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Alternative techniques used for confirmation of commercial immunodots
Source publication
Objective
To investigate the diagnostic yield of commercial immunodots to detect onconeural antibodies associated with paraneoplastic neurologic syndromes (PNSs), we analyzed the proportion of confirmed positive results using alternative techniques.
Methods
Sera (n = 5,300) of patients with suspected PNS were tested by PNS+2 blot (Ravo Diagnostika...
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SOX1 antibodies (SOX1-abs) are associated with paraneoplastic neurological syndromes (PNS) and small cell lung cancer (SCLC). In many clinical laboratories SOX1-abs are determined by commercial line blots without confirmation by cell-based assay (CBA) with HEK293 cells expressing SOX1. However, the diagnostic yield of commercial line blots is low a...
Detection of onconeural antibodies is important because establishes a definitive diagnosis of paraneoplastic neurological syndrome (PNS). The recommended method for diagnosis of onconeural antibodies is by immunohistochemistry on rodent brain sections and confirmation of results by immunoblot. However, in many diagnostic laboratories samples are on...
Citations
... This work also has some strengths, most importantly, all included cases were diagnosed or confirmed in a European Reference Network site for PNS, which uses in-house, highly reliable, diagnostic techniques. 38 This European bicentric study is to our knowledge the largest Northern country cohort of GABA B R-AE. ...
Background and objectives:
While patients with paraneoplastic autoimmune encephalitis (AE) with gamma-aminobutyric-acid B receptor antibodies (GABABR-AE) have poor functional outcomes and high mortality, the prognosis of nonparaneoplastic cases has not been well studied.
Methods:
Patients with GABABR-AE from the French and the Dutch Paraneoplastic Neurologic Syndromes Reference Centers databases were retrospectively included and their data collected; the neurologic outcomes of paraneoplastic and nonparaneoplastic cases were compared. Immunoglobulin G (IgG) isotyping and human leukocyte antigen (HLA) genotyping were performed in patients with available samples.
Results:
A total of 111 patients (44/111 [40%] women) were enrolled, including 84 of 111 (76%) paraneoplastic and 18 of 111 (16%) nonparaneoplastic cases (cancer status was undetermined for 9 patients). Patients presented with seizures (88/111 [79%]), cognitive impairment (54/111 [49%]), and/or behavioral disorders (34/111 [31%]), and 54 of 111 (50%) were admitted in intensive care unit (ICU). Nonparaneoplastic patients were significantly younger (median age 54 years [range 19-88] vs 67 years [range 50-85] for paraneoplastic cases, p < 0.001) and showed a different demographic distribution. Nonparaneoplastic patients more often had CSF pleocytosis (17/17 [100%] vs 58/78 [74%], p = 0.02), were almost never associated with KTCD16-abs (1/16 [6%] vs 61/70 [87%], p < 0.001), and were more frequently treated with second-line immunotherapy (11/18 [61%] vs 18/82 [22%], p = 0.003). However, no difference of IgG subclass or HLA association was observed, although sample size was small (10 and 26 patients, respectively). After treatment, neurologic outcome was favorable (mRS ≤2) for 13 of 16 (81%) nonparaneoplastic and 37 of 84 (48%) paraneoplastic cases (p = 0.03), while 3 of 18 (17%) and 42 of 83 (51%) patients had died at last follow-up (p = 0.008), respectively. Neurologic outcome no longer differed after adjustment for confounding factors but seemed to be negatively associated with increased age and ICU admission. A better survival was associated with nonparaneoplastic cases, a younger age, and the use of immunosuppressive drugs.
Discussion:
Nonparaneoplastic GABABR-AE involved younger patients without associated KCTD16-abs and carried better neurologic and vital prognoses than paraneoplastic GABABR-AE, which might be due to a more intensive treatment strategy. A better understanding of immunologic mechanisms underlying both forms is needed.
... However, as many antibodies lack specificity, it is essential to correlate the clinical presentation with established antibody-associated syndromes before making a diagnosis [8]. Furthermore, given the prevalence of false positives, the results should be validated using confirmatory tests [22,27]. Antinuclear antibody (ANA) testing is non-specific but sometimes advised, as it can suggest an autoimmune tendency [8,[14][15][16]. ...
