Lesnick, Connie

Gephyrin is a postsynaptic scaffold protein essential for inhibitory neurotransmission. Gephyrin‐immunoglobulin G (IgG) was reported, decades ago, in a single case of paraneoplastic stiff‐person‐like syndrome, but its broader clinical relevance remains unknown. While investigating unclassified synaptic IgGs mimicking amphiphysin‐IgG, we identified gephyrin‐IgG in 3 patients using human protein microarray. Gephyrin specificity was confirmed using confocal tissue‐based immunofluorescence assay, cell‐based assay, and Western blot. The 3 patients, all female, had: encephalitis (2) and myeloneuropathy and breast cancer (1). All had inflammatory cerebrospinal fluid and abnormal magnetic resonance imaging, and improved following immunotherapy. Gephyrin‐IgG is associated with a treatable autoimmune neurological disease, sometimes paraneoplastic. ANN NEUROL 2026

ABSTRACT Objective We sought to characterize the sixth most common finding in our neuroimmunological laboratory practice (tissue assay‐observed unclassified neural antibodies [UNAs]), combining protein microarray and phage immunoprecipitation sequencing (PhIP‐Seq). Methods Patient specimens (258; 133 serums; 125 CSF) meeting UNA criteria were profiled; October 2022–September 2023. Top‐ranking candidate antigens were validated in silico, by dual‐staining confocal microscopy, and ≥ 1 protein‐specific assay. Clinical data were reviewed. Results Among 21 patients, 11 autoantibodies were characterized (serum, 19; CSF, all 9 available). Autoantigens were CACNA1I, 1; CAMK2B, 2; CLIP2, 1; FMN2, 2; MAP1A, 2; MAP2, 5; NECAB1, 1; SNAP91, 3; SRCIN1, 1; SYNJ1, 1; SYT3, 2. Analytical validation was by confocal TIIFA (all), western blot (10/10 available), and cell‐based assay (5/5 performed). Clinical accompaniments were: encephalitis, 6; brainstem encephalitis, 2; encephalomyelitis, 2; cerebellar ataxia, 2; longitudinally extensive transverse myelitis (LETM), 2; sensory neuronopathy, 1; peripheral neuropathy, 4, and movement disorders, 2. Inflammatory MRI abnormalities were identified in 5/16 patients (31%) with CNS disorders: T2 signal change (2), LETM (2), leptomeningeal enhancement (1). Seven of 8 (88%) had inflammatory CSF (pleocytosis, 5 [median 25.5 cells, range 7–294]; elevated IgG index/synthesis rate, 4; CSF‐exclusive oligoclonal bands, 4). Six had paraneoplastic causation (lung cancer, 2; other, 4); 3 were postinfectious (1 each of COVID‐19, HSV‐1, and post‐Group A streptococcal infection). Of 9 immunotherapy‐treated patients, 5 improved. Interpretation UNAs are partly accounted for by a repertoire of diverse mostly intracellular synaptic antigens. Their characterization is expedited by protein arrays and PhIPSeq. Further individual studies are needed to assess them as disease biomarkers.

