication was registered. The majority of myasthenic patients with a thymoma went into remission, mostly within 12 to 24 months after thymectomy (81%). No statistical difference was found in the number of complications, conversions, incomplete resections, or deaths between patients with myasthenia gravis and nonmyasthenic patients. Thirty-six patients (27.7%) underwent postoperative radiotherapy. The recurrence rate was 9.1%, and the 5-year thymoma-related survival rate was 96.6%. CONCLUSIONS: Robotic thymectomy was found to be safe and feasible for early stage thymomas, most advanced-stage thymomas, and thymomatous myasthenia gravis. A national guideline could contribute to the improvement of the oncologic follow-up of thymic epithelial tumors in the Netherlands.

The association between anti-acetylcholine receptor antibody level and clinical improvement in myasthenia gravis.

BACKGROUND AND PURPOSE: Anti-acetylcholine receptor (AChR) antibodies (ab) in the serum are detected in most patients with generalized myasthenia gravis (MG) and used as a diagnostic tool. The aim of this study was to analyse a possible association between anti-AChR-ab serum levels and clinical improvement of MG. METHODS: The Maastricht University Medical Center is a centre of expertise for the treatment of MG. Between 1997 and 2020, more than 4000 anti-AChR-ab blood samples were measured for clinical care using a quantitative radioimmunoassay technique. These results, in combination with clinical status obtained from the patients' electronic patient files, were retrospectively analysed by a single blinded clinician. Symptoms of MG were classified using the Myasthenia Gravis Foundation of America (MGFA) scale. RESULTS: In total, 90 anti-AChR-ab-positive MG patients with 837 blood samples were included. The median follow-up time was 72 months. The majority of the included patients were women (61.1%), were on immunosuppressive drug therapy (88.9%), and underwent a thymectomy (54.4%). Multilevel logistic regression analysis showed a significantly inverse association between change in anti-AChR-ab level and the odds of MGFA improvement (per 10% decrease of anti-AChR-ab level: odds ratio 1.21, 95% confidence interval 1.12-1.31; p < 0.001). CONCLUSIONS: A change in anti-AChR-ab serum level is associated with clinical status in patients with MG. Analyses of anti-AChR-ab are not only useful for diagnostics but also in follow-up of adult symptomatic patients with MG. The use of repetitive anti-AChR-ab serum levels might be valuable in long-term monitoring for clinical improvement in patients with MG, however, further research is required for specific recommendations.

Subclinical myasthenia gravis in thymomas.

BACKGROUND: A proportion of thymoma-patients without a history of myasthenia gravis (MG) before thymectomy, appears to have positive anti-AChR-antibodies in the serum. These subclinical MG-patients could be underdiagnosed because analyzation of anti-AChR-antibodies in thymomas is not always performed in patients who did not experience neurological symptoms. The prevalence and long-term outcomes of subclinical MG are never described in literature yet. METHODS: We retrospectively analyzed 398 consecutive patients who underwent a robotic-assisted thoracoscopic surgery at the Maastricht University Medical Center+ (MUMC+) between April 2004 and December 2018. In the MUMC+, a robotic approach is the standard surgical approach in patients with thymic diseases. Inclusion criteria were thymomas, thymectomy performed in the MUMC + with a follow-up of at least one year and age above 18 years old. Exclusion criteria were patients with thymic carcinomas, refused participation, or those who were lost to follow-up. RESULTS: Of the 102 included thymoma-patients, 87 patients (85 %) were tested for anti-AChR-antibodies before thymectomy, of which 57 patients were diagnosed with clinical MG and seven subclinical MG-patients were found. Of the 15 patients who were not tested for anti-AChR-antibodies, four more subclinical MG-patients were discovered in the years after thymectomy. The median follow-up time was 62 months. In total, 11 subclinical MG-patients were found, with a mean age of 54 years and predominantly females (64 %). Ten subclinical MG-patients (91 %) developed clinical-MG, within six years after thymectomy. Immunosuppressive drugs were prescribed in five patients. Four patients were diagnosed with a recurrence of the thymoma. No surgical mortality was reported. Two patients died due to a myasthenic crisis. CONCLUSIONS: The prevalence of subclinical MG in thymomas was found to be 10.8 %. One in four patients who experienced no neurological symptoms before thymectomy, appeared to have anti-AChR-antibodies and 91 % of these patients developed clinical MG within six years after the thymectomy. Analyzing anti-AChR-antibodies in the serum is recommended in all suspected thymomas before a thymectomy is performed.

