SOD1 gene screening in ALS - frequency of mutations, patients' attitudes to genetic information and transition to tofersen treatment in a multi-center program.

Objective: To report the frequency of pathogenic SOD1 gene variants in a screening program in amyotrophic lateral sclerosis (ALS), and the clinical practice of transition to an expanded access program (EAP) of tofersen treatment. Methods: From October 2021 to February 2024, at 11 ALS centers in Germany genetic testing for SOD1, FUS, TARDBP, and C9orf72 was performed. Patients were offered to opt for notification either about all genetic variants or SOD1 variants relevant for tofersen therapy. The transition to the EAP with tofersen was assessed. Results: 1935 patients were screened (94.7% sporadic ALS). 48.8% (n = 928) opted for notification of treatment-relevant information. Genetic variants were found as follows: SOD1 (likely) pathogenic variants (class 4/5) 1.8% (n = 34), variants of unknown significance (class 3) 0.8% (n = 16), FUS (class 4/5) 0.9% (n = 17), TARDBP (class 4/5) 1.3% (n = 25), C9orf72 hexanucleotide repeat expansion 7.0% (n = 135). In SOD1-ALS (encompassing class 3-5 variants, n = 50), 68.0% (n = 34) reported a negative family history. 74.0% (n = 37) of SOD1-ALS patients - which represent 1.9% of all participants of the screening program - were transitioned to tofersen. Median duration from start of genetic testing to treatment was 94 days (57 to 295 days). Eight patients declined treatment whereas five individuals died before initiation of therapy. Conclusion: The finding of SOD1 variants in patients with a negative family history underscores the need for a broad genetic screening in ALS. In SOD1-ALS, the treatment option with tofersen was mostly utilized. The wide range in the transition time to tofersen calls for a SOD1-ALS management program.

Clinical characterization of common pathogenic variants of SOD1-ALS in Germany.

Pathogenic variants in the Cu/Zn superoxide dismutase (SOD1) gene can be detected in approximately 2% of sporadic and 11% of familial amyotrophic lateral sclerosis (ALS) patients in Europe. We analyzed the clinical phenotypes of 83 SOD1-ALS patients focusing on patients carrying the most frequent (likely) pathogenic variants (R116G, D91A, L145F) in Germany. Moreover, we describe the effect of tofersen treatment on ten patients carrying these variants. R116G patients showed the most aggressive course of disease with a median survival of 22.0 months compared to 198.0 months in D91A and 87.0 months in L145F patients (HR 7.71, 95% CI 2.89-20.58 vs. D91A; p < 0.001 and HR 4.25, 95% CI 1.55-11.67 vs. L145F; p = 0.02). Moreover, R116G patients had the fastest median ALSFRS-R progression rate with 0.12 (IQR 0.07-0.20) points lost per month. Median diagnostic delay was 10.0 months (IQR 5.5-11.5) and therefore shorter compared to 57.5 months (IQR 14.0-83.0) in D91A (p < 0.001) and 21.5 months (IQR 5.8-38.8) in L145F (p = 0.21) carriers. As opposed to D91A carriers (50.0%), 96.2% of R116G (p < 0.001) and 100.0% of L145F (p = 0.04) patients reported a positive family history. During tofersen treatment, all patients showed a reduction of neurofilament light chain (NfL) serum levels, independent of the SOD1 variant. Patients with SOD1-ALS carrying R116G, D91A, or L145F variants show commonalities, but also differences in their clinical phenotype, including a faster progression rate with shorter survival in R116G, and a comparatively benign disease course in D91A carriers.

A candidate reference measurement procedure for the quantification of α-synuclein in cerebrospinal fluid using an SI traceable primary calibrator and multiple reaction monitoring.