Introduction: Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of malignant lymphocytes in the lymph nodes, spleen, blood, and bone marrow. Although current clinical treatment approaches targeting Bruton Tyrosine Kinase (BTK) and Bcl-2, either as monotherapy or in combination are highly effective with high overall response, complete responses, and achievement of undetectable measurable residual disease (MRD), most patients eventually experience disease relapse. In this study, we utilized our unique mesenchymal stromal cells (MSCs) system, which mimics the in vivo stromal environment in CLL, to explore the combination interaction effect of the first-in-class BTK inhibitor, ibrutinib, with novel tumor microenvironment (TME)-directed drugs in CLL. Methods: Since 1999, patients with CLL seen at Mayo Clinic in Rochester, MN have been offered participation in a longitudinal study of CLL B-cell Biology (CLL Tissue Bank). This Tissue Bank is seamlessly integrated into the Mayo Clinic CLL Database where the clinical characteristics of all patients are prospectively maintained. Using this resource, we identified previously untreated CLL patients (n=10) for our experiments. Individuals with high risk CLL (defined as those with both unmutated IGHV gene mutations and either del17p- or del11q- by FISH) (n=5) and with low risk CLL (defined as those with both mutated IGHV genes and no del17p- or del11q- by FISH) (n=5) were studied. Annexin-V/PI apoptotic cell death assays were employed to evaluate the effects of TME-directed drugs including everolimus (mTOR inhibitor), MI-2 (MALT1 inhibitor), ruxolitinib (JAK1/2 inhibitor), S63845 (MCL-1 inhibitor), and venetoclax (Bcl-2 inhibitor) either as single agent, or in combination with ibrutinib on CLL B cells in direct contact with MSCs. Dose response curves and IC50 levels were generated for single agent killing effect in CLL. The combination interaction effects were determined by Combination Index (CI) values generated by CalcuSyn software indicating synergistic (CI < 0.9), additive (CI between 0.9 - 1.1), or antagonistic (CI > 1.1) interactions. Among drug combinations with synergistic CI values, the following indicated different levels: below 0.1 very strong, 0.1 - 0.3 strong, 0.3 - 0.7 good, and 0.7 - 0.9 moderate synergism. Results: All tested drugs exhibited different IC50 levels in CLL cells co-cultured with MSCs: MI-2 IC50 of 3.5uM, ruxolitinib IC50 of 18.5uM, S63845 IC50 of 0.115uM, venetoclax IC50 of 0.006uM, and both everolimus and ibrutinib did not reach IC50 levels at maximum tested doses. Of interest we observed more killing in high versus low-risk cohort when treated with everolimus (19uM vs. undetectable), MI-2 (2.5 vs. 4uM) and S63845 (0.09 vs. 0.14uM) as single agents (IC50, high vs. low, respectively), while ruxolitinib and venetoclax showed no differences. Combination interaction effect results showed highly synergistic interactions for all patients (good to strong synergism) when ibrutinib was combined with S63845 or venetoclax, moderate to strong synergism for ibrutinib and everolimus, moderate synergism to additive levels for ibrutinib and MI-2, and moderate synergism to antagonism for ibrutinib and ruxolitinib. We also observed more synergism in the low-risk cohort when treated with ibrutinib and MI-2/ruxolitinib combinations, but no difference with ibrutinib and everolimus/S63845/venetoclax combinations between low and high-risk cohort. Conclusions: In summary, we evaluated the pre-clinical combination effects of ibrutinib when used together with other novel agents on CLL B cells in the presence of TME, where patients were assigned to low or high-risk cohorts. Our study showed that ibrutinib exhibits highly synergistic effects with venetoclax, S63845 and everolimus in both cohorts. The observed difference in effectiveness between high and low-risk cohorts when treated with ibrutinib plus MI-2 or ruxolitinib suggests these combinations would be beneficial for a subset of patients with high risk CLL. These preclinical data offer preliminary insights that support the evaluation of therapeutic combinations in patient with high-risk CLL: a population with significant unmet medical needs. Funding Statement: This study was funded and supported by Pharmacyclics LLC, an AbbVie company.

AbstractBackgroundThere are more than 30 immunoglobulin G (IgG) autoantibody biomarkers of autoimmune cerebellar ataxia reported.ObjectiveTo describe a novel IgG biomarker targeting neurobeachin, a scaffolding protein integral to synapse formation and function.MethodsClinical and laboratory‐based assessment was used for a novel diagnostic biomarker of autoimmune ataxia.ResultsA 46‐year‐old woman had ataxia onset followed by bilateral knee pain, attributable to rheumatoid arthritis. She was negative for standard neural antibodies. Her ataxia stabilized and tremor initially improved after corticosteroid treatment, but she progressed to wheelchair‐dependence (on methotrexate and then rituximab) over 4 years, in keeping with the trajectory encountered by many with autoimmune ataxia. In contrast, her inflammatory arthritis remitted with immunotherapy. Neurobeachin‐IgG was unmasked in serum and cerebrospinal fluid using protein arrays and confirmed using neurobeachin‐specific confocal immunofluorescence and Western blot.ConclusionsNeurobeachin‐IgG is a new neural antibody biomarker of cerebellar ataxia among a growing antibody repertoire, collectively common but often individually rare. © 2024 International Parkinson and Movement Disorder Society.

