Validation of serum neurofilaments as prognostic and potential pharmacodynamic biomarkers for ALS

Abstract

Objective: To identify preferred neurofilament assays and clinically validate serum neurofilament light (NfL) and phosphorylated neurofilament heavy (pNfH) as prognostic and potential pharmacodynamic biomarkers relevant to amyotrophic lateral sclerosis (ALS) therapy development.

Methods: In this prospective, multicenter, longitudinal observational study of patients with ALS (n = 229), primary lateral sclerosis (n = 20), and progressive muscular atrophy (n = 11), biological specimens were collected, processed, and stored according to strict standard operating procedures (SOPs). Neurofilament assays were performed in a blinded manner by independent contract research organizations.

Results: For serum NfL and pNfH measured using the Simoa assay, there were no missing data (i.e., 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 for NfL in serum and CSF were both ∼3%. Mean coefficients of variation for pNfH in serum and CSF were ∼4%-5% and ∼2%-3%, respectively, in all assays. Baseline serum NfL concentration, but not pNfH, predicted the future Revised ALS Functional Rating Scale (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, has potential utility as a pharmacodynamic biomarker of treatment effect.

Figure 1. Kaplan-Meier survival curves

Kaplan-Meier survival curves show the prognostic value of (A) C9ORF72 repeat expansion, (B) ∆FRS (dichotomized at the median, 0.62 points/month), (C) baseline serum neurofilament light (NfL) (dichotomized at the median, 17 pg/mL), and (D) baseline serum phosphorylated neurofilament heavy (pNfH) (dichotomized at the median, 67 pg/mL). The presence of a C9ORF72 repeat expansion, higher ∆FRS, higher baseline serum NfL, and higher baseline serum pNfH are shown in red.

Figure 2. Longitudinal trajectories of serum neurofilaments

(A) Spaghetti plot of log-transformed neurofilament light (NfL) level, (B) spaghetti plot of log-transformed phosphorylated neurofilament heavy (pNfH) level, and (C) boxplot of the distribution of NfL and pNfH slopes. Slope estimates were obtained from a linear model with log-transformed neurofilament level as the outcome and time as the independent variable.

Figure 3. Change in serum neurofilaments over time, as compared to baseline level

(A) Change from baseline in serum neurofilament light (NfL) (pg/mL) and (B) change from baseline in serum phosphorylated neurofilament heavy (pNfH) (pg/mL).