The increasing use of immune checkpoint inhibitors (ICI) in cancer therapy has brought attention to their associated neurotoxicities, termed neurological immune-related adverse events (n-irAEs). Despite their relatively rare incidence, n-irAEs pose a significant risk, potentially leading to severe, long-lasting disabilities or even fatal outcomes. This narrative review aims to provide a comprehensive overview of n-irAEs, focusing on their recognition and management. The review addresses a spectrum of n-irAEs, encompassing myositis, myasthenia gravis, various neuropathies, and central nervous system complications, such as encephalitis, meningitis, and demyelinating diseases. The key features of n-irAEs are emphasized in this review, including their early onset after initiation of ICIs, potential association with non-neurological irAEs and/or concurrent oncological response, the significance of ruling out other etiologies, and the expected improvement upon discontinuation of ICIs and/or immunosuppression. Furthermore, this review delves into considerations for ICI re-challenge and the intricate nature of n-irAEs within the context of pre-existing autoimmune and paraneoplastic syndromes. It underscores the importance of a multidisciplinary approach to diagnosis and treatment, highlighting the pivotal role of severity grading in guiding treatment decisions.
... positivity, particularly of those targeting intracellular antigens [i.e., high-risk paraneoplastic neurological syndromes (PNS) antibodies [16]], should always be interpreted in the clinical context, as patients with certain cancers [e.g., small cell lung cancer (SCLC)] can asymptomatically harbour these antibodies (e.g. anti-Hu antibodies), and the commercially available tests have a high rate of false positive results [17,18]. To reduce this risk, two independent detection methods should be used, and dubious cases should be evaluated at reference centres [16]. ...
Purpose of review
Immune checkpoint inhibitors (ICI) may trigger immune-related adverse events which rarely affect the central nervous system (CNS-irAEs). Over the past few years, cumulative data have led to the characterization of well defined syndromes with distinct cancer and antibody associations as well as different outcomes.
Recent findings
The most frequent CNS-irAE is encephalitis, which includes three main groups: meningoencephalitis, a nonfocal syndrome usually responsive to corticosteroids; limbic encephalitis, associated with high-risk paraneoplastic neurological syndromes (PNS) antibodies (e.g. anti-Hu, anti-Ma2) and neuroendocrine cancers, characterized by poor treatment response and outcomes; and cerebellar ataxia, with variable outcomes (worse when high-risk PNS antibodies are detected). Additionally, a diffuse encephalopathy without inflammatory findings, with poor response to corticosteroids and high mortality has been described. The spectrum of CNS-irAEs also includes meningitis, myelitis, and rarer presentations. A subset of CNS-irAEs (i.e. limbic encephalitis and/or rapidly progressive cerebellar ataxia) is undistinguishable from ICI-naïve PNS.
Summary
The clinical and outcomes diversity of CNS-irAEs suggests different pathogenic mechanisms, which need to be understood to establish more effective and specific treatment modalities. It is crucial to identify biomarkers able to predict which patients will experience severe CNS-irAEs, to anticipate their diagnosis, and to predict long-term outcomes.
... On one hand, commercial assays are a practical and time-effective approach allowing simultaneous detection of numerous antibodies using a single assay. On the other hand, regarding the detection of intracellular antibodies, a high proportion of false positive antibody results not confirmable with alternative diagnostic assays and alternative explanations of neurological symptoms have been reported [5][6][7] . Caveats of commercial assays may be ameliorated with additional diagnostic modalities available in specialized research laboratories. ...
Detection of neuronal antibodies for autoimmune encephalitis and paraneoplastic neurological syndromes relies on commercially available cell-based assays and lineblots. However, lineblots may reveal the presence of neuronal antibodies in patients with various non-autoimmune etiologies. Herein we describe patients with non-autoimmune etiologies (cohort B) and detectable neuronal antibodies and compare them to definite cases of autoimmune encephalitis (cohort A) for differences in clinical data. All patients positive for at least one neuronal antibody were retrospectively evaluated for autoimmune encephalitis and/or paraneoplastic neurological syndrome between 2016 and 2022. 39 cases in cohort B and 23 in cohort A were identified. In cohort B, most common diagnoses were neurodegenerative disorders in 9/39 (23.1%), brain tumors in 6/39 (15.4%) while most common detected antibodies were anti–titin (N10), anti-recoverin (N11), anti-Yo (N8) and all were detected in serum only. Differential aspects between cohort A and B were CSF pleocytosis (14/23 (60.8%) vs 11/35 (31.4%), p = 0.042, respectively), MRI features suggestive of encephalitis (6/23 (26.1%) vs 0 (0%), p = 0.002, respectively) and epilepsy restricted to temporal lobes (14/23 (60.9%) vs 2/30 (6.7%), p = 0.0003, respectively). A large proportion of lineblot results were non-specific when only serum was tested and were frequently found in non-autoimmune neurological conditions.
