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Background: Regulatory guidance advises validation of patient-reported outcome (PRO) instruments prior to use in pivotal clinical studies, which may then be used to generate critical patient-centered evidence and support labelling claims. This targeted literature review aimed to determine if PRO instruments psychometrically validated in a phase 3 trial setting could support label claims from the same phase 3 study (i.e. PRO data were generated as an endpoint)., Methods: A targeted search of published studies (1 January 2006-3 June 2021) using the MEDLINE database identified PRO instruments validated during phase 3 trials. The search included instrument terms (e.g. patient-reported outcome measures, questionnaire, survey) and validation terms (e.g. reproducibility, minimal important difference), without filtering for therapeutic indications. Results were limited to phase 3 clinical trials or validation studies. The PROLABELS database was used to identify PROs validated in phase 3 trials and accepted in labelling claims., Results: Of 355 references identified, 68 studies with PRO psychometric validation in phase 3 studies were selected, covering 78 instruments. Of these, 20 were novel PRO instruments and 58 were existing instruments being validated for a new therapeutic indication/population. The psychometric properties most frequently validated were internal consistency reliability, known-group validity, responsiveness, minimal important difference, and concurrent validity. Five novel instruments obtained ten labelling claims for seven drugs/products., Conclusions: These results suggest that quantitative validation of novel PRO instruments, and existing PROs for new indications, can occur within the context of phase 3 trials, and these PROs can also support label claims.

Objective: Including qualitative research in clinical trial design is an innovative approach to understanding patients' perspective and incorporate the patient's voice in all stages of drug development and evaluation. This review aims to explore current practices, lessons learned from the literature, as well as how qualitative interviews are considered by health authorities for marketing authorization and reimbursement., Methods: A targeted literature review of Medline and Embase databases was conducted in February 2022 to identify publications on qualitative methods embedded in clinical trial of pharmaceutical products. An additional search of guidelines and labeling claims of approved products regarding qualitative research was performed in various sources of grey literature., Results: From the 24 publications and nine documents reviewed, we identified the research questions addressed with qualitative methods during clinical trials (e.g., change in quality of life, symptoms assessment, treatment benefit), preferred data collection methods (e.g., interviews), and data collection points (e.g., baseline and exit interviews). Moreover, the data from labels and HTAs demonstrate that qualitative data can play an important role in approval processes., Conclusion: The use of in-trial interviews is still emerging and is not yet common practice. Although the industry, scientific community, regulatory agencies and HTAs are showing an increasing interest in the use of evidence generated via in-trial interviews, guidance from regulators and HTAs would be helpful. Developing new methods and technologies to address the common challenges for such interviews is key to progress., Competing Interests: A-SM, AM, RA, CD-G, and SL are employees of ICON. PS is an employee of LAIFE REPLY. PK is an employee of Merck KgaA. JP and ES are employees of EMD Serono (a company of Merck KgaA). ICON and LAIFE REPLY were contracted with Merck KgaA as consultants on patient-centered research activities., (Copyright © 2023 Michel, Kamudoni, Marrel, Adiutori, Desvignes-Gleizes, Lanar, Schache, Spies and Park.)