ObjectivesTo report an autoimmune paraneoplastic encephalitis characterized by immunoglobulin G (IgG) antibody targeting synaptic protein calmodulin kinase‐like vesicle‐associated (CAMKV).MethodsSerum and cerebrospinal fluid (CSF) samples harboring unclassified antibodies on murine brain‐based indirect immunofluorescence assay (IFA) were screened by human protein microarray. In 5 patients with identical cerebral IFA staining, CAMKV was identified as top‐ranking candidate antigen. Western blots, confocal microscopy, immune‐absorption, and mass spectrometry were performed to substantiate CAMKV specificity. Recombinant CAMKV‐specific assays (cell‐based [fixed and live] and Western blot) provided additional confirmation.ResultsOf 5 CAMKV‐IgG positive patients, 3 were women (median symptom‐onset age was 59 years; range, 53–74). Encephalitis‐onset was subacute (4) or acute (1) and manifested with: altered mental status (all), seizures (4), hyperkinetic movements (4), psychiatric features (3), memory loss (2), and insomnia (2). Paraclinical testing revealed CSF lymphocytic pleocytosis (all 4 tested), electrographic seizures (3 of 4 tested), and striking MRI abnormalities in all (mesial temporal lobe T2 hyperintensities [all patients], caudate head T2 hyperintensities [3], and cortical diffusion weighted hyperintensities [2]). None had post‐gadolinium enhancement. Cancers were uterine adenocarcinoma (3 patients: poorly differentiated or neuroendocrine‐differentiated in 2, both demonstrated CAMKV immunoreactivity), bladder urothelial carcinoma (1), and non‐Hodgkin lymphoma (1). Two patients developed encephalitis following immune checkpoint inhibitor cancer therapy (atezolizumab [1], pembrolizumab [1]). All treated patients (4) demonstrated an initial response to immunotherapy (corticosteroids [4], IVIG [2]), though 3 died from cancer.InterpretationCAMKV‐IgG is a biomarker of immunotherapy‐responsive paraneoplastic encephalitis with temporal and extratemporal features and uterine cancer as a prominent oncologic association. ANN NEUROL 2024;96:21–33

Abstract Bruton tyrosine kinase inhibitors (BTKis) that target B-cell receptor signaling have led to a paradigm shift in chronic lymphocytic leukemia (CLL) treatment. BTKis have been shown to reduce abnormally high CLL-associated T-cell counts and the expression of immune checkpoint receptors concomitantly with tumor reduction. However, the impact of BTKi therapy on T-cell function has not been fully characterized. Here, we performed longitudinal immunophenotypic and functional analysis of pretreatment and on-treatment (6 and 12 months) peripheral blood samples from patients in the phase 3 E1912 trial comparing ibrutinib-rituximab with fludarabine, cyclophosphamide, and rituximab (FCR). Intriguingly, we report that despite reduced overall T-cell counts; higher numbers of T cells, including effector CD8+ subsets at baseline and at the 6-month time point, associated with no infections; and favorable progression-free survival in the ibrutinib-rituximab arm. Assays demonstrated enhanced anti-CLL T-cell killing function during ibrutinib-rituximab treatment, including a switch from predominantly CD4+ T-cell:CLL immune synapses at baseline to increased CD8+ lytic synapses on-therapy. Conversely, in the FCR arm, higher T-cell numbers correlated with adverse clinical responses and showed no functional improvement. We further demonstrate the potential of exploiting rejuvenated T-cell cytotoxicity during ibrutinib-rituximab treatment, using the bispecific antibody glofitamab, supporting combination immunotherapy approaches.

Abstract Monoclonal B-cell lymphocytosis (MBL) is a common hematological premalignant condition that is understudied in screening cohorts. MBL can be classified into low-count (LC) and high-count (HC) types based on the size of the B-cell clone. Using the Mayo Clinic Biobank, we screened for MBL and evaluated its association with future hematologic malignancy and overall survival (OS). We had a two-stage study design including discovery and validation cohorts. We screened for MBL using an eight-color flow-cytometry assay. Medical records were abstracted for hematological cancers and death. We used Cox regression to evaluate associations and estimate hazard ratios and 95% confidence intervals (CIs), adjusting for age and sex. We identified 1712 (17%) individuals with MBL (95% LC-MBL), and the median follow-up time for OS was 34.4 months with 621 individuals who died. We did not observe an association with OS among individuals with LC-MBL (P = .78) but did among HC-MBL (hazard ratio, 1.8; 95% CI, 1.1-3.1; P = .03). Among the discovery cohort with a median of 10.0 years follow-up, 31 individuals developed hematological cancers with two-thirds being lymphoid malignancies. MBL was associated with 3.6-fold risk of hematological cancer compared to controls (95% CI, 1.7-7.7; P < .001) and 7.7-fold increased risk for lymphoid malignancies (95% CI:3.1-19.2; P < .001). LC-MBL was associated with 4.3-fold risk of lymphoid malignancies (95% CI, 1.4-12.7; P = .009); HC-MBL had a 74-fold increased risk (95% CI, 22-246; P < .001). In this large screening cohort, we observed similar survival among individuals with and without LC-MBL, yet individuals with LC-MBL have a fourfold increased risk of lymphoid malignancies. Accumulating evidence indicates that there are clinical consequences to LC-MBL, a condition that affects 8 to 10 million adults in the United States.