... It is also important to consider the testing methodology as some commercial line blot kits have been associated with false positives. 19 Our patient was tested by an in-house Western blot for CRMP5-IgG twice at the Mayo Clinic Neuroimmunology laboratory (a reference laboratory). As PND may precede tumor recognition by months to years and studies have reported patients often have only early stage or microscopic tumor identified at the time of diagnosis, 20 we considered that is was possible that our patient had undiagnosed cancer (suspected lung). ...
We present a case of subacute onset progressive encephalomyelopathy in a 77-year-old man with symmetric lateral column signal abnormalities on spinal MRI. We discuss the differential and presumptive final diagnosis along with a review of the postulated disease immunopathogenesis.
... As such, the results reported are not confirmed with 2 testing methodologies, which may result in a high proportion of false-positive results. [60][61][62]65 Similarly, more limited testing can miss antibodies that are not included in, or are sub-optimally detected by, standard commercially available panels; such antibodies may only be detected if TIIF/IHC along with confirmatory assays available at specialized centers are used. 81,82 As such, it is highly recommended to involve a specialist in autoimmune neurology when a positive neural antibody result is found in a patient with an atypical clinical presentation or when no antibody is detected in a patient in whom AIE is strongly suspected, so they can review results and direct further testing at specialized laboratories. ...
... 86,87 With respect to particular MRI findings, T2/ FLAIR hyperintensities restricted to the medial temporal lobes are classical of autoimmune limbic encephalitis and are central to its diagnostic criteria 28 . Cortical-subcortical T2/FLAIRhyperintense lesions are characteristic of GABA(A)R antibody encephalitis, and have also been reported with several other neural antibodies including those targeting Hu. [88][89][90][91][92][93] Unilateral cortical T2/FLAIR-hyperintense lesions with hypointensity of the adjacent subcortical white matter have been described in patients with encephalitic presentations of MOG antibody-associated Requires expertise in interpretation of neural antibody tissue staining patterns Can be used to screen for rare/novel neural antibodies against intracellular/ extracellular antigens to maximize sensitivity (48)(49)(50)(51)(52)(53)(54)(55)(56)(57)(58) Can be used to corroborate positive immunoblot or CBA results to maximize specificity (59)(60)(61)(62)(63)(64) Cell-based assays (CBA) Anti-NMDAR, LGI1, CASPR2, GABA(B)R, AMPAR, DPPX, GAD65, IGLON5, MOG, GLYR CBA reported to have higher sensitivity than TIIF/IHC for certain neural antibodies (e.g., LGI1, CASPR2); however, higher sensitivity may come at cost to specificity (46,21) Specificity of isolated positivity by CBA varies across analytes and is lower in the absence of corresponding positivity by second assay (e.g., TIIF/IHC); for weak/low isolated serum positivity by CBA, discuss further evaluation with testing laboratory (e.g., testing at higher dilution for anti-CASPR2) (63,(65)(66)(67) Note that CBAs for anti-MOG and anti-GlyR are not routinely incorporated in neural antibody panels for autoimmune encephalitis, but should be ordered in patients with compatible disease phenotypes (e.g., ADEM and unilateral cerebral cortical encephalitis/FLAMES for anti-MOG, PERM for anti-GlyR); restricting testing of these antibodies to patients with compatible disease phenotypes reduces proportion of false-positives, which usually occur as low levels of positivity in serum ( disease. 73,74,94,95 T2/FLAIR hyperintensities restricted to the claustrum have been reported to be useful markers of seizures related to autoimmune encephalitis. ...
Autoimmune encephalitis is increasingly recognized as a neurologic cause of acute mental status changes with similar prevalence to infectious encephalitis. Despite rising awareness, approaches to diagnosis remain inconsistent and evidence for optimal treatment is limited. The following Canadian guidelines represent a consensus and evidence (where available) based approach to both the diagnosis and treatment of adult patients with autoimmune encephalitis. The guidelines were developed using a modified RAND process and included input from specialists in autoimmune neurology, neuropsychiatry and infectious diseases. These guidelines are targeted at front line clinicians and were created to provide a pragmatic and practical approach to managing such patients in the acute setting.