Plasma proteome composition reflects the inflammatory and metabolic state of the organism and can be predictive of system-level and organ-specific pathologies. Circulating protein aggregates are enriched with neurofilament heavy chain-axonal proteins involved in brain aggregate formation and recently identified as biomarkers of the fatal neuromuscular disorder amyotrophic lateral sclerosis. Using unbiased proteomic methods, we have fully characterized the content in neuronal proteins of circulating protein aggregates from amyotrophic lateral sclerosis patients and healthy controls, with reference to brain protein aggregate composition. We also investigated circulating protein aggregate protein aggregation propensity, stability to proteolytic digestion and toxicity for neuronal and endothelial cell lines. Circulating protein aggregates separated by ultracentrifugation are visible as electron-dense macromolecular particles appearing as either large globular or as small filamentous formations. Analysis by mass spectrometry revealed that circulating protein aggregates obtained from patients are enriched with proteins involved in the proteasome system, possibly reflecting the underlying basis of dysregulated proteostasis seen in the disease, while those from healthy controls show enrichment of proteins involved in metabolism. Compared to the whole human proteome, proteins within circulating protein aggregates and brain aggregates show distinct chemical features of aggregation propensity, which appear dependent on the tissue or fluid of origin and not on the health status. Neurofilaments' two high-mass isoforms (460 and 268 kDa) showed a strong differential expression in amyotrophic lateral sclerosis compared to healthy control circulating protein aggregates, while aggregated neurofilament heavy chain was also partially resistant to enterokinase proteolysis in patients, demonstrated by immunoreactive bands at 171 and 31 kDa fragments not seen in digested healthy controls samples. Unbiased proteomics revealed that a total of 4973 proteins were commonly detected in circulating protein aggregates and brain, including 24 expressed from genes associated with amyotrophic lateral sclerosis. Interestingly, 285 circulating protein aggregate proteins (5.7%) were regulated ( P  < 0.05) and are present in biochemical pathways linked to disease pathogenesis and protein aggregation. Biologically, circulating protein aggregates from both patients and healthy controls had a more pronounced effect on the viability of hCMEC/D3 endothelial and PC12 neuronal cells compared to immunoglobulins extracted from the same plasma samples. Furthermore, circulating protein aggregates from patients exerted a more toxic effect than healthy control circulating protein aggregates on both cell lines at lower concentrations ( P : 0.03, in both cases). This study demonstrates that circulating protein aggregates are significantly enriched with brain proteins which are representative of amyotrophic lateral sclerosis pathology and a potential source of biomarkers and therapeutic targets for this incurable disorder., (© The Author(s) (2021). Published by Oxford University Press on behalf of the Guarantors of Brain.)

Most proteins in cell and tissue lysates are soluble. We show here that in lysate from human neurons, more than 1,300 proteins are maintained in a soluble and functional state by association with endogenous RNA, as degradation of RNA invariably leads to protein aggregation. The majority of these proteins lack conventional RNA-binding domains. Using synthetic oligonucleotides, we identify the importance of nucleic acid structure, with single-stranded pyrimidine-rich bulges or loops surrounded by double-stranded regions being particularly efficient in the maintenance of protein solubility. These experiments also identify an apparent one-to-one protein-nucleic acid stoichiometry. Furthermore, we show that protein aggregates isolated from brain tissue from Amyotrophic Lateral Sclerosis patients can be rendered soluble after refolding by both RNA and synthetic oligonucleotides. Together, these findings open new avenues for understanding the mechanism behind protein aggregation and shed light on how certain proteins remain soluble., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

The lack of biomarkers for an early diagnosis of neurodegenerative disorders (NDs) has hampered the development of therapeutics whose effect would be enhanced by a timely intervention. Neurofilaments light chain (Nf-L), an integral part of the axonal structure, has emerged as a robust fluid biomarker for fatal neurodegenerative disorders like amyotrophic lateral sclerosis (ALS). To facilitate large-scale studies into early-stage neurodegeneration, reduce costs of samples collection/processing and cold-chain storage, we describe the measurement of Nf-L in blood fractions obtained from dry blood spots (DBS) and dry plasma spots (DPS), two filter paper-based remote blood collection tools. To test the feasibility of using this approach, Nf-L analysis in DBS/DPS is compared to that in plasma obtained from the same blood sample, looking at Nf-L discriminatory power in the clinical stratification of ALS compared to healthy controls. With the best pre-analytical treatment for total protein recovery and using highly sensitive immunoassays, we report the detection of different Nf-L levels in DBS elute compared to reference plasma and DPS from the same blood samples. However, Nf-L measurement in DBS elutes provides a very good discrimination of ALS from healthy controls which is comparable to that obtained using plasma Nf-L assays. With the available immunodetection methods, we show that Nf-L measurement based on DPS microsampling is similar to that in plasma. The filter-paper biophysical characteristics and the interference of high haemoglobin concentration released by erythrocyte lysis is likely to perturb Nf-L detection in DBS elute. Further studies into DBS-based Nf-L detection and its analytical optimization are needed to make this method suitable for routine Nf-L blood analyses in neurodegeneration.

