Clinical value of cell-based assays in the characterisation of seronegative myasthenia gravis

Abstract

ObjectivePatients with myasthenia gravis without acetylcholine receptor (AChR) or muscle-specific kinase (MuSK) antibodies detected by radioimmunoprecipitation assays (RIAs) are classified as seronegative myasthenia gravis (SNMG). Live cell-based assays (l-CBAs) can detect additional antibodies to clustered AChR, MuSK and low-density lipoprotein receptor-related protein 4 (LRP4), but positivity rates are variable and both clinical relevance and utility of CBA platforms remain unclear.MethodsSera from 82 patients with SNMG were tested by l-CBAs. Human embryonic kidney cells were transfected to individually express clustered AChR, MuSK or LRP4; or transfected to jointly express both clustered adult AChR and MuSK. Sera from 30 and 20 patients positive by RIA for AChR or MuSK antibodies were used as comparators.Results53 of 82 (72%) patients with SNMG had generalised and 29 (28%) had ocular disease. The clustered AChR CBA detected antibodies in 16 of 82 patients (19.5%; including 4 patients with solely fetal AChR antibodies), while 7 of 82 (8.5%) patients had MuSK antibodies. A novel exploratory combined adult AChR-MuSK l-CBA efficiently detected all these antibodies in a subset of the SNMG cohort. No LRP4 antibodies were identified. Overall, patients with SNMG with clustered AChR antibodies, CBA-positive MuSK-MG or triple seronegative were younger, had less severe disease than patients with RIA-positive MG and had a better clinical outcome when immunotherapy was started soon after disease onset, although the time interval from onset to immunotherapy was not different when compared with patients with RIA-positive MG.ConclusionAround one-third of patients with SNMG had AChR or MuSK antibodies by l-CBAs, which were efficiently detected with a combined l-CBA. The results in this large and unselected cohort of patients with MG demonstrate the diagnostic usefulness of performing CBAs and the importance of making these tests more widely available.

Comparison of Fixed and Live Cell-Based Assay for the Detection of AChR and MuSK Antibodies in Myasthenia Gravis

“…It is important that all RIA-dSN but CBA-positive patients had typical MG clinical features and treatment response, with an overall milder disease severity and higher frequency of purely ocular forms, as already described in patients with clustered AChR MG or with MuSK Abs solely detected by CBA. 5 , 8 Notably, 2 patients, both with purely ocular MG, had Abs targeting only the fetal isoform of AChR, which highlights the importance of testing for these Abs to maximize the CBA sensitivity.…”

“…Afterward, all sera were tested for Abs to the adult/fetal AChR and MuSK with an in-house live CBA and with the commercial fixed CBA (Euroimmun, Lubeck, Germany). 8 Samples were tested in the laboratories of the 2 institutions (n = 206 UCSC, Rome, Italy; n = 86 IRCCS Mondino Foundation, Pavia, Italy), using the same protocols briefly described as follows. Fixed CBA was performed according to the manufacturer's instructions (serum dilution 1:10).…”

“… 4 , 7 , 8 Indeed, the high-density expression of the antigen in its native conformation and the advantage of cotransfecting the antigen with rapsyn to maintain AChR clustering on the cell surface facilitate the binding of low-affinity AChR Abs. 9 This advance in the Ab testing allowed a significant improvement in the serologic diagnosis of MG, as recently confirmed by a study from our group finding clustered AChR or MuSK Abs in around one-third of patients with RIA-dSN MG. 8 However, the use of live CBA is currently limited to specialized research settings because it requires expertise and cell-culture facilities. A commercial fixed CBA for adult and fetal AChR and MuSK Ab detection has recently become available, widening the possibility to improve MG serologic diagnosis and providing a reliable test without the need for radioactive ligands.…”

“…A commercial fixed CBA for adult and fetal AChR and MuSK Ab detection has recently become available, widening the possibility to improve MG serologic diagnosis and providing a reliable test without the need for radioactive ligands. 8 Recently, the performance of fixed CBA for AChR Ab detection was compared with RIA (cutoff for positivity 0.5 nmol/L), showing 4% increased sensitivity with 99% specificity. 10 To date, there are no studies comparing fixed and live CBAs, which could clarify their respective role in the MG diagnostic algorithm.…”

Immunological response after SARS-CoV-2 infection and mRNA vaccines in patients with myasthenia gravis treated with Rituximab

“…Several reports showed that multiple sclerosis (MS) patients treated with anti-CD20 therapies were able to mount spike-specific T-cell responses comparable to patients not under B cell-depleting therapies. [ 13 , 14 ] To date, a single study reported the humoral and cell-mediated response (by an interferon gamma (IFN-γ) release assay in plasma) to mRNA vaccines in MG patients under different immunotherapies, including rituximab (RTX), [15] an anti-CD20 monoclonal antibody used as second-line treatment in MG. Here we employed a comprehensive immune profiling approach to determine the innate and adaptive immune response, including humoral and cellular response, to SARS-CoV-2 infection and mRNA vaccines in MG patients receiving RTX.…”