... Cases with (1) findings in serum CBAs without confirmation in CSF and/or TBAs, (2) findings in CSF TBAs without confirmation in CBAs or live hippocampal neuron indirect immunofluorescence assay, or (3) borderline or low line-blot positivity in serum without tissue-based confirmation and considered unrelated to the clinical picture (e.g., borderline positivity of anti-Yo only on line blot) were labeled antibody-negative. 32 ...
Objective
Refractory epilepsy may have an underlying autoimmune etiology. Our aim was to assess the prevalence of neural autoantibodies in a multicenter national prospective cohort of patients with drug‐resistant epilepsy undergoing epilepsy surgery utilizing comprehensive clinical, serologic, and histopathological analyses.
Methods
We prospectively recruited patients undergoing epilepsy surgery for refractory focal epilepsy not caused by a brain tumor from epilepsy surgery centers in the Czech Republic. Perioperatively, we collected cerebrospinal fluid (CSF) and/or serum samples and performed comprehensive commercial and in‐house assays for neural autoantibodies. Clinical data were obtained from the patients' medical records, and histopathological analysis of resected brain tissue was performed.
Results
Seventy‐six patients were included, mostly magnetic resonance imaging (MRI)‐lesional cases (74%). Mean time from diagnosis to surgery was 21 ± 13 years. Only one patient (1.3%) had antibodies in the CSF and serum (antibodies against glutamic acid decarboxylase 65) in relevant titers; histology revealed focal cortical dysplasia (FCD) III (FCD associated with hippocampal sclerosis [HS]). Five patients' samples displayed CSF‐restricted oligoclonal bands (OCBs; 6.6%): three cases with FCD (one with FCD II and two with FCD I), one with HS, and one with negative histology. Importantly, eight patients (one of them with CSF‐restricted OCBs) had findings on antibody testing in individual serum and/or CSF tests that could not be confirmed by complementary tests and were thus classified as nonspecific, yet could have been considered specific without confirmatory testing. Of these, two had FCD, two gliosis, and four HS. No inflammatory changes or lymphocyte cuffing was observed histopathologically in any of the 76 patients.
Significance
Neural autoantibodies are a rare finding in perioperatively collected serum and CSF of our cohort of mostly MRI‐lesional epilepsy surgery patients. Confirmatory testing is essential to avoid overinterpretation of autoantibody‐positive findings.
... The identification of paraneoplastic neurological syndromes requires high clinical suspicion and should prompt neural antibody testing. 19,45 However, the available commercial tests carry a high risk of false positive results, 88,89 and paraneoplastic neurological syndromes antibodies might be detected in patients with some malignant diseases (eg, small cell lung cancer), regardless of the presence of neurological symptoms. 7,44,77,78 Therefore, antibody results must always be interpreted in the context of the clinical presentation, and complex cases should be assessed at reference centres with at least two different techniques, as recommended for the diagnosis of spontaneous paraneoplastic neurological syndromes. ...
... 25,70 However, in the absence of neurological symptoms, positivity of neural antibodies must be interpreted with caution. 88,89 There is no evidence supporting the exclusion of asymptomatic neural antibody-positive patients from receiving checkpoint inhibitor treatment, and prospective studies are needed to assess the role of antibody positivity before treatment. In patients who are antibody-positive, antibody titre variation might be a predictive biomarker of immune checkpoint inhibitor-induced paraneoplastic neurological syndromes, especially in patients with cancer types usually associated with paraneoplastic syndromes. ...
Immune related adverse events of immune checkpoint inhibitors and paraneoplastic neurologic syndromes
... Furthermore, commercial immunoblotting was used for detecting 12 antineuronal antibodies (AMPH, CV2/CRMP5, Ma2/PNMA2, Ri/ANNA-2, Yo/PCA-1, Hu/ANNA-1, Recoverin, SOX1, Titin, Zic4, GAD65, and Tr/DNER) (EUROLINE PNS 12 Ag [Euroimmun, Lubeck, Germany]). 21 Serum samples were analyzed according to the manufacturer's instructions. Specifically, immunoblot assays were performed on the EUROBlotOne system (Euroimmun), and bands were scanned and analyzed using EUROLineScan (Euroimmun), giving an arbitrary unit of intensity. ...
