CDR2 and CDR2L line blot performance in PCA-1/anti-Yo paraneoplastic autoimmunity

BackgroundPurkinje cytoplasmic autoantibody type 1 (PCA-1)/anti-Yo autoimmunity is a common high-risk paraneoplastic neurological disorder, traditionally attributed antigenically to cerebellar degeneration–related protein 2 (CDR2), predominantly affecting women with gynecologic or breast adenocarcinoma. Single-modality CDR2 testing may produce false-positive results. We assessed the performance characteristics of the more recently purported major PCA-1/Yo antigen, CDR2-like (CDR2L), side by side with CDR2, in a line blot format.MethodsCDR2 and CDR2L were tested in six specimen groups (serum and cerebrospinal fluid (CSF)). Group 1, PCA-1/Yo mouse brain indirect immunofluorescence assay (IFA) positives; Group 2, PCA-1/Yo IFA mimics; Group 3, suspected CDR2 line blot false positives; Group 4, consecutive patient samples tested for neural antibodies over 1 year; Group 5, healthy subject serums; and Group 6, polyclonal (non-specific) immunoglobulin G (IgG)-positive serums.ResultsGroup 1: Of 64 samples tested, all but two were CDR2 positive (both CSF samples) and all were CDR2L positive. In individual patients, CDR2L values were always higher than CDR2. The two “CDR2L-only” positives were CSF samples with low titer PCA-1/Yo by IFA with serum negativity but with typical clinical phenotype. Group 2: All 51 PCA-1/Yo mimics were CDR2/CDR2L negative. Group 3: Nine samples [six of 1289 (0.47%) serums and three of 700 CSF samples (0.43%) were PCA-1/Yo IFA negative/CDR2 positive; two of the six available (serums from the same patient) were also CDR2L positive; the other four CDR2L negative had low CDR2 values (17–22). Group 4: Twenty-two patients had unexpected CDR2 or CDR2L positivity; none had tissue IFA positivity. Eleven of the 2,132 serum (0.5%) and three of the 677 CSF (0.4%) samples were CDR2 positive; median value was 19 (range, 11–48). Seven of the 2,132 serum (0.3%) and three of the 677 CSF (0.4%) samples were CDR2L positive; median value was 18 (range, 11–96). Group 5: All 151 healthy serum samples were negative. Group 6: One of the 46 polyclonal serum samples was CDR2L positive. Optimum overall performance was accomplished by requiring both CDR2 and CDR2L positivity in serum (sensitivity, 100%; and specificity, 99.9%) and positivity for CDR2L in CSF (sensitivity, 100%; and specificity, 99.6%).ConclusionCDR2L provides additional PCA-1/anti-Yo sensitivity in CSF, and dual positivity with CDR2 provides additional specificity assurance in serum. Combining antigen-specific and tissue-based assays optimizes PCA-1/anti-Yo testing

