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Background: Molecular components in blood, such as proteins, are used as biomarkers to detect or predict disease states, guide clinical interventions and aid in the development of therapies. While multiplexing proteomics methods promote discovery of such biomarkers, their translation to clinical use is difficult due to the lack of substantial evidence regarding their reliability as quantifiable indicators of disease state or outcome. To overcome this challenge, a novel orthogonal strategy was developed and used to assess the reliability of biomarkers and analytically corroborate already identified serum biomarkers for Duchenne muscular dystrophy (DMD). DMD is a monogenic incurable disease characterized by progressive muscle damage that currently lacks reliable and specific disease monitoring tools., Methods: Two technological platforms are used to detect and quantify the biomarkers in 72 longitudinally collected serum samples from DMD patients at 3 to 5 timepoints. Quantification of the biomarkers is achieved by detection of the same biomarker fragment either through interaction with validated antibodies in immuno-assays or through quantification of peptides by Parallel Reaction Monitoring Mass Spectrometry assay (PRM-MS)., Results: Five, out of ten biomarkers previously identified by affinity-based proteomics methods, were confirmed to be associated with DMD using the mass spectrometry-based method. Two biomarkers, carbonic anhydrase III and lactate dehydrogenase B, were quantified with two independent methods, sandwich immunoassays and PRM-MS, with Pearson correlations of 0.92 and 0.946 respectively. The median concentrations of CA3 and LDHB in DMD patients was elevated in comparison to those in healthy individuals by 35- and 3-fold, respectively. Levels of CA3 vary between 10.26 and 0.36 ng/ml in DMD patients whereas those of LDHB vary between 15.1 and 0.8 ng/ml., Conclusions: These results demonstrate that orthogonal assays can be used to assess the analytical reliability of biomarker quantification assays, providing a means to facilitate the translation of biomarkers to clinical practice. This strategy also warrants the development of the most relevant biomarkers, markers that can be reliably quantified with different proteomics methods., (© 2023. The Author(s).)

Longitudinal and high-dimensional measurements have become increasingly common in biomedical research. However, methods to predict survival outcomes using covariates that are both longitudinal and high-dimensional are currently missing. In this article, we propose penalized regression calibration (PRC), a method that can be employed to predict survival in such situations. PRC comprises three modeling steps: First, the trajectories described by the longitudinal predictors are flexibly modeled through the specification of multivariate mixed effects models. Second, subject-specific summaries of the longitudinal trajectories are derived from the fitted mixed models. Third, the time to event outcome is predicted using the subject-specific summaries as covariates in a penalized Cox model. To ensure a proper internal validation of the fitted PRC models, we furthermore develop a cluster bootstrap optimism correction procedure that allows to correct for the optimistic bias of apparent measures of predictiveness. PRC and the CBOCP are implemented in the R package pencal, available from CRAN. After studying the behavior of PRC via simulations, we conclude by illustrating an application of PRC to data from an observational study that involved patients affected by Duchenne muscular dystrophy, where the goal is predict time to loss of ambulation using longitudinal blood biomarkers., (© 2021 The Authors. Statistics in Medicine published by John Wiley & Sons Ltd.)

Rimeporide, a first-in-class sodium/proton exchanger Type 1 inhibitor (NHE-1 inhibitor) is repositioned by EspeRare for patients with Duchenne Muscular Dystrophy (DMD). Historically, NHE-1 inhibitors were developed for cardiac therapeutic interventions. There is considerable overlap in the pathophysiological mechanisms in Congestive Heart Failure (CHF) and in cardiomyopathy in DMD, therefore NHE-1 inhibition could be a promising pharmacological approach to the cardiac dysfunctions observed in DMD. Extensive preclinical data was collected in various animal models including dystrophin-deficient (mdx) mice to characterise Rimeporide's anti-fibrotic and anti-inflammatory properties and there is evidence that NHE-1 inhibitors could play a significant role in modifying DMD cardiac and also skeletal pathologies, as the NHE-1 isoform is ubiquitous. We report here the first study with Rimeporide in DMD patients. This 4-week treatment, open label phase Ib, multiple oral ascending dose study, enrolled 20 ambulant boys with DMD (6-11 years), with outcomes including safety, pharmacokinetic (PK) and pharmacodynamic (PD) biomarkers. Rimeporide was safe and well-tolerated at all doses. PK evaluations showed that Rimeporide was well absorbed orally reaching pharmacological concentrations from the lowest dose, with exposure increasing linearly with dose and with no evidence of accumulation upon repeated dosing. Exploratory PD biomarkers showed positive effect upon a 4-week treatment, supporting its therapeutic potential in patients with DMD, primarily as a cardioprotective treatment, and provide rationale for further efficacy studies., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Duchenne Muscular Dystrophy is a severe, incurable disorder caused by mutations in the dystrophin gene. The disease is characterized by decreased muscle function, impaired muscle regeneration and increased inflammation. In a clinical context, muscle deterioration, is evaluated using physical tests and analysis of muscle biopsies, which fail to accurately monitor the disease progression., Objectives: This study aims to confirm and asses the value of blood protein biomarkers as disease progression markers using one of the largest longitudinal collection of samples., Methods: A total of 560 samples, both serum and plasma, collected at three clinical sites are analyzed using a suspension bead array platform to assess 118 proteins targeted by 250 antibodies in microliter amount of samples., Results: Nine proteins are confirmed as disease progression biomarkers in both plasma and serum. Abundance of these biomarkers decreases as the disease progresses but follows different trajectories. While carbonic anhydrase 3, microtubule associated protein 4 and collagen type I alpha 1 chain decline rather constantly over time, myosin light chain 3, electron transfer flavoprotein A, troponin T, malate dehydrogenase 2, lactate dehydrogenase B and nestin plateaus in early teens. Electron transfer flavoprotein A, correlates with the outcome of 6-minutes-walking-test whereas malate dehydrogenase 2 together with myosin light chain 3, carbonic anhydrase 3 and nestin correlate with respiratory capacity., Conclusions: Nine biomarkers have been identified that correlate with disease milestones, functional tests and respiratory capacity. Together these biomarkers recapitulate different stages of the disorder that, if validated can improve disease progression monitoring.

There is a need for standardized validation methods for antibody specificity and selectivity. Recently, five alternative validation pillars were proposed to explore the specificity of research antibodies using methods with no need for prior knowledge about the protein target. Here, we show that these principles can be used in a streamlined manner for enhanced validation of research antibodies in Western blot applications. More than 6,000 antibodies were validated with at least one of these strategies involving orthogonal methods, genetic knockdown, recombinant expression, independent antibodies, and capture mass spectrometry analysis. The results show a path forward for efforts to validate antibodies in an application-specific manner suitable for both providers and users.

Numerous biomarkers have been unveiled in the rapidly evolving biomarker discovery field, with an aim to improve the clinical management of disorders. In rare diseases, such as Duchenne muscular dystrophy, this endeavor has created a wealth of knowledge that, if effectively exploited, will benefit affected individuals, with respect to health care, therapy, improved quality of life and increased life expectancy. The most promising findings and molecular biomarkers are inspected in this review, with an aim to provide an overview of currently known biomarkers and the technological developments used. Biomarkers as cells, genetic variations, miRNAs, proteins, lipids and/or metabolites indicative of disease severity, progression and treatment response have the potential to improve development and approval of therapies, clinical management of DMD and patients' life quality. We highlight the complexity of translating research results to clinical use, emphasizing the need for biomarkers, fit for purpose and describe the challenges associated with qualifying biomarkers for clinical applications., Competing Interests: Disclosure The authors report no conflicts of interest in this work.