Introduction
Although pretreatment autoantibodies have been associated with immune-related adverse events (irAEs) and immune checkpoint inhibitor treatment efficacy in some types of cancer, their importance has not been evaluated in patients with SCLC.
Methods
A multicenter prospective observational study was conducted on a total of 52 patients with extensive-disease SCLC who received immune checkpoint inhibitors in combination with chemotherapy as the first-line treatment at either of the six participating centers in Japan. Pretreatment serum samples were collected and analyzed for autoantibodies (rheumatoid factor, antinuclear antibodies, and antithyroid). Moreover, 12 antineuronal antibodies (AMPH, CV2, PNMA2, Ri, Yo, Hu, Recoverin, SOX1, Titin, Zic4, GAD65, and Tr) were analyzed using immunoblot assays. The primary end point was the incidence of irAEs with or without autoantibodies. The secondary end points were progression-free survival (PFS) and overall survival (OS) on the basis of the presence or absence of autoantibodies.
Results
PFS and OS were 4.4 and 25.3 months, respectively. Autoantibodies (rheumatoid factor, antinuclear antibodies, and antithyroid antibodies) were detected in 29 patients (56%). In total, irAEs were observed in 18 patients (35%); irAE incidence was 48% in the autoantibody-positive group and 17% in the autoantibody-negative group (p = 0.039). There was no difference in PFS or OS between patients with and without autoantibodies (4.4 mo versus 4.6 mo, p = 0.36; 15.3 mo versus 18.2 mo, p = 0.36). Antineuronal antibodies were detected in 16 patients (31%). However, the development of neurologic irAEs was not observed in both groups.
Conclusions
Vigilance is required against the development of irAEs in pretreatment antibody-positive patients.
... anti-Yo in a male with LE) were considered antibody-negative. 27 All samples were kept in aliquots frozen at −80°C until further analysis. Patients without samples (obtained during the active phase of disease) available for further testing were excluded from the study. ...
Background and Objectives
Autoimmune encephalitis (AE) refers to a heterogenous group of inflammatory CNS diseases. Subgroups with specified neural autoantibodies are more homogeneous in presentation, trigger factors, outcome, and response to therapy. However, a considerable fraction of patients has AE features but does not harbor detectable autoantibodies and is referred to as antibody-negative AE. Our aim was to describe clinical features, trigger factors, treatments, and outcome of a cohort of comprehensively tested antibody-negative AE patients.
Methods
This retrospective monocentric study recruited adult patients whose serum and/or CSF was sent to our tertiary center for neural antibody testing between 2011 and 2020, who entered the diagnostic algorithm as possible antibody-negative AE and had the following: (1) probable antibody-negative AE, definite antibody-negative acute disseminated encephalomyelitis (ADEM), or definite autoimmune limbic encephalitis (LE) according to diagnostic criteria; (2) available data on MRI of the brain, CSF, and EEG; and (3) stored serum and/or CSF samples. These samples were reanalyzed using a comprehensive combination of cell-based and tissue-based assays.
Results
Of 2,250 patients tested, 33 (1.5%) were classified as possible antibody-negative AE. Of these, 5 were found to have antibodies by comprehensive testing, 5 fulfilled the criteria of probable AE (3F:2M, median age 67, range 42–67), 4 of definite autoimmune LE (2F:2M, median age 45.5, range 27–60 years), one of definite antibody-negative ADEM, 2 of Hashimoto encephalopathy, one had no samples available for additional testing, and 15 had no further categorization. Of 10 probable/definite AE/LE/ADEM, one had a malignancy and none of them received an alternative diagnosis until the end of follow-up (median 18 months). In total, 80% (8/10) of patients received immunotherapy including corticosteroids, and 6/10 (60%) patients received rituximab, azathioprine, cyclophosphamide, plasma exchange, or IV immunoglobulins. Five (50%) patients improved, one (10%) stabilized, one (10%) worsened, and 3 (30%) died. All deaths were considered to be related to encephalitis. We did not observe differences of immunotherapy-treated patients in likelihood of improvement with or without nonsteroidal immunotherapy (with 2/6, without 1/2).
Discussion
Antibody-negative AE should be diagnosed only after comprehensive testing. Diagnostic effort is important because many patients benefit from immunotherapy and some have malignancies.