The rise of neurodegenerative disorders (NDD) in the ageing population is increasing demand on already stretched Health Services and is a major financial burden for society (1). The development of tools for early diagnosis is one of the responses to this problem (2). Cerebrospinal fluid (CSF) is the main repository of by-products of neuronal destruction (3); as serial lumbar punctures to procure CSF may be impractical in advanced patients (4), blood may represent the ideal source to track any meaningful disease signal of neurodegeneration.High-costs and low energy efficiency have pushed alternative means of samples collection and storage. Noviplex dried plasma spot (Np-DPS) and dried blood spot (DBS) on filter paper can be a remote, quick and inexpensive way of obtaining blood microsamples for the measurement of a large number of analytes in non-hospitalized, public health settings (5).We have used commercially-available and highly sensitive immunodetection assays (6) to test neurofilament light chain (Nf-L) expression in elute from DBS and from Np-DPS and compared these to standard Nf-L plasma measurement of healthy controls and patients with amyotrophic lateral sclerosis (ALS).We show that DBS and Np-DP cards can be used for remote blood collection and measurement of Nf-L. Nf-L measurement using elute from Noviplex DPS cards is equivalent to that obtained in plasma from the same blood samples, while levels of the same analyte in DBS elute are different, but provide discrimination between controls and ALS patients. Normalization using known loading protein concentration may also be needed for DBS analysis to adjust for the chromatographic effect of retained haemoglobin in the spot elute. Conversely, a simple elution step is required for Np-DPS, which reconstitutes a test fluid which is indistinguishable from plasma originated by conventional blood processing.

The lack of biomarkers for early diagnosis, clinical stratification and to monitor treatment response has hampered the development of new therapies for amyotrophic lateral sclerosis (ALS), a clinically heterogeneous neurodegenerative disorder with a variable site of disease initiation and rate of progression. To identify new biomarkers and therapeutic targets, two separate proteomic workflows were applied to study the immunological response and the plasma/brain proteome in phenotypic variants of ALS. Conventional multiplex (TMT) proteomic analysis of peripheral blood mononuclear cells (PBMCs) was performed alongside a recently introduced method to profile neuronal-derived proteins in plasma using brain tissue-enhanced isobaric tagging (TMTcalibrator). The combined proteomic analysis allowed the detection of regulated proteins linked to ALS pathogenesis (RNA-binding protein FUS, superoxide dismutase Cu-Zn and neurofilaments light polypeptide) alongside newly identified candidate biomarkers (myosin-9, fructose-bisphosphate aldolase and plectin). In line with the proteomic results, orthogonal immunodetection showed changes in neurofilaments and ApoE in bulbar versus limb onset fast progressing ALS. Functional analysis of significantly regulated features showed enrichment of pathways involved in regulation of the immune response, Rho family GTPases, semaphorin and integrin signalling. Our cross-phenotype investigation of PBMCs and plasma/brain proteins provides a more sensitive biomarker exploratory platform than conventional case-control studies in a single matrix. The reported regulated proteins may represent novel biomarker candidates and potentially druggable targets.

Background: It is unclear to what extent pre-clinical studies in genetically homogeneous animal models of amyotrophic lateral sclerosis (ALS), an invariably fatal neurodegenerative disorder, can be informative of human pathology. The disease modifying effects in animal models of most therapeutic compounds have not been reproduced in patients. To advance therapeutics in ALS, we need easily accessible disease biomarkers which can discriminate across the phenotypic variants observed in ALS patients and can bridge animal and human pathology. Peripheral blood mononuclear cells alterations reflect the rate of progression of the disease representing an ideal biological substrate for biomarkers discovery., Methods: We have applied TMTcalibrator™, a novel tissue-enhanced bio fluid mass spectrometry technique, to study the plasma proteome in ALS, using peripheral blood mononuclear cells as tissue calibrator. We have tested slow and fast progressing SOD1G93A mouse models of ALS at a pre-symptomatic and symptomatic stage in parallel with fast and slow progressing ALS patients at an early and late stage of the disease. Immunoassays were used to retest the expression of relevant protein candidates., Results: The biological features differentiating fast from slow progressing mouse model plasma proteomes were different from those identified in human pathology, with only processes encompassing membrane trafficking with translocation of GLUT4, innate immunity, acute phase response and cytoskeleton organization showing enrichment in both species. Biological processes associated with senescence, RNA processing, cell stress and metabolism, major histocompatibility complex-II linked immune-reactivity and apoptosis (early stage) were enriched specifically in fast progressing ALS patients. Immunodetection confirmed regulation of the immunosenescence markers Galectin-3, Integrin beta 3 and Transforming growth factor beta-1 in plasma from pre-symptomatic and symptomatic transgenic animals while Apolipoprotein E differential plasma expression provided a good separation between fast and slow progressing ALS patients., Conclusions: These findings implicate immunosenescence and metabolism as novel targets for biomarkers and therapeutic discovery and suggest immunomodulation as an early intervention. The variance observed in the plasma proteomes may depend on different biological patterns of disease progression in human and animal model.