Neuronal intermediate filament IgGs in CSF: Autoimmune Axonopathy Biomarkers

Objectives: To describe CSF-defined neuronal intermediate filament (NIF) autoimmunity. Methods: NIF-IgG CSF-positive patients (41, 0.03% of 118599 tested, 1996-2019) were included (serum was neither sensitive nor specific). Criteria-based patient NIF-IgG staining of brain and myenteric NIFs was detected by indirect immunofluorescence assay (IFA); NIF-specificity was confirmed by cell-based assays (CBAs, alpha internexin, neurofilament light [NF-L]), heavy-[NF-H] chain). Results: Sixty-one percent of 41 patients were men, median age, 61 years (range, 21-88). Syndromes were encephalopathy predominant (23), cerebellar ataxia predominant (11), or myeloradiculoneuropathies (7). MRI abnormalities (T2 hyperintensities of brain, spinal cord white matter tracts. and peripheral nerve axons) and neurophysiologic testing (EEG, EMG, evoked potentials) co-localized with clinical neurological phenotypes (multifocal in 29%). Thirty patients (73%) had 65 1 immunological perturbation: cancer (paraneoplastic), 22; systemic infection (parainfectious [including ehrlichosis, 3] or HIV), 7; checkpoint-inhibitor cancer immunotherapy, 4; other, 5. Cancers were as follows: neuroendocrine-lineage carcinomas, 12 (small cell, 6; Merkel cell, 5; pancreatic, 1 [11/12 had NF-L-IgG detected, versus 8/29 others, P = 0.0005]) and other, 11. Onset was predominantly subacute (92%) and accompanied by inflammatory CSF (75%), and immunotherapy response (77%). In contrast, CSF controls (15684 total) demonstrated NIF-IgG negativity (100% of test validation controls), and low frequencies of autoimmune diagnoses (20% of consecutively referred clinical specimens) and neuroendocrine-lineage carcinoma diagnosis (3.1% vs. 30% of NIF cases), P < 0.0001. Median NF-L protein concentration was higher in 8 NF-L-IgG-positive patients (median, 6718 ng/L) than 16 controls. Interpretation: Neurological autoimmunity, defined by CSF-detected NIF-IgGs, represents a continuum of treatable axonopathies, sometimes paraneoplastic or parainfectious

Ανάλυση αυτοαντισωμάτων έναντι του νευρικού συστήματος σε ορούς και αντιγόνων σε όγκους σε ασθενείς με νευρολογική παρανεοπλασματική νόσο

