Neurodegenerative biomarkers outperform neuroinflammatory biomarkers in amyotrophic lateral sclerosis

Figures & data

Table 1 List of ALS mimics.

Mimic number	Diagnosis
1	Progressive multifocal leukoencephalopathy.
2	Motor neuropathy.
3	Inclusion body myositis, multiple system atrophy (parkinsonian type) and polyneuropathy.
4	Spinal stenosis and neuroborreliosis.
5	Dysarthria and dysphagia of unknown origin.
6	Isolated unilateral hypoglossal nerve paralysis, possibly due to atlanto-occipital dislocation.
7	Polyneuropathy.
8	Vasculitic neuropathy.
9	Degenerative spine (cervical and especially lumbar).
10	Dysarthria and dysphagia due to radiation therapy after base of tongue cancer.
11	Neuroborreliosis.
12	Dysarthria and dysphagia. Halted progression. Unknown cause.
13	Paraneoplastic process resulting in lower motor neuron involvement in one arm. Colon cancer with liver metastases and prostate cancer.
14	Cervical spinal stenosis and polyneuropathy.
15	Spastic paraparesis.
16	Spastic paraparesis.
17	Dysarthria and dysphagia due to base of tongue cancer.
18	Parkinson’s disease.
19	Progressive supranuclear palsy.
20	Polyneuropathy and dementia.
21	Benign fasciculations, axonal polyneuropathy and lumbar degeneration.
22	Familial prion disease (probably fatal familial insomnia).
23	Neuroborreliosis, cervical spondylosis and polyneuropathy.
24	Myelopathy, neuropathy and myopathy partly explained by excessive alcohol consumption.
25	Pronounced degenerative cervical spine.
26	Polymyositis.
27	Myelopathy due to radiation therapy after breast cancer.
28	Parkinson’s disease.
29	Lumbar spondylosis.
30	Drug induced polyneuropathy.
31	Cervical radiculopathy.
32	Benign fasciculations.
33	Multiple system atrophy (parkinsonian type).
34	Benign fasciculations.
35	Unknown myopathy.
36	Adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP).
37	Psychological stress and fatigue.
38	Multiple sclerosis.
39	Benign fasciculations.
40	Benign fasciculations.
41	Bulbar dystonia.
42	Suspected mitochondrial disease (motor neuropathy and leukodystrophy).

Mimics were evaluated at the time of the diagnostic work-up, and their medical records were reviewed again in November 2022, to ensure that they did not convert to ALS or to other motor neuron diseases.

Table 2 Characteristics of study participants and biomarker concentrations at diagnosis (in ng/L).

Characteristics	ALS patients	ALS mimics	Inflammatory neurological controls	Non-inflammatory neurological controls	Healthy controls
n (CSF)	192	42	80	34	23
n (serum)	157	30	80	34	117
Female, n (%)*	86 (44.8%)	10 (23.8%)	41 (51.2%)	20 (58.8%)	9 (39.1%)
Age at first sample, median (IQR)*	67.6 (59.1–74.0)	71.2 (63.1–75.4)	50.0 (46.0–57.0)	51.0 (41.0–63.0)	50.5 (41.0–60.2)
pNfH, median (IQR)	2,124 (1,350–3,092)	411 (258–568)	198 (157–339)	177 (62–330)	194 (143–225)
CSF NfL, median (IQR)	6,300 (3,764–10,234)	1,373 (881–2,407)	706 (404–1,305)	536 (354–990)	455 (314–662)
Serum NfL, median (IQR)	106.0 (74.5–167.6)	24.6 (15.7–52.4)	14.4 (10.4–21.8)	11.3 (7.3–19.5)	20.4 (14.6–27.3)
CHIT1, median (IQR)	7,290 (3,038–17,398)	2,320 (1,277–4,745)	NA	NA	1,295 (631–1,837)
MCP-1, median (IQR)	601 (514–700)	592 (436–685)	NA	NA	423 (347–499)
Site of onset, n (%)	Spinal: 114 (59.4%) Bulbar: 70 (36.5%) Other†: 8 (4.2%)
Months from onset to diagnosis, median (IQR)	11.7 (7.6-16.6)
ALSFRS-R at diagnosis, median (IQR)	39.5 (35.0-43.0)
Progression rate at diagnosis, median (IQR)	0.7 (0.4-1.3)
Longitudinal progression rate, median (IQR)	1.2 (0.7-2.0)
No. of deaths during follow-up, n (%)‡	165 (85.9%)

Note: * Sex and age based on the study participants providing CSF samples, not serum. ^† 4 patients with frontal lobe onset and 4 patients with respiratory onset. ^‡ Death or invasive ventilation. n: number of study participants. IQR: interquartile range. pNfH: phosphorylated neurofilament heavy. NfL: neurofilament light. CHIT1: chitotriosidase-1. MCP-1: monocyte chemoattractant protein-1.

Figure 1 Box plots for biomarker concentrations in ALS patients, ALS mimics, and different control groups. Note: Biomarker concentrations are shown on a logarithmic scale in ng/L. Differences are calculated using Mann–Whitney U test. *** p < 0.001. NS: Not statistically significant. INC: Inflammatory neurological controls. NINC: Non-inflammatory neurological controls. HC: Healthy controls. pNfH: phosphorylated neurofilament heavy. NfL: neurofilament light. CHIT1: chitotriosidase-1. MCP-1: monocyte chemoattractant protein-1.

