Urinary biomarkers for amyotrophic lateral sclerosis: candidates, opportunities and considerations.

Amyotrophic lateral sclerosis is a relentless neurodegenerative disease that is mostly fatal within 3-5 years and is diagnosed on evidence of progressive upper and lower motor neuron degeneration. Around 15% of those with amyotrophic lateral sclerosis also have frontotemporal degeneration, and gene mutations account for ∼10%. Amyotrophic lateral sclerosis is a variable heterogeneous disease, and it is becoming increasingly clear that numerous different disease processes culminate in the final degeneration of motor neurons. There is a profound need to clearly articulate and measure pathological process that occurs. Such information is needed to tailor treatments to individuals with amyotrophic lateral sclerosis according to an individual's pathological fingerprint. For new candidate therapies, there is also a need for methods to select patients according to expected treatment outcomes and measure the success, or not, of treatments. Biomarkers are essential tools to fulfil these needs, and urine is a rich source for candidate biofluid biomarkers. This review will describe promising candidate urinary biomarkers of amyotrophic lateral sclerosis and other possible urinary candidates in future areas of investigation as well as the limitations of urinary biomarkers.

Urinary neopterin: A novel biomarker of disease progression in amyotrophic lateral sclerosis.

BACKGROUND AND PURPOSE: The aim was to evaluate urinary neopterin, a marker of pro-inflammatory state, as a potential biomarker of disease prognosis and progression in amyotrophic lateral sclerosis (ALS); and to compare its utility to urinary neurotrophin receptor p75 extracellular domain (p75ECD ). METHODS: This was an observational study including 21 healthy controls and 46 people with ALS, 29 of whom were sampled longitudinally. Neopterin and p75ECD were measured using enzyme-linked immunoassays. Baseline and longitudinal changes in clinical measures, neopterin and urinary p75ECD were examined, and prognostic utility was explored by survival analysis. RESULTS: At baseline, urinary neopterin was higher in ALS compared to controls (181.7 ± 78.9 µmol/mol creatinine vs. 120.4 ± 60.8 µmol/mol creatinine, p = 0.002, Welch's t test) and correlated with the Revised ALS Functional Rating Scale (r = -0.36, p = 0.01). Combining previously published urinary p75ECD results from 22 ALS patients with a further 24 ALS patients, baseline urinary p75ECD was also higher compared to healthy controls (6.0 ± 2.7 vs. 3.2 ± 1.0 ng/mg creatinine, p < 0.0001) and correlated with the Revised ALS Functional Rating Scale (r = -0.36, p = 0.01). Urinary neopterin and p75ECD correlated with each other at baseline (r = 0.38, p = 0.009). In longitudinal analysis, urinary neopterin increased on average (±SE) by 6.8 ± 1.1 µmol/mol creatinine per month (p < 0.0001) and p75ECD by 0.19 ± 0.02 ng/mg creatinine per month (p < 0.0001) from diagnosis in 29 ALS patients. CONCLUSION: Urinary neopterin holds promise as marker of disease progression in ALS and is worthy of future evaluation for its potential to predict response to anti-inflammatory therapies.

DSP-01 Barriers to the diagnosis of motor neuron disease - a South Australian study.

Background: MND is a progressive neurodegenerative disease characterised by death of upper and lower motor neurons, leading to progressive weakness of the bulbar, limb, thoracic and abdominal muscles. MND has a fairly stereotypical course, with death from respiratory failure occurring 2-4 years after symptom onset in most cases (1). Making the diagnosis of MND can be straightforward when key clinical criteria are met; however, at first presentation, rarely do patients meet these criteria, neurological changes may be subtle and disease progression slow. Thus, diagnosis poses a significant challenge, particularly in general practice, where patients are most likely to first present. Not surprisingly, there is usually a delay of 10-18 months between symptom onset and MND diagnosis (2). Importantly, early assessment by a neurologist is associated with a shorter time to MND diagnosis (3), which has significant implications for access to healthcare, including Riluzole and multidisciplinary clinics, which improve survival (4). Although delay in diagnosis is well documented, there have been no studies that have sought to identify factors associated with time to diagnosis, thereby enabling targeted implementation of a public health intervention.Objectives: To characterise the clinical factors that influence time to diagnosis of MND.Methods: 112 patients with MND attending the Southern Adelaide MND Clinic enrolled between January 2016 - 2018 were retrospectively recruited in to a cohort study. Information pertaining to the patient's demographics and their journey to diagnosis collected by a specialist physician and stored in the Australian MND Registry during clinic review were analysed to identify factors associated with time to diagnosis.Results: Mean time to diagnosis was 13 ± 1 months (range 1 - 38 months) from symptom onset. 41% of patients were classified as having fast disease progression; compared to those with slow disease progression, these patients were diagnosed earlier (8 ± 1 months vs 16 ± 2 months) (p < 0.0001, t = 34.6, df =220), were less likely to undergo multiple specialist opinions prior to referral to a neurologist (53% vs 73%) (p < 0.05, Chi-squared = 9.5, df =1), and were more disabled at time of diagnosis (mean ALSFRS-R 33 ± 5 vs ALSFRS-R 41 ± 5) (p < 0.0001, t = 12.4, df =220).Discussion and conclusions: Fast disease progression identifies a dichotomy of MND patients diagnosed earlier, although more disabled at diagnosis, likely mediated by a more efficient referral process. A greater awareness of MND is required to shorten time to diagnosis.