The use of immunotherapies to treat malignancies is currently in the forefront of cancer management. Immune checkpoint inhibitors are monoclonal antibodies that are proving highly successful in therapeutically targeting and blocking “stop signs” that limit the progression of a patient’s immune responses against cancer. Their use has recently been extended to treating malignancies that have high association with spontaneous neurological paraneoplastic autoimmunity, namely SCLC and thymoma. Knowledge of the spontaneous neurological and serological accompaniments of paraneoplastic autoimmunity as well as understanding the initial trigger of the autoimmune response is crucial in the era of immune-oncology. For this reason, my doctoral thesis will first present the results of my investigation of potential antigenic triggers of paraneoplastic autoimmunity in a patient who had paraneoplastic myasthenia gravis by analyzing onconeural antigen expression in the patient’s tumor. Secondly, I will present the results of two studies in which I documented the clinical manifestations of neurological paraneoplastic syndromes associated with thymoma and SCLC, as well as the neural autoantibodies detected in the sera of patients with these cancers, regardless of neurological manifestations. The serological studies utilized the database of the Neuroimmunology Laboratory at Mayo Clinic, Rochester. The studies were approved by the Mayo Clinic Institutional Review Board. Each experimental chapter has a brief abstract summarizing the results of the respective study.Η αλληλεπίδραση του ανοσοποιητικού συστήματος με τον καρκίνο είναι αντικείμενο μελέτης στην ογκολογία εδώ και πολλές δεκαετίες. Τα τελευταία χρόνια, ανοσοθεραπείες για τη θεραπεία του καρκίνου που δρουν αναστέλλοντας την λειτουργία αρνητικών ρυθμιστικών παραγόντων στην ενεργοποίηση του ανοσοποιητικού συστήματος και οδηγούν στην ενίσχυση της λειτουργίας των Τ-λεμφοκυττάρων, έχουν αποδείξει τον ευεργετικό ρόλο ενός ενεργοποιημένου ανοσοποιητικού συστήματος και την προστατευτική δράση μιας ανοσολογικής αντίδρασης για την αντιμετώπιση του καρκίνου. Τα νευρολογικά παρανεοπλασματικά νοσήματα είναι ένδειξη μιας αυθόρμητης ανοσολογικής αντίδρασης κατά αντιγόνων που υπό κανονικές συνθήκες εκφράζονται μόνο στο νευρικό σύστημα ενώ στους ασθενείς με παρανεοπλασματικά νοσήματα εκφράζονται και από τα καρκινικά κύτταρα. Οι όγκοι που συχνότερα συνοδεύονται από νευρολογική παρανεοπλασματική ανοσία είναι ο μικροκυτταρικός καρκίνος του πνεύμονα (ΜΚΠ) και το θύμωμα. Τα αυτοαντισώματα έναντι πρωτεϊνών του νευρικού συστήματος βρίσκονται συχνά σε ασθενείς με νευρολογική παρανεοπλασματική νόσο και βοηθούν στην διάγνωση της αυτοανοσίας και του καρκίνου. Έγκαιρη αναγνώριση των νευρολογικών παρανεοπλασματικών νοσημάτων, γνώση των νευρολογικών αυτοαντισωμάτων στον ορό αυτών των ασθενών και των συνοδών συμπτωμάτων, και εν τω βάθει κατανόηση της παθοφυσιολογίας είναι ζωτικής σημασίας σε αυτήν την τρέχουσα περίοδο της Ογκοανοσολογίας. Για τους παραπάνω λόγους, σε αυτή τη διδακτορική διατριβή μελέτησα την έκφραση αντιγόνων του νευρικού συστήματος και συγκεκριμένα του μυϊκού υποδοχέα ακετυλοχολίνης στον όγκο μίας ασθενούς με μυασθένια και ΜΚΠ σε επίπεδο πρωτεΐνης, mRNA έκφρασης και πεπτιδίων που παρουσιάζονται από τα μόρια του μείζονος συμπλέγματος ιστοσυμβατότητας τύπου 1 στην επιφάνεια των καρκινικών κυττάρων και μπορούν να αναγνωριστούν από τα Τ-λεμφοκύτταρα. Ο όγκος της ασθενούς εξέφραζε μυϊκό υποδοχέα ακετυλοχολίνης όπως μελετήσαμε με ραδιοανοσολογική μέθοδο, πολλαπλά mRNA του μυϊκού υποδοχέα ακετυλοχολίνης (κάποια που μπορούν να μεταφραστούν σε μεταλλαγμένες μορφές πρωτεΐνης) και πεπτίδια της ίδιας πρωτεΐνης στο μείζον συμπλέγμα ιστοσυμβατότητας τύπου 1 του όγκου της. Επίσης, συνέκρινα τα αποτελέσματα με τα πεπτίδια στα μόρια του μείζονος συμπλέγματος ιστοσυμβατότητας τύπου 1 στην επιφάνεια των καρκινικών κυττάρων από μία ασθενή με ΜΚΠ και αντισώματα αντι-Hu και αισθητική νευρονοπάθεια στον όγκο της οποίας δεν βρέθηκαν πεπτίδια από τον μυϊκό υποδοχέα ακετυλοχολίνης αλλά ανιχνεύσαμε από την πρωτεΐνη HuC. Στο δεύτερο μέρος αναφέρομαι στα αποτελέσματα δύο μελετών που περιγράφουν τις κλινικές εκδηλώσεις των νευρολογικών παρανεοπλασματικών συνδρόμων σε ασθενείς με θύμωμα και ΜΚΠ καθώς και των αυτοαντισωμάτων που ανιχνεύονται στους ορούς αυτών των ασθενών. Αυτές οι μελέτες έγιναν με τη χρήση της βάσης δεδομένων του εργαστηρίου Νευροανοσολογίας της Mayo Clinic, στο Ρότσεστερ της Μινεσότας, όπου έγινε και το πειραματικό μέρος της διατριβής και όλα τα αντισώματα μελετήθηκαν με τις τεχνικές που χρησιμοποιούνται για κλινική διάγνωση (ραδιοανοσολογική μέθοδος, ανοσοφθορισμός χρησιμοποιώντας ιστούς ή κύτταρα, Western blot και ELISA). Κάθε πειραματικό κεφάλαιο έχει μια συνοπτική περίληψη των αποτελεσμάτων της μελέτης

Paraneoplastic autoimmunity and small‐cell lung cancer: Neurological and serological accompaniments

