Supplemental data

Tables and figures

Table e-1 Demographics of the training cohort.

pNfH NfLMotor neuron diseases m/f Age n Conc. (pg/mL) n Conc. (pg/mL)

Amyotrophic lateral sclerosis 90/61 63 (27-84) 151 2319 (114-18089) 151 8902 (370-93574)Primary lateral sclerosis 10/6 62 (53-69) 16 440 (95-3365) 16 2189 (570-10180)

Progressive muscular atrophy 3/0 60 (46-77) 3 692 (259-878) 3 3223 (1802-4489)   

Disease controls            Guillain-Barré syndrome 42/27 57 (8-86) 68 260 (24-13296) 67 1754 (320-38388)Frontotemporal dementia 20/23 66 (48-79) 43 345 (41-1633) 42 4977 (523-12175)

CIDP 24/9 58 (16-75) 33 212 (24-6343) 33 2212 (345-35384)Cognitive problems 4/5 69 (43-81) 9 278 (30-401) 6 1221 (849-2300)

Polyneuropathy 0/7 67 (39-78) 7 773 (125-1467) 7 1510 (1071-6700)Myopathy 3/1 61 (53-67) 4 456 (125-8763) 4 2634 (836-16051)

Sensory polyneuropathy 1/3 63 (30-73) 4 649 (24-18740) 4 3848 (897-53677)Multiple sclerosis 2/1 55 (48-63) 3 317 (24-326) 3 1419 (840-3168)

Leukoencephalopathy 2/1 39 (38-50) 3 92 (29-728) 3 983 (857-1602)Fasciculations 1/2 47 (27-47) 3 127 (125-159) 3 394 (315-875)

Dementia 0/2 66 (51-82) 2 173 (85-262) 2 1381 (626-2137)Paresthesia 1/1 48 (47-49) 2 99 (90-108) 2 1072 (1024-1119)

Myelopathy 1/0 58 1 807 1 7610Alzheimer disease 1/0 52 1 308 1 1976

Epilepsy 1/0 66 1 96 1 1310Stroke 1/0 49 1 753 1 1502

Post-operative neuropathic pain 1/0 40 1 88 1 2148Normal pressure hydrocephalus 0/1 80 1 441 1 2650

Corticobasal degenaration 0/1 69 1 291 1 4059Atypical Parkinson syndrome 1/0 62 1 628 1 6763

Cramps 1/0 54 1 125 1 779Schizophrenia 1/0 27 1 125 1 354

Obstructive sleep apnea 0/1 57 1 94 1 906Chronic pain 0/1 43 1 75 1 752

Prevertebral space tumor 1/0 51 1 9294 1 31653Normal controls 4/6 49 (27-71) 10 108 (24-453) 9 1604 (455-2384)Disease controls 113/92 62 (8-86) 204 269 (24-18740) 198 1995 (315-53677)

Median values and ranges are given. m: male, f: female, pNfH: phosphorylated neurofilament

heavy chain, NfL: neurofilament light chain, Conc.: concentration, CIDP: Chronic

inflammatory demyelinating polyneuropathy.

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Table e-2 Demographics of the disease mimics.

pNfH NfLMotor neuron diseases m/f Age n Conc. (pg/mL) n Conc. (pg/mL)

Amyotrophic lateral sclerosis 90/61 63 (27-84) 151 2319 (114-18089) 151 8902 (370-93574)     

Disease mimics            Hereditary spastic paraplegia 10/7 48 (25-72) 17 222 (24-3752) 17 1075 (143-36597)

Radiculo-, Plexopathy 9/2 54 (37-71) 11 296 (139-1037) 11 1300 (667-4436)Spinal muscular atrophy 2/3 62 (53-67) 5 409 (148-719) 5 1495 (1041-4364)

Multifocal motor neuropathy 3/1 46 (28-68) 4 364 (117-485) 4 934 (692-2281)Cervical stenosis 2/0 64 (63-66) 2 214 (122-306) 2 1284 (744-1824)