α-synuclein aggregation is an important hallmark of neurodegenerative diseases such as Parkinson's disease (PD) and Lewy body dementia. α-synuclein has been increasingly used as a diagnostic biomarker in PD and other synucleinopathies. Current clinical assays rely on antibody-based immunoassays to detect α-synuclein, which possess high sensitivity, afford high throughput and require small sample volumes. The utility of these assays, however, may be compounded by the specificity, selectivity and batch-to-batch heterogeneity of the antibody used, which can lead to deviations in the total amount of the protein measured when comparing results among different laboratories. Similarly, current mass spectrometry-based quantification methods for α-synuclein lack well-defined, value assigned calibrators to ensure comparability of measurements. Therefore, there is still an unmet need for the standardisation of clinical measurements for α-synuclein that can be achieved by the development of reference measurement procedures (RMPs) utilising calibrators traceable to the SI (International System of Units). Here, we report a candidate RMP for α-synuclein, using an SI traceable primary calibrator and an isotope dilution mass spectrometry (IDMS) approach to address this need. The gravimetrically prepared primary calibrator was traceably quantified utilising a combination of amino acid analysis (AAA) and quantitative nuclear magnetic resonance (qNMR) for value assignment. An optimised targeted sample clean-up procedure involving a non-denaturing Lys-C digestion and solid-phase extraction strategy was devised, followed by the development of a targeted multiple reaction monitoring (MRM) method for the quantification of α-synuclein in cerebrospinal fluid (CSF). This candidate RMP was then deployed for the sensitive detection and accurate quantification of multiple proteotypic α-synuclein peptides in patient derived CSF samples. The LC-MS based results were subsequently compared to immunoassay data to assess the overall performance of our approach. The development and adoption of this candidate RMP, along with the availability of the SI traceable primary calibrator will allow for reliable quantifications of α-synuclein in CSF by an LC-MS based assay. The RMP will potentially contribute towards the standardisation of this important biomarker and may lead to future interlaboratory comparisons.

Clinical and patient-reported outcomes and neurofilament response during tofersen treatment in SOD1-related ALS-A multicenter observational study over 18 months.

INTRODUCTION/AIMS: In amyotrophic lateral sclerosis (ALS) caused by SOD1 mutations (SOD1-ALS), tofersen received accelerated approval in the United States and is available via expanded access programs (EAP) outside the United States. This multicenter study investigates clinical and patient-reported outcomes (PRO) and serum neurofilament light chain (sNfL) during tofersen treatment in an EAP in Germany. METHODS: Sixteen SOD1-ALS patients receiving tofersen for at least 6 months were analyzed. The ALS progression rate (ALS-PR), as measured by the monthly change of the ALS functional rating scale-revised (ALSFRS-R), slow vital capacity (SVC), and sNfL were investigated. PRO included the Measure Yourself Medical Outcome Profile (MYMOP2), Treatment Satisfaction Questionnaire for Medication (TSQM-9), and Net Promoter Score (NPS). RESULTS: Mean tofersen treatment was 11 months (6-18 months). ALS-PR showed a mean change of -0.2 (range 0 to -1.1) and relative reduction by 25%. Seven patients demonstrated increased ALSFRS-R. SVC was stable (mean 88%, range -15% to +28%). sNfL decreased in all patients except one heterozygous D91A-SOD1 mutation carrier (mean change of sNfL -58%, range -91 to +27%, p < .01). MYMOP2 indicated improved symptom severity (n = 10) or yet perception of partial response (n = 6). TSQM-9 showed high global treatment satisfaction (mean 83, SD 16) although the convenience of drug administration was modest (mean 50, SD 27). NPS revealed a very high recommendation rate for tofersen (NPS +80). DISCUSSION: Data from this EAP supported the clinical and sNfL response to tofersen in SOD1-ALS. PRO suggested a favorable patient perception of tofersen treatment in clinical practice.

Tofersen decreases neurofilament levels supporting the pathogenesis of the SOD1 p.D91A variant in amyotrophic lateral sclerosis patients.