Globe Subluxation following Long-Term High-Dose Steroid Treatment for Myasthenia Gravis.

This case report describes the unusual presentation of a globe subluxation following long-term high-dose oral steroid treatment for myasthenia gravis (MG). The patient presented initially with fluctuating vertical diplopia. Auto-antibodies against the acetylcholine receptor were weakly positive, confirming the diagnosis of MG. After initial treatment with pyridostigmine, the disease evolved to generalized MG. Plasmapheresis and high-dose steroids were started subsequently. As a side effect of this treatment the patient gained about 30 kg in weight and developed steroid myopathy and a prominent cushingoid facies with bilateral exophthalmos. A year after his initial diagnosis he experienced a spontaneous globe subluxation on the left eye. He was able to immediately reposition the globe manually himself. Four months later a new subluxation occurred. Because of these aforementioned severe side effects of the steroid treatment, the methylprednisolone was tapered and replaced by tacrolimus. After about 6 weeks the patient went into remission. We believe, that the spontaneous globe subluxations were caused by a weakness of the extraocular muscles in combination with a significant gain of intraorbital fat tissue, both induced by cumulative, excessive steroids. Steroids are often necessary in the treatment of MG; however, most of the time a high dose of 64 mg is not needed for ocular MG and especially the continuation of a dose of 58 mg or more for a long period is not recommended. Careful follow-up is obligatory to timely recognize side effects. In case of severe side effects or the need for long-term treatment, the use of other immunosuppressive therapies should be considered. Extra care and caution is recommended in patients who are anatomically predisposed with proptosis.

Low prevalence of Merkel cell polyomavirus in human epithelial thymic tumors.

BACKGROUND: The etiology of thymic epithelial tumors is unknown. Murine polyomavirus strain PTA has been shown to induce thymomas in mice. Recently, using diverse molecular techniques, we reported the presence of human polyomavirus 7 (HPyV7) in thymic epithelial tumors. In the present study, we investigated the prevalence of Merkel cell polyomavirus (MCPyV) in thymic epithelial tumors. METHODS: Thirty-six thymomas were screened for MCPyV by PCR and subsequently tested by DNA and RNA in situ hybridization and immunohistochemistry. Twenty-six thymomas were diagnosed with myasthenia gravis (MG). RESULTS: MCPyV DNA was detected by PCR in 7 (19.4%) of the 36 thymic epithelial tumors and in six of these, the presence of MCPyV was confirmed by fluorescence situ hybridization. Of these, 3 (28.6%) revealed weak MCPyV LT-antigen protein expression. In addition, one of the MCPyV positive thymomas tested positive for MCPyV LT RNA with RNAscope. Of interest, two out of the three thymomas that previously tested positive for MCPyV by immunohistochemistry also tested positive for HPyV7. One of the 11 MG-negative and 2 of the 25 MG-positive were positive for MCPyV. CONCLUSIONS: MCPyV DNA and MCPyV protein expression can be detected in human epithelial thymoma; however, to a far lesser extent than HPyV7. Our data strongly indicate that because of its infrequent detection and weak expression, MCPyV is unlikely to play an important role in the etiopathogenesis of human thymomas.

Characterization of the thymus in Lrp4 myasthenia gravis: Four cases.

Myasthenia gravis (MG) is an autoimmune disease of the neuromuscular junction. Most patients have pathogenic autoantibodies against the acetylcholine receptor (AChR). In the last years a novel subpopulation of MG patients has been described that harbors antibodies against low-density lipoprotein receptor-related protein 4 (Lrp4), another postsynaptic neuromuscular antigen. In early-onset AChR MG (EOMG), the thymus plays an important role in immunopathogenesis, and early thymectomy is beneficial. It is still unknown if the thymus plays any role in Lrp4-MG. In this pilot study, we compared thymus samples from four patients with Lrp4-MG (one pre-treated with immunosuppressive drugs), four non-MG controls and five EOMG patients (not pretreated with immunosuppressive drugs). Immunohistochemistry of the Lrp4-MG thymi revealed normal architecture, with normal numbers and distribution of B-cells, lymphoid follicles and Hassall's corpuscles. Primary CD23+ lymphoid follicles were similarly infrequent in Lrp4-MG and control thymic sections. In none of the control or Lrp4-MG thymi did we find secondary follicles with CD10+ germinal centers. These were evident in 2 of the 5 EOMG thymi, where primary lymphoid follicles were also more frequent on average, thus showing considerable heterogeneity between patients. Even if characteristic pathological thymic changes were not observed in the Lrp4 subgroup, we cannot exclude a role for the thymus in Lrp4-MG pathogenesis, since one Lrp4-MG patient went into clinical remission after thymectomy alone (at one year follow-up) and one more improved after thymectomy in combination with immunosuppressive therapy.

