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Project

Blood based biomarkers for early diagnosis and prognosis of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a rare progressive neurodegenerative disease characterized by the degeneration of upper motor neurons and lower motor neurons. The first symptoms associated with the disease are muscle weakness, muscle atrophy and fasciculations. To date, ALS is a fatal disease as no cure is available. The diagnosis of ALS is based on clinical symptoms, electrophysiological examinations, progression of the symptoms and exclusion of other diseases. However, it takes about one year to diagnose a patient with ALS after symptom onset. This period, which is defined as the diagnostic delay, represents a significant proportion of the average survival of 2-5 years after symptom onset in ALS.

Biomarkers have been studied to facilitate the diagnosis of ALS. Until now, the most promising biofluid markers in ALS are the neurofilaments (NF). These proteins are the building blocks of the neuronal cytoskeleton and are released into the extracellular space when neurons are damaged. The NF are heteropolymers and mainly consist out of three subunits, the neurofilament heavy, neurofilament median and neurofilament light (NfL) chain, all class IV intermediate filaments. Later, a fourth member was discovered, namely α-internexin, which is replaced by peripherin in the peripheral nervous system. In the past, we and others have demonstrated that the phosphorylated neurofilament heavy (pNfH) and NfL levels in cerebrospinal fluid (CSF) could differentiate patients with ALS from ALS mimics (patients with a disease that mimics the clinical phenotype of ALS) with high accuracy. The aim of this PhD project was to investigate if NF are increased in blood and whether NF are already elevated before diagnosis at a tertiary ALS center. Next, I investigated if NF assessed at an early disease stage can decrease the diagnostic delay in patients with ALS. As a secondary outcome, I determined the added value of NF in blood to predict survival in patients with ALS.

First, in chapter one I demonstrated that pNfH levels in blood (i.e. serum) of patients with ALS and controls correlated with pNfH levels in CSF. Serum pNfH levels were approximately 12-fold lower than CSF levels. Following this observation, pNfH levels were significantly increased in serum of patients with ALS compared to ALS mimics. Serum pNfH levels showed a good specificity of 85% and positive predictive value of 94%. Yet, lower discriminatory performance was observed than CSF pNfH levels.  In meanwhile, my host laboratory and others reported similar findings for NfL levels in serum of patients with ALS. Next, in the second and third chapter I performed two independent retrospective studies to investigate if NF in blood are increased before diagnosis at a tertiary ALS center. Therefore, patients diagnosed with ALS at the tertiary ALS center were included. For patients to be included, a residual serum sampled for routine purposes before a diagnosis of ALS had to be available. In total, 95 patients with ALS could be included. Among them, 58 percent had increased serum pNfH levels before diagnosis, based on a threshold of 111 pg/mL representing the 95th percentile of serum pNfH levels in 85 healthy controls. In those patients with ALS for whom longitudinal samples were available before diagnosis I noticed an increase of serum pNfH over time that was significantly higher than seen in patients suffering from another neurodegenerative disease. This indicates that the sharp rise of serum pNfH levels before diagnosis of ALS might be related to motor neuron degeneration. In the second retrospective study, I included patients with a suspicion of ALS whose CSF was collected by local neurologists at regional hospitals. In follow-up these patients were diagnosed with ALS at the tertiary ALS center or were classified as a disease control. When measuring these CSF samples for NfL and pNfH levels, I was able to demonstrate that NF at an early ALS disease stage, i.e. before diagnosis, are significantly increased in patients with ALS compared to disease controls. Subsequently, I showed that when NF were measured by the local neurologist before diagnosis at a tertiary ALS center, the diagnostic delay was three months shorter compared to the delay of patients with ALS whose NF were assessed at time of diagnosis at a tertiary ALS center, at least when covariates including the presence of a genetic mutation and disease progression rate were not taken into account. Finally, as a secondary outcome, I demonstrated that NfL in serum was an independent predictor in a multivariate survival model including other established prognostic markers in ALS.

In conclusion, this PhD project provided new insights on the discriminatory performance of NF in blood at an early disease stage before diagnosis at a tertiary ALS center. Furthermore, it provides the first evidence that NF might decrease the diagnostic delay in patients with ALS when determined at an early disease stage. A multicenter study was initiated to prospectively validate the effect of determining NF at an early disease on the diagnostic delay in ALS.

Date:1 Nov 2016 →  1 Apr 2022
Keywords:Amyotrophic lateral sclerosis, Biomarkers, Neurofilaments
Disciplines:Other biological sciences not elsewhere classified, Neurological and neuromuscular diseases
Project type:PhD project