BACKGROUND: Since the antisense oligonucleotide tofersen has recently become available for the treatment of amyotrophic lateral sclerosis (ALS) caused by mutations in SOD1, determining the causality of the over 230 SOD1 variants has become even more important. The most common SOD1 variant worldwide is p.D91A (c.272A > C), whose causality for ALS is contested when in a heterozygous state. The reason is the high allele frequency of SOD1D91A in Europe, exceeding 1% in Finno-Scandinavia. METHODS: We present the clinical disease course and serum neurofilament light chain (NfL) results of treating 11 patients either homo- or heterozygous for the SOD1D91A allele for up to 16 months with tofersen. RESULTS: Tofersen decreases serum neurofilament levels (sNFL), which are associated with the ALS progression rate, in the 6 ALS patients homozygous for SOD1D91A. We observe significantly lower sNfL levels in the 5 patients heterozygous for SOD1D91A. The results indicate that both mono- and bi-allelic SOD1D91A are causally relevant targets, with a possibly reduced effect size of SOD1D91Ahet. CONCLUSIONS: The finding is relevant for decision making regarding tofersen treatment, patient counseling and inclusion of SOD1D91A patients in drug trials. As far as we are aware, the approach is conceptually new since it provides evidence for the causality of an ALS variant based on a biomarker response to gene-specific treatment.

Amyotrophic lateral sclerosis (ALS) is a disease that can be inherited which affects nerve cells in the brain and spinal cord. Changes within a gene called SOD1 that result in a mutation named p.D91A can lead to the development of ALS. People have two copies of the SOD1 gene. It has been unclear whether the presence of only one copy of p.D91A can cause ALS. We treated ALS patients with the p.D91A variant of SOD1 with a drug called tofersen. We found that a marker of disease progression was reduced in patients with one or two copies of the p.D91A mutation. This suggests that the presence of just one p.D91A variant of SOD1 contributes to disease development. This information could be used to improve treatment decisions for people with ALS.

Serum neurofilament light chain in distinct phenotypes of amyotrophic lateral sclerosis: A longitudinal, multicenter study.

OBJECTIVE: To assess the performance of serum neurofilament light chain (sNfL) in clinical phenotypes of amyotrophic lateral sclerosis (ALS). METHODS: In 2949 ALS patients at 16 ALS centers in Germany and Austria, clinical characteristics and sNfL were assessed. Phenotypes were differentiated for two anatomical determinants: (1) upper and/or lower motor involvement (typical, typMN; upper/lower motor neuron predominant, UMNp/LMNp; primary lateral sclerosis, PLS) and (2) region of onset and propagation of motor neuron dysfunction (bulbar, limb, flail-arm, flail-leg, thoracic onset). Phenotypes were correlated to sNfL, progression, and survival. RESULTS: Mean sNfL was - compared to typMN (75.7 pg/mL, n = 1791) - significantly lower in LMNp (45.1 pg/mL, n = 413), UMNp (58.7 pg/mL n = 206), and PLS (37.6 pg/mL, n = 84). Also, sNfL significantly differed in the bulbar (92.7 pg/mL, n = 669), limb (64.1 pg/mL, n = 1305), flail-arm (46.4 pg/mL, n = 283), flail-leg (53.6 pg/mL, n = 141), and thoracic (74.5 pg/mL, n = 96) phenotypes. Binary logistic regression analysis showed highest contribution to sNfL elevation for faster progression (odds ratio [OR] 3.24) and for the bulbar onset phenotype (OR 1.94). In contrast, PLS (OR 0.20), LMNp (OR 0.45), and thoracic onset (OR 0.43) showed reduced contributions to sNfL. Longitudinal sNfL (median 12 months, n = 2862) showed minor monthly changes (<0.2%) across all phenotypes. Correlation of sNfL with survival was confirmed (p < 0.001). CONCLUSIONS: This study underscored the correlation of ALS phenotypes - differentiated for motor neuron involvement and region of onset/propagation - with sNfL, progression, and survival. These phenotypes demonstrated a significant effect on sNfL and should be recognized as independent confounders of sNfL analyses in ALS trials and clinical practice.

Leucine-Rich Glioma-Inactivated 1 (LGI1) Protein Stimulates Proliferation and IL-10 Production in Peripheral Blood Mononuclear Cells of Patients with LGI1 Antibody-Mediated Autoimmune Encephalitis In Vitro.