Alpha7 acetylcholine receptor autoantibodies are rare in sera of patients diagnosed with schizophrenia or bipolar disorder.

The α7 acetylcholine receptor (AChR) has been linked with the onset of psychotic symptoms and we hypothesized therefore that it might also be an autoimmune target. Here, we describe a new radioimmunoassay (RIA) using iodine 125-labelled α-bungarotoxin and membrane extract from transfected HEK293 cells expressing human α7 AChR. This RIA was used to analyze sera pertaining to a cohort of 711 subjects, comprising 368 patients diagnosed with schizophrenia spectrum disorders, 140 with bipolar disorder, 58 individuals diagnosed of other mental disorders, and 118 healthy comparison subjects. We identified one patient whose serum tested positive although with very low levels (0.2 nM) for α7 AChR-specific antibodies by RIA. Three out of 711 sera contained antibodies against iodine 125-labelled α-bungarotoxin, because they precipitated with it in the absence of α7 AChR. This first evidence suggests that autoantibodies against α7 AChR are absent or very rare in these clinical groups.

Characterization of an anti-fetal AChR monoclonal antibody isolated from a myasthenia gravis patient.

We report here the sequence and functional characterization of a recombinantly expressed autoantibody (mAb 131) previously isolated from a myasthenia gravis patient by immortalization of thymic B cells using Epstein-Barr virus and TLR9 activation. The antibody is characterized by a high degree of somatic mutations as well as a 6 amino acid insertion within the VHCDR2. The recombinant mAb 131 is specific for the γ-subunit of the fetal AChR to which it bound with sub-nanomolar apparent affinity, and detected the presence of fetal AChR on a number of rhabdomyosarcoma cell lines. Mab 131 blocked one of the two α-bungarotoxin binding sites on the fetal AChR, and partially blocked the binding of an antibody (mAb 637) to the α-subunit of the AChR, suggesting that both antibodies bind at or near one ACh binding site at the α/γ subunit interface. However, mAb 131 did not reduce fetal AChR ion channel currents in electrophysiological experiments. These results indicate that mAb 131, although generated from an MG patient, is unlikely to be pathogenic and may make it a potentially useful reagent for studies of myasthenia gravis, rhabdomyosarcoma and arthrogryposis multiplex congenita which can be caused by fetal-specific AChR-blocking autoantibodies.

Methylome and transcriptome profiling in Myasthenia Gravis monozygotic twins.

OBJECTIVE: To identify novel genetic and epigenetic factors associated with Myasthenia gravis (MG) using an identical twins experimental study design. METHODS: The transcriptome and methylome of peripheral monocytes were compared between monozygotic (MZ) twins discordant and concordant for MG, as well as with MG singletons and healthy controls, all females. Sets of differentially expressed genes and differentially methylated CpGs were validated using RT-PCR for expression and target bisulfite sequencing for methylation on additional samples. RESULTS: >100 differentially expressed genes and ∼1800 differentially methylated CpGs were detected in peripheral monocytes between MG patients and controls. Several transcripts associated with immune homeostasis and inflammation resolution were reduced in MG patients. Only a relatively few genes differed between the discordant healthy and MG co-twins, and both their expression and methylation profiles demonstrated very high similarity. INTERPRETATION: This is the first study to characterize the DNA methylation profile in MG, and the expression profile of immune cells in MZ twins with MG. Results suggest that numerous small changes in gene expression or methylation might together contribute to disease. Impaired monocyte function in MG and decreased expression of genes associated with inflammation resolution could contribute to the chronicity of the disease. Findings may serve as potential new predictive biomarkers for disease and disease activity, as well as potential future targets for therapy development. The high similarity between the healthy and the MG discordant twins, suggests that a molecular signature might precede a clinical phenotype, and that genetic predisposition may have a stronger contribution to disease than previously assumed.

Hinge-deleted IgG4 blocker therapy for acetylcholine receptor myasthenia gravis in rhesus monkeys.

