Objective. Identify preferred neurofilament assays, and clinically validate serum NfL and pNfH as prognostic and potential pharmacodynamic biomarkers relevant to ALS therapy development. 

Methods. Prospective, multi-center, longitudinal observational study of patients with ALS (n=229), primary lateral sclerosis (PLS, n=20) and progressive muscular atrophy (PMA, n=11). Biological specimens were collected, processed and stored according to strict standard operating procedures (SOPs) ¹. Neurofilament assays were performed in a blinded manner by independent contract research organizations (CROs). 

Results. For serum NfL and pNfH measured using the Simoa assay, there were no missing data (i.e. both technical replicates below the lower limit of detection were not encountered). For the Iron Horse and Euroimmun pNfH assays, such missingness was encountered in ~4% and ~10% of serum samples respectively. Mean coefficients of variation (CVs) for pNfH in serum and CSF were ~4-5% and ~2-3% respectively in all assays. Baseline NfL concentration, but not pNfH, predicted the future ALSFRS-R slope and survival. Incorporation of baseline serum NfL into mixed effects models of ALSFRS-R slopes yields an estimated sample size saving of ~8%. Depending on the method used to estimate effect size, use of serum NfL (and perhaps pNfH) as pharmacodynamic biomarkers, instead of the ALSFRS-R slope, yields significantly larger sample size savings.

Conclusions. Serum NfL may be considered a clinically validated prognostic biomarker for ALS. Serum NfL (and perhaps pNfH), quantified using the Simoa assay, have potential utility as pharmacodynamic biomarkers of treatment effect.