Factors contributing to delays in the diagnosis of motor neuron disease - A South Australian study.

OBJECTIVE(S): To characterize the clinical factors that influence time to diagnosis of motor neuron disease (MND) in a cohort of patients living in South Australia. DESIGN: A retrospective study. SETTING: Single centre study of patients managed at a tertiary referral hospital. PARTICIPANTS: Patients with MND living in South Australia enrolled in the Australian MND Registry between January 2016 and January 2018. One participant was excluded as study variables of interest were missing. RESULTS: The mean time to diagnosis was 13 ±â€¯1 months (median 11 months; range 1-38 months) from symptom onset. 41% of patients were classified as having fast disease progression; mean age of disease onset of those with fast disease progression was significantly later in life compared to those with slow disease progression (68 ±â€¯10 years vs 64 ±â€¯8 years) (P < .05, t = -3.921, df = 220). Patients with fast disease progression were diagnosed significantly earlier than those with slow disease progression (8 ±â€¯1 months vs 16 ±â€¯2 months) (P < .0001, t = 34.6, df = 220), were less likely to undergo multiple specialist opinions prior to referral to a neurologist (53% vs 73%) (P < .05, Chi-squared = 9.5, df = 1), and were significantly more disabled at time of diagnosis (mean ALSFRS-R 33 ±â€¯5) than those with slow disease progression (mean ALSFRS-R 41 ±â€¯5) (P < .0001, t = 12.4, df = 220). CONCLUSION(S): Fast disease progression identifies a dichotomy of MND patients that are diagnosed earlier, probable because they are more disabled at diagnosis, likely mediated by a more efficient referral process. A greater awareness of the disease and increased accessibility to neurologists is required to shorten time to diagnosis.

The Influence of Electrostatic Controls on MDI Size Distribution Measurements.

Cascade impactor testing is widely used to characterize the aerodynamic particle-size distribution of metered dose inhaler aerosols. Charge is often imparted to MDI aerosols by triboelectrification as formulation rapidly travels through the valve stem and actuator during atomization. The presence of charge on MDI aerosols can impact the accuracy and reproducibility of APSD measurements made using cascade impactors. The aerodynamic particle size distribution of three different experimental MDI formulations were evaluated using the Next Generation Impactor with and without incorporating static controls during testing. The static controls included grounding the analyst and the equipment, using an ionizing air blower and anti-static gun, rinsing and allowing the actuator to air dry prior to testing, and having the analyst not wear gloves or touch the USP throat during testing. For all three formulations, tests that used static controls had lower actuator and throat deposition and correspondingly higher deposition on the impactor stages. While static controls influenced the amount of drug entering into the impactor during testing, the static controls did not otherwise change the aerodynamic particle size distribution of these particles. Static controls had the greatest impact on the ethanol-free HFA-227 formulation. For this formulation, there was a 15% difference in throat deposition for the tests that did or did not incorporate static controls. These results demonstrated that electrostatic effects can lead to meaningful variability in cascade impactor test results. Static controls should be considered when developing cascade impactor test methods for MDI products in order to eliminate variability in test results.

Motor neurone disease: progress and challenges.