Autoantibodies against ion channels are the cause of numerous neurologic autoimmune disorders. Frequently, such pathogenic autoantibodies have a restricted epitope-specificity. In such cases, competing antibody formats devoid of pathogenic effector functions (blocker antibodies) have the potential to treat disease by displacing autoantibodies from their target. Here, we have used a model of the neuromuscular autoimmune disease myasthenia gravis in rhesus monkeys (Macaca mulatta) to test the therapeutic potential of a new blocker antibody: MG was induced by passive transfer of pathogenic acetylcholine receptor-specific monoclonal antibody IgG1-637. The effect of the blocker antibody (IgG4Δhinge-637, the hinge-deleted IgG4 version of IgG1-637) was assessed using decrement measurements and single-fiber electromyography. Three daily doses of 1.7 mg/kg IgG1-637 (cumulative dose 5 mg/kg) induced impairment of neuromuscular transmission, as demonstrated by significantly increased jitter, synaptic transmission failures (blockings) and a decrease in the amplitude of the compound muscle action potentials during repeated stimulations (decrement), without showing overt symptoms of muscle weakness. Treatment with three daily doses of 10 mg/kg IgG4Δhinge-637 significantly reduced the IgG1-637-induced increase in jitter, blockings and decrement. Together, these results represent proof-of principle data for therapy of acetylcholine receptor-myasthenia gravis with a monovalent antibody format that blocks binding of pathogenic autoantibodies.

IgG4 autoantibodies against muscle-specific kinase undergo Fab-arm exchange in myasthenia gravis patients.

Autoimmunity mediated by IgG4 subclass autoantibodies is an expanding field of research. Due to their structural characteristics a key feature of IgG4 antibodies is the ability to exchange Fab-arms with other, unrelated, IgG4 molecules, making the IgG4 molecule potentially monovalent for the specific antigen. However, whether those disease-associated antigen-specific IgG4 are mono- or divalent for their antigens is unknown. Myasthenia gravis (MG) with antibodies to muscle specific kinase (MuSK-MG) is a well-recognized disease in which the predominant pathogenic IgG4 antibody binds to extracellular epitopes on MuSK at the neuromuscular junction; this inhibits a pathway that clusters the acetylcholine (neurotransmitter) receptors and leads to failure of neuromuscular transmission. In vitro Fab-arm exchange-inducing conditions were applied to MuSK antibodies in sera, purified IgG4 and IgG1-3 sub-fractions. Solid-phase cross-linking assays were established to determine the extent of pre-existing and inducible Fab-arm exchange. Functional effects of the resulting populations of IgG4 antibodies were determined by measuring inhibition of agrin-induced AChR clustering in C2C12 cells. To confirm the results, κ/κ, λ/λ and hybrid κ/λ IgG4s were isolated and tested for MuSK antibodies. At least fifty percent of patients had IgG4, but not IgG1-3, MuSK antibodies that could undergo Fab-arm exchange in vitro under reducing conditions. Also MuSK antibodies were found in vivo that were divalent (monospecific for MuSK). Fab-arm exchange with normal human IgG4 did not prevent the inhibitory effect of serum derived MuSK antibodies on AChR clustering in C2C12 mouse myotubes. The results suggest that a considerable proportion of MuSK IgG4 could already be Fab-arm exchanged in vivo. This was confirmed by isolating endogenous IgG4 MuSK antibodies containing both κ and λ light chains, i.e. hybrid IgG4 molecules. These new findings demonstrate that Fab-arm exchanged antibodies are pathogenic.

Silencing of Dok-7 in Adult Rat Muscle Increases Susceptibility to Passive Transfer Myasthenia Gravis.

Myasthenia gravis (MG) is an autoimmune disease mediated by autoantibodies that target proteins at the neuromuscular junction, primarily the acetylcholine receptor (AChR) and the muscle-specific kinase. Because downstream of kinase 7 (Dok-7) is essential for the full activation of muscle-specific kinase and consequently for dense clustering of AChRs, we hypothesized that reduced levels of Dok-7 increase the susceptibility to passive transfer MG. To test this hypothesis, Dok-7 expression was reduced by transfecting shRNA-coding plasmids into the tibialis anterior muscle of adult rats by in vivo electroporation. Subclinical MG was subsequently induced with a low dose of anti-AChR monoclonal antibody 35. Neuromuscular transmission was significantly impaired in Dok-7-siRNA-electroporated legs compared with the contralateral control legs, which correlated with a reduction of AChR protein levels at the neuromuscular junction (approximately 25%) in Dok-7-siRNA-electroporated muscles, compared with contralateral control muscles. These results suggest that a reduced expression of Dok-7 may play a role in the susceptibility to passive transfer MG, by rendering AChR clusters less resistant to the autoantibody attack.