Myasthenia gravis 1/1 73 (72-74) 2 507 (297-718) 2 3064 (1364-4765)Kennedy's disease 1/0 75 1 337 1 1442Fewdon syndrome 0/1 25 1 427 1 1441

Paraneoplastic ganglionopathy 0/1 69 1 7049 1 28228Sphenoid meningioma 0/1 55 1 296 1 613Paralysis upper limbs 1/0 52 1 428 1 1979

Primary progressive aphasia 0/1 80 1 556 1 2396Dysphasia 1/0 74 1 768 1 2893

Corticobasal syndrome 0/1 79 1 370 1 3521Functional neurological syndrome 1/0 67 1 245 1 1591

Disease mimics 31/19 57 (25-80) 50 296 (24-7049) 50 1407 (143-36597)

Median values and ranges are given. m: male, f: female, pNfH: phosphorylated neurofilament

heavy chain, NfL: neurofilament light chain, Conc.: concentration.

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Table e-3 Demographics of the validation cohort.

pNfH NfLMotor neuron diseases m/f Age n Conc. (pg/mL) n Conc. (pg/mL)

Amyotrophic lateral sclerosis34/3

564 (36-

85) 69 2413 (196-11233) 69 9817 (748-108909)   

Non-ALS            

Cognitive problems 7/563 (51-

86) 12 333 (79-917) 12 1416 (746-5536)

Polyneuropathy 6/566 (43-

83) 11 460 (108-1080) 11 1754 (816-4824)

Alzheimer’s disease 6/565 (51-

78) 11 312 (184-4601) 11 2326 (1402-37568)

Leukoencephalopathy 2/539 (35-

82) 7 179 (52-865) 7 718 (488-18155)

Fasciculations 4/150 (28-

67) 5 222 (113-614) 5 816 (483-1502)

Multiple sclerosis 3/239 (28-

70) 5 242 (24-653) 5 1098 (735-2222)

Parkinson’s Disease 4/178 (53-

80) 5 1270 (180-1584) 5 4960 (821-7604)

Epilepsy 1/367 (49-

79) 4 368 (61-619) 4 2019 (584-3373)

Unspecified dementia 1/267 (63-

84) 3 285 (95-1332) 3 1395 (900-6855)

Frontotemporal dementia 1/265 (64-

82) 3 690 (268-774) 3 4827 (1282-5669)Idiopathic intracranial

hypertension 1/236 (33-

50) 3 208 (170-453) 3 1681 (788-2829)

Monomyelic atrophy 3/023 (20-

59) 3 72 (24-328) 3 328 (262-1218)Neuralgia 1/2 52 (47-66 3 193 (59-722) 3 989 (722-1353)

Stroke 3/059 (61-

33) 3 188 (141-3899) 3 2912 (772- 18639)

Headache 1/238 (37-

45) 3 158 (27-350) 3 983 (806-1116)

CIDP 2/068 (55-

80) 2 2920 (2344-3496) 2 9065 (7393-10737)

Hydrocephalus 1/178 (74-

81) 2 616 (603-630) 2 2796 (2318-3275)

Myelopathy 1/156 (46-

66) 2 571 (149-995) 2 1593 (526-2660)

Primary progressive aphasia 1/168 (67-

68) 2 668 (585-751) 2 7238 (5380-9095)

Encephalopathy 1/176 (75-

78) 2 562 (437-686) 2 2092 (1893-2291)Hypersomnolence 1/0 51 1 119 1 795

Cerebral palsy 1/0 21 1 77 1 338Facial palsy 0/1 17 1 98 1 491

Glioma 0/1 57 1 63 1 839Ulnar neuropathy 0/1 44 1 160 1 665

Myasthenia gravis 1/0 67 1 651 1 3171Myopathy 1/0 52 1 473 1 1116

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Optic papillitis 0/1 33 1 377 1 715Atypical Parkinson’s syndrome 0/1 71 1 504 1 1625