Major progress has been made over the past decade in the understanding of motor neurone disease (MND), changing the landscape of this complex disease. Through identifying positive prognostic factors, new evidence-based standards of care have been established that improve patient survival, reduce burden of disease for patients and their carers, and enhance quality of life. These factors include early management of respiratory dysfunction with non-invasive ventilation, maintenance of weight and nutritional status, as well as instigation of a multidisciplinary team including neurologists, general practitioners and allied health professionals. Advances in technology have enhanced our understanding of the genetic architecture of MND considerably, with implications for patients, their families and clinicians. Recognition of extra-motor involvement, particularly cognitive dysfunction, has identified a spectrum of disease from MND through to frontotemporal dementia. Although riluzole remains the only disease-modifying medication available in clinical practice in Australia, several new therapies are undergoing clinical trials nationally and globally, representing a shift in treatment paradigms. Successful translation of this clinical research through growth in community funding, awareness and national MND research organisations has laid the foundation for closing the research-practice gap on this debilitating disease. In this review, we highlight these recent developments, which have transformed treatment, augmented novel therapeutic platforms, and established a nexus between research and the MND community. This era of change is of significant relevance to both specialists and general practitioners who remain integral to the care of patients with MND.

The extracellular domain of neurotrophin receptor p75 as a candidate biomarker for amyotrophic lateral sclerosis.

Objective biomarkers for amyotrophic lateral sclerosis would facilitate the discovery of new treatments. The common neurotrophin receptor p75 is up regulated and the extracellular domain cleaved from injured neurons and peripheral glia in amyotrophic lateral sclerosis. We have tested the hypothesis that urinary levels of extracellular neurotrophin receptor p75 serve as a biomarker for both human motor amyotrophic lateral sclerosis and the SOD1(G93A) mouse model of the disease. The extracellular domain of neurotrophin receptor p75 was identified in the urine of amyotrophic lateral sclerosis patients by an immuno-precipitation/western blot procedure and confirmed by mass spectrometry. An ELISA was established to measure urinary extracellular neurotrophin receptor p75. The mean value for urinary extracellular neurotrophin receptor p75 from 28 amyotrophic lateral sclerosis patients measured by ELISA was 7.9±0.5 ng/mg creatinine and this was significantly higher (p<0.001) than 12 controls (2.6±0.2 ng/mg creatinine) and 19 patients with other neurological disease (Parkinson's disease and Multiple Sclerosis; 4.1±0.2 ng/mg creatinine). Pilot data of disease progression rates in 14 MND patients indicates that p75NTR(ECD) levels were significantly higher (p = 0.0041) in 7 rapidly progressing patients as compared to 7 with slowly progressing disease. Extracellular neurotrophin receptor p75 was also readily detected in SOD1(G93A) mice by immuno-precipitation/western blot before the onset of clinical symptoms. These findings indicate a significant relation between urinary extracellular neurotrophin receptor p75 levels and disease progression and suggests that it may be a useful marker of disease activity and progression in amyotrophic lateral sclerosis.

EPITHET: Positive Result After Reanalysis Using Baseline Diffusion-Weighted Imaging/Perfusion-Weighted Imaging Co-Registration.

BACKGROUND AND PURPOSE: the Echoplanar Imaging Thrombolytic Evaluation Trial (EPITHET) was a prospective, randomized, double-blinded, placebo-controlled, phase II trial of alteplase between 3 and 6 hours after stroke onset. The primary outcome of infarct growth attenuation on MRI with alteplase in mismatch patients was negative when mismatch volumes were assessed volumetrically, without coregistration, which underestimates mismatch volumes. We hypothesized that assessing the extent of mismatch by coregistration of perfusion and diffusion MRI maps may more accurately allow the effects of alteplase vs placebo to be evaluated. METHODS: patients were classified as having mismatch if perfusion-weighted imaging divided by coregistered diffusion-weighted imaging volume ratio was >1.2 and total coregistered mismatch volume was ≥ 10 mL. The primary outcome was a comparison of infarct growth in alteplase vs placebo patients with coregistered mismatch. RESULTS: of 99 patients with baseline diffusion-weighted imaging and perfusion-weighted imaging, coregistration of both images was possible in 95 patients. Coregistered mismatch was present in 93% (88/95) compared to 85% (81/95) with standard volumetric mismatch. In the coregistered mismatch patients, of whom 45 received alteplase and 43 received placebo, the primary outcome measure of geometric mean infarct growth was significantly attenuated by a ratio of 0.58 with alteplase compared to placebo (1.02 vs 1.77; 95% CI, 0.33-0.99; P=0.0459). CONCLUSIONS: when using coregistration techniques to determine the presence of mismatch at study entry, alteplase significantly attenuated infarct growth. This highlights the necessity for a randomized, placebo-controlled, phase III clinical trial of alteplase using penumbral selection beyond 3 hours.