Myasthenia gravis with muscle specific kinase antibodies mimicking amyotrophic lateral sclerosis.

Muscle-specific kinase (MuSK) myasthenia gravis (MG) is hallmarked by the predominant involvement of bulbar muscles and muscle atrophy. This might mimic amyotrophic lateral sclerosis (ALS) presenting with bulbar weakness. We encountered four cases of MuSK MG patients with an initial misdiagnosis of ALS. We analyzed the clinical data of the four misdiagnosed MuSK MG patients, and investigated the presence of MuSK autoantibodies in a group of 256 Dutch bulbar-onset ALS patients using a recombinant MuSK ELISA and a standard MuSK radioimmunorecipitation assay. Clues for changing the diagnosis were slow progression, clinical improvement, development of diplopia and absence of signs of upper motor neuron involvement. No cases of MuSK MG were identified among a group of 256 bulbar ALS patients diagnosed according to the revised El Escorial criteria. A misdiagnosis of ALS in patients with MuSK MG is rare. We recommend to carefully consider the diagnosis of MuSK MG in patients presenting with bulbar weakness without clear signs of upper motor neuron dysfunction.

Expression of pRb and p16INK4 in human thymic epithelial tumors in relation to the presence of human polyomavirus 7.

BACKGROUND: We have recently reported the presence of the Human polyomavirus 7 (HPyV7) in human thymic epithelial tumors as assessed by diverse molecular techniques. Here we report on the co-expression of p16, retinoblastoma protein (pRb) and phosphorylated retinoblastoma protein (phospho-Rb) in human thymic epithelial tumors in relation to HPyV7. METHODS: PRB, phospho-RB and p16 expression was assessed by immuno-histochemistry in 37 thymomas and 2 thymic carcinomas. 17 thymomas (46 %) and 1 thymic carcinoma (50 %) were recently tested positive for HPyV7. In addition, 20 follicular hyperplasias were tested. RESULTS: Expression of pRb was observed in 35 thymomas (94.6 %), in 16 thymomas (43.2 %) the expression was strong. Phospho-Rb was observed in 31 thymomas (83.8 %). 19 thymomas (51.4 %) showed immunoreactivity for p16 of which 8 thymomas revealed very strong p16 expression. No p16 expression was detected in thymic carcinomas. In addition, no significant correlation between the presence of HPyV7 and pRb-, phospho-Rb- and p16-expression could be established. No correlation between pRb, phospho-Rb, p16 and WHO staging, Masaoka-Koga staging or the presence of MG was found. All 20 follicular hyperplasias showed expression of pRb and less expression of phospho-Rb. CONCLUSIONS: Although polyomaviruses have been shown to interact with cell cycle proteins no correlation between the presence of HPyV7 and the expression of pRb, phospho-Rb and p16 in human thymic epithelial tumors was observed. In as much HPyV7 contributes to human thymomagenesis remains to be established. Our data indicate pRb, phospho-Rb and p16 expression are rather unlikely to be involved in HPyV7 related thymomagenesis.

Do associated auto-antibodies influence the outcome of myasthenia gravis after thymectomy?

Myasthenia gravis (MG) is a neuromuscular autoimmune disease, where antibodies against the acetylcholine receptor destroy this receptor. The role of thymectomy in the treatment of MG remains controversial. Because of the frequent association with other autoimmune diseases, we hypothesized that patients with multiple autoantibodies (autoAbs) might have a lower chance of reaching complete stable remission after thymectomy. We analyzed sera of 85 MG patients who underwent a thymectomy between April 2004 and December 2012. We used four different immunodot kits (D-Tek, Mons, Belgium): ANA25 Quantrix, Synthetase 10 Diver, Myositis 7 Diver and Liver 10 profile Diver, all automatized on the BlueDiver Instrument (D-Tek). The Myasthenia Gravis Foundation of America (MGFA) postintervention status was used to determine the outcome after thymectomy. AutoAbs other than anti-acetylcholine receptor (AChR) antibodies were detected in 29.4% of the patients of whom 16.5% clinically had a second autoimmune disease. In none of the seronegative patients other autoAbs were detected. No significant difference was observed in the 3-years remission rate after thymectomy in patients with or without antibodies other than anti-AChR antibodies. Although these autoAbs do not predict outcome in our MG patient cohort, screening for multiple autoAbs in MG patients might be warranted to identify patients with additional autoimmune diseases.