Multiple system atrophy 0/1 77 1 1605 1 5564Sarcoidosis 0/1 51 1 71 1 1186

Syncope 1/0 71 1 228 1 967Vogt-Koyanagi-Harada syndrome 0/1 57 1 718 1 2946

Normal controls 4/348 (39-

72) 7 105 (24-330) 7 842 (559-1268)

Non-ALS59/5

260 (17-

86)111 333 (24-4601)

111 1502 (262-37568)

Median values and ranges are given. m: male, f: female, pNfH: phosphorylated neurofilament

heavy chain, NfL: neurofilament light chain, Conc.: concentration, CIDP: Chronic

inflammatory demyelinating polyneuropathy.

Table e-4 pNfH cutoff applied on validation cohort

ALS Non-ALS Total>618 pg/mL pNfH 65 28 93<618 pg/mL pNfH 4 83 87

Total 69 111 180

Contingency table of the applied pNfH cutoff on the validation cohort (Fisher’s exact test,

p<0.0001). ALS: amyotrophic lateral sclerosis, pNfH: phosphorylated neurofilament heavy.

Table e-5 NfL cutoff applied on validation cohort

ALS Non-ALS Total>3189 pg/mL NfL 61 17 78<3819 pg/mL NfL 8 94 102

Total 69 111 180

Contingency table of the applied NfL cutoff on the validation cohort (Fisher’s exact test,

p<0.0001). ALS: amyotrophic lateral sclerosis, NfL: neurofilament light.

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Figure e-1 Neurofilaments in different disease groups and correlation with GBS disease

severity scale.

Scatter plots show CSF pNfH (A) and NfL (B) concentrations of amyotrophic lateral sclerosis

(ALS) patients and other disease groups: Primary lateral sclerosis (PLS), Frontal temporal

dementia (FTD), Chronic inflammatory demyelinating polyneuropathy (CIDP), Guillain-

Barré syndrome (GBS) and normal controls (NC). Median and interquartile values are shown

on top of the plot. Red dashed line represents the calculated cut-off value. Kruskal-Wallis test

with Dunn’s post hoc correction (*p<0.0001, **p=0.0002, ***p=0.0062). Scatterplot C and D

show GBS patients (n=58) classified according to their score for the Guillain-Barré disease

severity (GDS) scale by Hughes et al..1 Kruskal-Wallis test with Dunn’s post hoc correction.

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Figure e-2 CSF neurofilaments levels correlate with the extent of upper and lower motor

neuron degeneration

CSF NfL and pNfH levels correlate with the extent of motor neuron disease as shown for CSF

NfL (in red, left Y-axis of every panel) or pNfH (in blue, right Y-axis of every panel) with the

extent of upper motor neuron disease (A) the number of regions with LMN involvement on

EMG according to the revised El Escorial criteria (B), the number of regions with LMN

involvement on EMG according to the revised the Awaji criteria (C) and the number of

regions with LMN involvement assessed by clinical examination. For counting regions with

an aberrant EMG or with clinical LMN involvement, patients with 0 and 1 regions were

grouped into 1, and patients with 3 and 4 affected regions were grouped into group 3, as

indicated on the X-axis.

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Figure e-3 ALS onset and correlation with diagnostic delay and FVC.

ALS patients classified in a scatter plot according to disease onset (spinal versus bulbar) for

pNfH (A) and NfL (B). Median value and interquartile range are shown on top of the plot. p-

value obtained with Mann-Whitney U test. Correlation between diagnostic delay and FVC at

LP (median: 92%, range: 38-144%) with pNfH (C,E) and NfL (D,F).

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Figure e-4 ALS disease progression rate and ROC analysis.