Neurofilament proteins (Nf) are a biomarker of disease progression in amyotrophic lateral sclerosis (ALS). This study investigated whether there are major differences in expression from in vivo measurements of neurofilament isoforms, from the light chain, NfL (68 kDa), compared with larger proteins, the medium chain (NfM, 150 kDa) and the heavy (NfH, 200-210 kDa) chains in ALS patients and healthy controls. New immunological methods were combined with Nf subunit stoichiometry calculations and Monte Carlo simulations of a coarse-grained Nf brush model. Based on a physiological Nf subunit stoichiometry of 7 : 3 : 2 (NfL:NfM:NfH), we found an 'adaptive' Nf subunit stoichiometry of 24 : 2.4 : 1.6 in ALS. Adaptive Nf stoichiometry preserved NfL gyration radius in the Nf brush model. The energy and time requirements for Nf translation were 56 ± 27k ATP (5.6 h) in control subjects compared to 123 ± 102k (12.3 h) in ALS with 'adaptive' (24:2.4:1.6) Nf stoichiometry (not significant) and increased significantly to 355 ± 330k (35.5 h) with 'luxury' (7:3:2) Nf subunit stoichiometry (p < 0.0001 for each comparison). Longitudinal disease progression-related energy consumption was highest with a 'luxury' (7:3:2) Nf stoichiometry. Therefore, an energy and time-saving option for motor neurons is to shift protein expression from larger to smaller (cheaper) subunits, at little or no costs on a protein structural level, to compensate for increased energy demands., (© 2018 The Authors. Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of International Society for Neurochemistry.)

Background: Although the aetiology of multiple sclerosis (MS) remains elusive, it is clear that Epstein Barr virus (EBV) and possibly other viruses play a role in the pathogenesis of MS. Laboratory evidence suggests that human endogenous retroviruses (HERVs) could also have a role, but no interventional therapy has determined what will happen if HERVs are suppressed. Recent epidemiological evidence indicates patients with HIV infection have a significantly lower risk of developing MS and that HIV antiretroviral therapies may be coincidentally inhibiting HERVs, or other retroelements, that could be implicated in MS., Objectives: To systematically investigate the effects of an HIV integrase strand inhibitor, raltegravir, on the number of gadolinium (Gd)-enhanced MRI lesions in people with active relapsing MS., Methods: This is a Phase 2a clinical trial where twenty participants were enrolled in a 3 month baseline phase followed by 3 months of treatment with raltegravir 400 mg twice a day. Patients had monthly Gd-enhanced MRI, saliva collection to test for EBV shedding, blood sampling for safety monitoring, virology (including HERVs), measurement of immunological and inflammatory markers; and physical, neurological and quality-of-life assessments., Results: All patients completed the six months trial period.The primary outcome measure of MS disease activity was the number of Gd-enhancing lesions observed, and raltegravir had no significant effect on the rate of development of Gd-enhancing lesions during the treatment phase compared with the baseline phase. Additionally, there was no change in secondary outcomes of either disability or quality-of-life measures that could reasonably be attributed to the intervention. There was a significant positive between HERV-W/MSRV (multiple sclerosis related virus) Gag Flix (Fluorescence index) B cells and the number of Gd-enhanced lesions at any visit (p = 0.029), which was independent of any potential influence of the trial drug administration. Regarding EBV shedding, there was no significant correlation between the amount of EBV shedding and the number of lesions. No change was detected in inflammatory markers (IL-8, IL-1β, IL-6, IL-10, TNF, IL-12p70 and HCRP), which were all within normal limits both before and after the intervention. Serum CD163 expression was also unchanged by raltegravir., Conclusions: Raltegravir did not have any impact on MS disease activity. This could be due to the choice of antiretroviral agent used in this study, the need for a combination of agents, as used in treating HIV infection, the short treatment period or dosing regimen, or the lack of a role of HERV expression in MS once the disease is established. Borderline significance for the association between EBV shedding and the total number of lesions, probably driven by new lesion development, may indicate EBV shedding as a marker of inflammatory disease activity. In conclusion, interesting correlations between HERV-W markers, EBV shedding and new MRI lesions, independent from treatment effects, were found., (Copyright © 2018. Published by Elsevier B.V.)