Limbic encephalitis (LE) due to anti-leucine-rich glioma-inactivated 1 (LGI1) antibodies is an autoimmune disease characterized by distinct clinical features unique to LGI1 LE, such as faciobrachial dystonic seizures. However, it is unclear whether an additional disease-related LGI1 antigen-specific T cell response is involved in the pathogenesis of this disease. To address this question, we studied the effect of recombinant LGI1 on the proliferation and effector-specific cytokine production (IFN-γ, IL-5, IL-10, and IL-17) of peripheral blood mononuclear cells (PBMCs) from patients with LGI1 LE and healthy controls. We observed that recombinant LGI1 stimulated the proliferation of PBMCs from patients with LGI1 LE, but not from healthy controls. Cytokine measurement of cell culture supernatants from PBMCs incubated with recombinant LGI1 revealed a highly significant increase in IL-10 release in PBMCs from patients with LGI1 LE in comparison with healthy controls. These results suggest that LGI1-mediated stimulation of PBMCs from patients with LGI1 LE leads to the establishment of an IL-10-dominated immunosuppressive cytokine milieu, which may inhibit Th1 differentiation and support B cell proliferation, IgG production, and IgG subclass switching.

Different pain phenotypes are associated with anti-Caspr2 autoantibodies.

Autoantibodies against contactin-associated protein 2 (Caspr2) not only induce limbic autoimmune encephalitis but are also associated with pain conditions. Here, we analyzed clinical data on pain in a large cohort of patients included into the German Network for Research in Autoimmune Encephalitis. Out of 102 patients in our cohort, pain was a frequent symptom (36% of all patients), often severe (63.6% of the patients with pain) and/or even the major symptom (55.6% of the patients with pain). Pain phenotypes differed between patients. Cluster analysis revealed two major phenotypes including mostly distal-symmetric burning pain and widespread pain with myalgia and cramps. Almost all patients had IgG4 autoantibodies and some additional IgG1, 2, and/or 3 autoantibodies, but IgG subclasses, titers, and presence or absence of intrathecal synthesis were not associated with the occurrence of pain. However, certain pre-existing risk factors for chronic pain like diabetes mellitus, peripheral neuropathy, or preexisting chronic back pain tended to occur more frequently in patients with anti-Caspr2 autoantibodies and pain. Our data show that pain is a relevant symptom in patients with anti-Caspr2 autoantibodies and support the idea of decreased algesic thresholds leading to pain. Testing for anti-Caspr2 autoantibodies needs to be considered in patients with various pain phenotypes.

Effects of tofersen treatment in patients with SOD1-ALS in a "real-world" setting - a 12-month multicenter cohort study from the German early access program.

Background: In April 2023, the antisense oligonucleotide tofersen was approved by the U.S. Food and Drug Administration (FDA) for treatment of SOD1-amyotrophic lateral sclerosis (ALS), after a decrease of neurofilament light chain (NfL) levels had been demonstrated. Methods: Between 03/2022 and 04/2023, 24 patients with SOD1-ALS from ten German ALS reference centers were followed-up until the cut-off date for ALS functional rating scale revised (ALSFRS-R), progression rate (loss of ALSFRS-R/month), NfL, phosphorylated neurofilament heavy chain (pNfH) in cerebrospinal fluid (CSF), and adverse events. Findings: During the observation period, median ALSFRS-R decreased from 38.0 (IQR 32.0-42.0) to 35.0 (IQR 29.0-42.0), corresponding to a median progression rate of 0.11 (IQR -0.09 to 0.32) points of ALSFRS-R lost per month. Median serum NfL declined from 78.0 pg/ml (IQR 37.0-147.0 pg/ml; n = 23) to 36.0 pg/ml (IQR 22.0-65.0 pg/ml; n = 23; p = 0.02), median pNfH in CSF from 2226 pg/ml (IQR 1061-6138 pg/ml; n = 18) to 1151 pg/ml (IQR 521-2360 pg/ml; n = 18; p = 0.02). In the CSF, we detected a pleocytosis in 73% of patients (11 of 15) and an intrathecal immunoglobulin synthesis (IgG, IgM, or IgA) in 9 out of 10 patients. Two drug-related serious adverse events were reported. Interpretation: Consistent with the VALOR study and its Open Label Extension (OLE), our results confirm a reduction of NfL serum levels, and moreover show a reduction of pNfH in CSF. The therapy was safe, as no persistent symptoms were observed. Pleocytosis and Ig synthesis in CSF with clinical symptoms related to myeloradiculitis in two patients, indicate the potential of an autoimmune reaction. Funding: No funding was received towards this study.