Paraneoplastic neurological autoimmunity is often associated with small‐cell lung cancer (SCLC), a highly malignant neuroendocrine tumor. Paraneoplastic autoimmunity often correlates with longer survival. We describe the paraneoplastic neurological manifestations of patients with SCLC with and without SCLC‐predictive autoantibodies and the correlation between autoimmunity and survival. We reviewed the records of 116 patients (51% male) from the Mayo Clinic with histopathologically confirmed SCLC for whom stored serum was available for neural autoantibody testing. Cancer was limited stage in 41%; the median age at diagnosis was 64 years. Paraneoplastic neurological manifestations were recorded in 61% (decreasing frequency: peripheral neuropathy, dysautonomia, cognitive decline, cerebellar ataxia, neuromuscular junction disorder, seizures, cranial neuropathy, movement disorder, brainstem disorder, or myelopathy). Neural autoantibodies, some with pathogenic potential, were detected in the sera of SCLC patients with and without neurological autoimmunity. The most frequent among patients with neurological manifestations were: anti‐neuronal nuclear antibody‐type 1, voltage‐gated calcium channel (VGCC)‐N‐type, VGCC‐P/Q‐type, glutamic acid decarboxylase 65 (GAD65), SOX1, and muscle acetylcholine receptor (AChR); while the most common in patients without neurological manifestations were: GAD65, muscle‐AChR, and VGCC‐P/Q‐type. Neither cancer stage at diagnosis nor survival correlated with neurological manifestations or autoantibody‐positivity, except for shorter survival in patients with myelopathy. The only predictor of longer survival was limited‐stage disease at diagnosis

GAD65 autoimmunity after treatment with nivolumab: a multifocal presentation

Introduction: Neurological disorders are considered rare complications of immune-checkpoint inhibitor. Case description: We report a 63-year-old man with recurrence of melanoma who presented epilepsy, limbic encephalitis, cerebellar ataxia, and stiff person syndrome soon after treatment with nivolumab, an immune-checkpoint inhibitor. On autoimmune screening, serum and CSF GAD65 were detected. Significant response to steroids and intravenous immunoglobulins were observed, but cancer recurred after nivolumab discontinuation in parallel with epileptic seizure and worsening of cognitive impairment and the patient died. Discussion: This case expands the spectrum of GAD65-associated conditions induced by immune-checkpoint inhibitor and underlines treatment complexity when both neurological complications and tumour recurrence occur

HLA-B7-Restricted EBV-Specific CD8+ T Cells Are Dysregulated in Multiple Sclerosis.

It was hypothesized that the EBV-specific CD8(+) T cell response may be dysregulated in multiple sclerosis (MS) patients, possibly leading to a suboptimal control of this virus. To examine the CD8(+) T cell response in greater detail, we analyzed the HLA-A2-, HLA-B7-, and HLA-B8-restricted EBV- and CMV-specific CD8(+) T cell responses in a high number of MS patients and control subjects using tetramers. Content in cytolytic granules, as well as cytotoxic activity, of EBV- and CMV-specific CD8(+) T cells was assessed. We found that MS patients had a lower or a higher prevalence of HLA-A2 and HLA-B7, respectively. Using HLA class I tetramers in HLA-B7(+) MS patients, there was a higher prevalence of MS patients with HLA-B*0702/EBV(RPP)-specific CD8(+) T cells ex vivo. However, the magnitude of the HLA-B*0702/EBV(RPP)-specific and HLA-B*0702/CMV(TPR)-specific CD8(+) T cell response (i.e., the percentage of tetramer(+) CD8(+) T cells in a study subject harboring CD8(+) T cells specific for the given epitope) was lower in MS patients. No differences were found using other tetramers. After stimulation with the HLA-B*0702/EBV(RPP) peptide, the production of IL-2, perforin, and granzyme B and the cytotoxicity of HLA-B*0702/EBV(RPP)-specific CD8(+) T cells were decreased. Altogether, our findings suggest that the HLA-B*0702-restricted viral (in particular the EBV one)-specific CD8(+) T cell response is dysregulated in MS patients. This observation is particularly interesting knowing that the HLA-B7 allele is more frequently expressed in MS patients and considering that EBV is associated with MS