Protein aggregation in biofluids is a poorly understood phenomenon. Under normal physiological conditions, fluid-borne aggregates may contain plasma or cell proteins prone to aggregation. Recent observations suggest that neurofilaments (Nf), the building blocks of neurons and a biomarker of neurodegeneration, are included in high molecular weight complexes in circulation. The composition of these Nf-containing hetero-aggregates (NCH) may change in systemic or organ-specific pathologies, providing the basis to develop novel disease biomarkers. We have tested ultracentrifugation (UC) and a commercially available protein aggregate binder, Seprion PAD-Beads (SEP), for the enrichment of NCH from plasma of healthy individuals, and then characterised the Nf content of the aggregate fractions using gel electrophoresis and their proteome by mass spectrometry (MS). Western blot analysis of fractions obtained by UC showed that among Nf isoforms, neurofilament heavy chain (NfH) was found within SDS-stable high molecular weight aggregates. Shotgun proteomics of aggregates obtained with both extraction techniques identified mostly cell structural and to a lesser extent extra-cellular matrix proteins, while functional analysis revealed pathways involved in inflammatory response, phagosome and prion-like protein behaviour. UC aggregates were specifically enriched with proteins involved in endocrine, metabolic and cell-signalling regulation. We describe the proteome of neurofilament-containing aggregates isolated from healthy individuals biofluids using different extraction methods.

Objective: To perform a cross-sectional study to determine whether plasma neurofilament light chain (NfL) concentration is elevated in patients with Charcot-Marie-Tooth disease (CMT) and if it correlates with disease severity., Methods: Blood samples were collected from 75 patients with CMT and 67 age-matched healthy controls over a 1-year period. Disease severity was measured using the Rasch modified CMT Examination and neuropathy scores. Plasma NfL concentration was measured using an in-house-developed Simoa assay., Results: Plasma NfL concentration was significantly higher in patients with CMT (median 26.0 pg/mL) compared to healthy controls (median 14.6 pg/mL, p < 0.0001) and correlated with disease severity as measured using the Rasch modified CMT examination ( r = 0.43, p < 0.0001) and neuropathy ( r = 0.37, p = 0.044) scores. Concentrations were also significantly higher when subdividing patients by genetic subtype ( CMT1A , SPTLC1 , and GJB1 ) or into demyelinating or axonal forms compared to healthy controls., Conclusion: There are currently no validated blood biomarkers for peripheral neuropathy. The significantly raised plasma NfL concentration in patients with CMT and its correlation with disease severity suggest that plasma NfL holds promise as a biomarker of disease activity, not only for inherited neuropathies but for peripheral neuropathy in general., (© 2018 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)