Table 3 Correlation matrix of biomarkers measured in ALS patients at the time of diagnosis.

	pNfH	CSF NfL	Serum NfL	CHIT1	MCP-1
CSF NfL	0.84***	1.00
- Spinal patients	0.91***
- Bulbar patients	0.73***
Serum NfL	0.75***	0.78***	1.00
- Spinal patients	0.82***	0.83***
- Bulbar patients	0.57***	0.65***
CHIT1	0.43***	0.44***	0.28**	1.00
- Spinal patients	0.54***	0.57***	0.44***
- Bulbar patients	0.22	0.18	−0.14
MCP-1	0.22**	0.28***	0.19	0.28***	1.00
- Spinal patients	0.28**	0.36***	0.27*	0.21*
- Bulbar patients	0.19	0.20	0.01	0.37**

Note: Correlations are estimated using Spearman rank order correlation (rho). For paired CSF: n = 192, including 114 onset spinal patients and 70 bulbar onset patients. For paired CSF-serum: n = 105, including 69 spinal onset patients and 31 bulbar onset patients. Paired biomarkers were sampled on the same day. *p < 0.05, **p < 0.01, and ***p < 0.001. pNfH: phosphorylated neurofilament heavy. NfL: neurofilament light. CHIT1: chitotriosidase-1. MCP-1: monocyte chemoattractant protein-1.

Table 4 Area under the curve (AUC) for differentiating ALS patients from ALS mimics, adjusted for age at measurement, together with optimal cutoffs (estimated with Youden index) and their sensitivity and specificity.

Biomarker	AUC (95% CI)	Cutoff (ng/L)	Sensitivity	Specificity
pNfH	0.92 (0.86–0.98)	726	94.3%	83.3%
CSF NfL	0.86 (0.78–0.94)	2,588	90.6%	76.2%
serum NfL	0.91 (0.84–0.97)	56.4	91.7%	76.7%
CHIT1	0.71 (0.63–0.80)	3,379	75.5%	64.3%
MCP-1	0.56 (0.44–0.67)	535	84.4%	28.6%

Note: pNfH, CSF NfL, CHIT1 and MCP-1 were measured in CSF of the 192 ALS patients and 42 mimics. Serum NfL was measured in 157 ALS patients and 30 mimics. CI: Confidence interval. pNfH: phosphorylated neurofilament heavy. NfL: neurofilament light. CHIT1: chitotriosidase-1. MCP-1: monocyte chemoattractant protein-1.

Figure 2 Predicting longitudinal progression rate and survival among ALS patients. (A-B) Quantile regression models predicting median longitudinal progression rate in relation to biomarker levels (medium and high versus low), in simple (A) and multivariable (B) analyses. (C-D) Cox proportional hazards regression models predicting survival with biomarker levels (medium and high versus low), in simple (C) and multivariable (D) analyses. Note: 192 patients with CSF and 157 patients with serum. In multivariable analyses the following variables were adjusted for: sex, age at diagnosis, BMI at diagnosis, site of onset, El Escorial diagnostic criteria, diagnostic delay, baseline ALSFRS-R, baseline progression rate, and sign of FTD at diagnosis. Biomarker concentrations were divided into tertiles, with low/medium/high concentrations. pNfH: phosphorylated neurofilament heavy. NfL: neurofilament light. CHIT1: chitotriosidase-1. MCP-1: monocyte chemoattractant protein-1.

Figure 3 Temporal patterns per biomarker for each ALS patient from date of first sampling and with mean change (assuming a linear model, from the longitudinal analysis in Table 5), for spinal and bulbar onset patients separately (all measured in ng/L). pNfH: phosphorylated neurofilament heavy. NfL: neurofilament light. CHIT1: chitotriosidase-1. MCP-1: monocyte chemoattractant protein-1.

Table 5 Number of samples (a) and temporal changes of biomarker concentrations per year after ALS diagnosis (b), for all patients and for spinal and bulbar onset patients (mean annual change in ng/L per year).

4 (a) Number of samples	pNfH	CSF NfL	Serum NfL	CHIT1	MCP-1
Sample 1, n	41	43	44	41	41
Sample 2, n	41	43	44	41	41
Sample 3, n	10	11	6	10	10
Sample 4, n	3	3	1	3	3
4 (b) Linear regression	pNfH			CSF NfL			Serum NfL			CHIT1			MCP-1
	Mean annual change	95% CI	R^2	Mean annual change	95% CI	R^2	Mean annual change	95% CI	R^2	Mean annual change	95% CI	R^2	Mean annual change	95% CI	R^2
All patients	−194	−473 − 85	0.02	−163	−1,288 - 962	0.002	1.96	−16.43 − 20.35	0.05	848	−1,964 − 3,659	0.03	23	−19 - 66	0.02
Spinal	−397*	−700 - −95	0.10	−854	−2,080 − 371	0.08	−8.90	−23.87 − 6.08	0.03	−1,071	−4,073 − 1,930	0.01	16	−35 - 67	0.01
Bulbar	494*	85 – 903	0.28	2,329*	558 − 4,100	0.31	43.54*	8.42 − 78.66	0.34	7,932**	2,219 − 13,645	0.18	80	−18 − 178	0.05

Note: Linear regression using cluster-robust standard errors, adjusted for age at diagnosis. R²: coefficient of determination. * p < 0.05, and ** p < 0.01. pNfH: phosphorylated neurofilament heavy. NfL: neurofilament light. CHIT1: chitotriosidase-1. MCP-1: monocyte chemoattractant protein-1.

Data availability statement

Anonymized data will be made available upon reasonable requests to the corresponding author. The data are not publicly available due to privacy or ethical restrictions.