Continuous relation between the progression rate and the CSF pNfH (A) and NfL (B)

concentrations. ALS disease progression rate less than 0.365 (<25% percentile), between

0.365 and 1.056, and more than 1.056 (>75% percentile) points/month was defined as slow,

intermediate and fast disease progression. pNfH (C) and NfL (D) concentrations of slow

progressors were significantly lower than intermediate and fast progressors. Mann-Whitney U

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test (*p<0.005). Receiver operating curves (ROC) to discriminate slow disease progressors

from fast disease progressors for pNfH (E) and NfL (F). AUC: Area under the curve.

Figure e-5 Representative simulated ALS disease curve and correlation between the model

parameters.

Diagram (A) shows the real ALSFRS data (black rhombs) ascertained during follow up visits

and the model curve fitted by the simulation model (grey squares) of one representative ALS

patient. The model calculates the disease parameters D50 = time point when ALSFRS-R

drops by 50% (here: 9.67 month) and dx = time constant representing the disease progression

(here: 4.12) (blue lines). Scatter plot (B) shows the linear correlation between both parameters

(p<0.001) in the training cohort.

Appendix e-1

Development of a simulation model for disease monitoring.

The progression of ALS is not linear 2,3 and we were interested to relate CSF neurofilament

levels taken at different time points to the total disease course. We therefore developed a

model to describe the disease course mathematically for each individual ALS patient which

can be estimated from regularly ascertained ALSFRS-R scores. The model is based on the

observation that after symptom onset the ALSFRS-R does not drop immediately but decays

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slowly first. Then a period of uniform progression follows which is captured in most clinical

trials due to a relatively late inclusion requiring at least laboratory supported ALS according

to EL Escorial / Awaji criteria. With increasing disability, ALSFRS-R seems to reach a

plateau again.

Thus we used a function which describes the transition between two states, i.e. full health to

maximum disease:

y= Amax+( Amin−Amax )

1+e( x−D50 )

dx

or in a simpler form with the known limits of the ALSFRS-R:

y= 48

1+e( x−D50)

dx

We set the following constraints: Amax = 48 (maximum ALSFRS-R before onset); Amin = 0

(theoretical minimum ALSFRS-R); D50 = time point when ALSFRS-R drops to 24; dx =

slope of ALSFRS-R decrease. D50 and dx are unknown and are obtained by iteration from a

minimum set of two ALSFRS-R values sufficiently different from Amax. Based on the

ALSFRS-R scores and the disease duration from onset to ALSFRS-R date from an

independent cohort of 200 patients in our database, we have been able to determine A50 and

dx in 98% of patients using the Microsoft® Excel Add-In Solver tool with dynamic presets

derived from the conventional estimation of ALSFRS-R progression. Here, only patients with

at least 2 ALSFRS-R scores were included, with an average number of 4.4±2.8 (mean±SD)

observations, first ALSFRS-R score of 39.6±5.6 and last score of 28.1±9.6. A representative

ALS disease course of one patient is shown in figure e-4A. The relationship between D50 and

dx was highly linear (figure e-4B), so that using this model the whole disease course can be

described using only one of these two parameters. D50 is more accessible to the end user as

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the number of months passed to reach an ALSFRS-R of 24 along the model based trajectory.

In addition, any sampling taken at any given time point can be correlated to one parameter

which allows the exploration of early prognostic markers.

References

1. Hughes RA, Newsom-Davis JM, Perkin GD, Pierce JM. Controlled trial prednisolone in acute polyneuropathy. Lancet. 1978;2:750–753.

2. Gordon PH, Cheng B, Salachas F, et al. Progression in ALS is not linear but is curvilinear. J Neurol. 2010;257:1713–1717.

3. Proudfoot M, Jones A, Talbot K, Al-Chalabi A, Turner MR. The ALSFRS as an outcome measure in therapeutic trials and its relationship to symptom onset. Amyotroph Lateral Scler Frontotemporal Degener. Epub 2016 Feb 11.:1–12.

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