Background: Neurofilament light chain (NfL) represents a promising biomarker for axonal injury. We present the first exploratory study on serum NfL in patients with a clinically isolated syndrome (CIS) and healthy controls., Methods: We investigated serum NfL levels in 100 patients with CIS with a short conversion interval to clinically definite multiple sclerosis (MS) (fast converters (FC), median (IQR) conversion time: 110 days (79-139)); 98 patients with non-converting CIS (non-converters (NC), follow-up: 6.5 years (5.3-7.9)); and 92 healthy controls., Results: NfL levels were higher in FC (24.1 pg/mL (13.5-51.8)) and NC (19.3 pg/mL (13.6-35.2)) than in healthy controls (7.9 pg/mL (5.6-17.2)) (OR=5.85; 95% CI 2.63 to 13.02; p = 1.5 × 10(-5) and OR = 7.03; 95% CI 2.85 to 17.34; p = 2.3 × 10(-5), respectively). When grouping FC and NC, increased serum NfL concentration was also associated with increasing numbers of T2 hyperintense MRI lesions (OR = 2.36; 95% CI 1.21 to 4.59; p = 0.011), gadolinium-enhancing lesions (OR = 2.69; 95% CI 1.13 to 6.41; p=0.026) and higher disability scores (OR = 2.54; 95% CI 1.21 to 5.31; p = 0.013) at CIS diagnosis., Conclusions: If replicated in future studies, serum NfL may represent a reliable and easily accessible biomarker of early axonal damage in CIS and MS., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

Background and Objective: We explored which clinical and biochemical variables predict conversion from clinically isolated syndrome (CIS) to clinically definite multiple sclerosis (CDMS) in a large international cohort., Methods: Thirty-three centres provided serum samples from 1047 CIS cases with at least two years' follow-up. Age, sex, clinical presentation, T2-hyperintense lesions, cerebrospinal fluid (CSF) oligoclonal bands (OCBs), CSF IgG index, CSF cell count, serum 25-hydroxyvitamin D3 (25-OH-D), cotinine and IgG titres against Epstein-Barr nuclear antigen 1 (EBNA-1) and cytomegalovirus were tested for association with risk of CDMS., Results: At median follow-up of 4.31 years, 623 CIS cases converted to CDMS. Predictors of conversion in multivariable analyses were OCB (HR = 2.18, 95% CI = 1.71-2.77, p < 0.001), number of T2 lesions (two to nine lesions vs 0/1 lesions: HR = 1.97, 95% CI = 1.52-2.55, p < 0.001; >9 lesions vs 0/1 lesions: HR = 2.74, 95% CI = 2.04-3.68, p < 0.001) and age at CIS (HR per year inversely increase = 0.98, 95% CI = 0.98-0.99, p < 0.001). Lower 25-OH-D levels were associated with CDMS in univariable analysis, but this was attenuated in the multivariable model. OCB positivity was associated with higher EBNA-1 IgG titres., Conclusions: We validated MRI lesion load, OCB and age at CIS as the strongest independent predictors of conversion to CDMS in this multicentre setting. A role for vitamin D is suggested but requires further investigation., (© The Author(s), 2015.)

A recent genome wide association study (GWAS) demonstrated that more than 100 genetic variants influence the risk of multiple sclerosis (MS). We investigated what proportion of the general population can be considered at high genetic risk of MS, whether genetic information can be used to predict disease development and how the recently found genetic associations have influenced these estimates. We used summary statistics from GWAS in MS to estimate the distribution of risk within a large simulated general population. We profiled MS associated loci in 70 MS patients and 79 healthy controls (HC) and assessed their position within the distribution of risk in the simulated population. The predictive performance of a weighted genetic risk score (wGRS) on disease status was investigated using receiver operating characteristic statistics. When all known variants were considered, 40.8% of the general population was predicted to be at reduced risk, 49% at average, 6.9% at elevated and 3.3% at high risk of MS. Fifty percent of MS patients were at either reduced or average risk of disease. However, they showed a significantly higher wGRS than HC (median 13.47 vs 12.46, p = 4.08×10(-10)). The predictive performance of the model including all currently known MS associations (area under the curve = 79.7%, 95%CI = 72.4%-87.0%) was higher than that of models considering previously known associations. Despite this, considering the relatively low prevalence of MS, the positive predictive value was below 1%. The increasing number of known associated genetic variants is improving our ability to predict the development of MS. This is still unlikely to be clinically useful but a more complete understanding of the complexity underlying MS aetiology and the inclusion of environmental risk factors will aid future attempts of disease prediction.