Robotic thymectomy in patients with myasthenia gravis: neurological and surgical outcomes.

OBJECTIVES: Thymectomy is frequently used in the treatment of myasthenia gravis (MG). But indication, timing or surgical approach remain controversial. This study reports our experiences with robotic thymectomy and surgical and neurological outcomes in a large cohort of patients with MG. METHODS: We retrospectively analysed the outcome of 125 patients with MG who underwent a robotic thymectomy using the da Vinci Surgical System (Intuitive Surgical, Inc., Sunnyvale, CA, USA) between 2004 and 2012. The Myasthenia Gravis Foundation of America (MGFA) Classification was used to determine preoperative and postintervention status. RESULTS: Ninety-five women and 30 men underwent a robotic thymectomy. One hundred patients had a neurological follow-up of more than 12 months. Preoperative most severe MGFA classification was Stage I in 11 patients (8.8%), Stage IIA in 18 patients (14.4%), Stage IIB in 18 patients (14.4%), Stage IIIA in 7 patients (5.6%), Stage IIIB in 29 patients (23.2%), Stage IVA in 10 patients (8.0%), Stage IVB in 29 patients (23.2%) and Stage V in 3 patients (2.4%). Median surgical procedure time was 123 min (range 45-353 min). There were no major perioperative complications or deaths. The median postoperative hospital stay was 3 days (range 2-24 days). Histological analysis showed thymic remnant tissue in 41 patients (32.8%), follicular hyperplasia in 52 patients (41.6%), thymoma in 31 patients (24%), lipoma in 1 patient (0.8%) and a cyst in 1 patient (0.8%). Patients with thymic remnant tissue were significantly more preoperative steroid users compared with the follicular hyperplasia group (P = 0.02). With a median follow-up of 33 months (range 12-104 months), 77% of the patients showed neurological improvement. Three-year probability remission rate [complete stable remission (CSR) and pharmacological remission] is 28.2%. Patients who were not treated with prednisolone preoperatively showed a significant higher CSR than patients who did take prednisolone (P = 0.014). No significant difference was observed regarding timing of surgery (P = 0.37). CONCLUSIONS: Robotic thymectomy in patients with MG is safe and feasible. A neurological benefit and decreased use of steroids can be obtained in the majority of patients. No significant difference in neurological outcome was observed as the result of timing of robot thymectomy after the onset of MG.

Detection of human polyomavirus 7 in human thymic epithelial tumors.

INTRODUCTION: Although the molecular genetics possibly underlying the pathogenesis of human thymoma have been extensively studied, its etiology remains poorly understood. Because murine polyomavirus consistently induces thymomas in mice, we assessed the presence of the novel human polyomavirus 7 (HPyV7) in human thymic epithelial tumors. METHODS: HPyV7-DNA Fluorescence in situ hybridization (FISH), DNA-polymerase chain reaction (PCR), and immunohistochemistry (IHC) were performed in 37 thymomas. Of these, 26 were previously diagnosed with myasthenia gravis (MG). In addition, 20 thymic hyperplasias and 20 fetal thymic tissues were tested. RESULTS: HPyV7-FISH revealed specific nuclear hybridization signals within the neoplastic epithelial cells of 23 thymomas (62.2%). With some exceptions, the HPyV7-FISH data correlated with the HPyV7-DNA PCR. By IHC, large T antigen expression of HPyV7 was detected, and double staining confirmed its expression in the neoplastic epithelial cells. Eighteen of the 26 MG-positive and 7 of the 11 MG-negative thymomas were HPyV7-positive. Of the 20 hyperplastic thymi, 40% were HPyV7-positive by PCR as confirmed by FISH and IHC in the follicular lymphocytes. All 20 fetal thymi tested HPyV7-negative. CONCLUSIONS: The presence of HPyV7-DNA and large T antigen expression in the majority of thymomas possibly link HPyV7 to human thymomagenesis. Further investigations are needed to elucidate the possible associations of HPyV7 and MG.