Amyotrophic Lateral Sclerosis and Frontotemporal

Degeneration

ISSN: 2167-8421 (Print) 2167-9223 (Online) Journal homepage: http://www.tandfonline.com/loi/iafd20

Phosphoneurofilament heavy chain and vascular

endothelial growth factor as cerebrospinal fluid
biomarkers for ALS

Margarida Gonçalves, Mamede De Carvalho, Cristina Peixoto, Paula Alves,

Carmo Barreto, Abel Oliva, Susana Pinto, Ana Laborinho-Pronto, Marta
Gromicho & Júlia Costa

To cite this article: Margarida Gonçalves, Mamede De Carvalho, Cristina Peixoto, Paula Alves,
Carmo Barreto, Abel Oliva, Susana Pinto, Ana Laborinho-Pronto, Marta Gromicho & Júlia
Costa (2016): Phosphoneurofilament heavy chain and vascular endothelial growth factor as
cerebrospinal fluid biomarkers for ALS, Amyotrophic Lateral Sclerosis and Frontotemporal
Degeneration, DOI: 10.1080/21678421.2016.1212894

To link to this article: http://dx.doi.org/10.1080/21678421.2016.1212894

View supplementary material

Published online: 11 Aug 2016.

Submit your article to this journal

View related articles

View Crossmark data

Full Terms & Conditions of access and use can be found at

http://www.tandfonline.com/action/journalInformation?journalCode=iafd20

Download by: [Flinders University of South Australia] Date: 16 August 2016, At: 15:50
Amyotrophic Lateral Sclerosis and Frontotemporal Degeneration, 2016; 1–3

REPORT

Phosphoneurofilament heavy chain and vascular endothelial growth

factor as cerebrospinal fluid biomarkers for ALS

MARGARIDA GONÇALVES1, MAMEDE DE CARVALHO2,3, CRISTINA PEIXOTO1,4,

PAULA ALVES1,2,3,4, CARMO BARRETO1,4, ABEL OLIVA1,4, SUSANA PINTO3,
ANA LABORINHO-PRONTO3, MARTA GROMICHO3 & JÚLIA COSTA1
1
Instituto de Tecnologia Quı́mica e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal,
2
Hospital de Santa Maria, Centro Hospitalar Lisboa-Norte, Lisbon, Portugal, 3Instituto de Medicina Molecular,
Faculty of Medicine, University of Lisbon, Portugal, and 4Instituto de Biologia Experimental e Tecnológica, Oeiras,
Portugal

Introduction
The most promising molecular markers for ALS are
neurofilaments, namely phosphorylated neurofila-
ment heavy chain (pNFH). In ALS, increased level
of pNFH has been reported in the CSF (1,2), in
particular in fast progressors (3), with a negative
correlation with disease duration (4).
VEGF has been associated with ALS pathogen-
esis (5). Decreased levels were described in the CSF
and plasma of ALS patients (6), but the results are
inconsistent (1).
Here we explore the diagnostic potential of
combining pNFH and VEGF as ALS biomarker.
Population and methods
Groups of 36 ALS patients with probable or definite
disease (revised El Escorial criteria) and 15 patients
with other neurological disorders were studied
(Table I). All patients gave written consent; the
protocol was approved by the ethics committee.
CSF was collected by lumbar puncture into
polypropylene tubes and kept at 80  C (7). pNFH
(8) and VEGF (9) were quantified by ELISA
(BioVendor Research and Diagnostic Products,
Czech Republic, no. RD191138300R and R&D
Systems, QuantiGlo, no. QVE00B, respectively).
Statistical analysis was performed using
GraphPad Prism 6.
Results
pNFH level was higher in ALS patients than in
controls (median 1739 pg/ml vs. 417.4 pg/ml,
respectively; p ¼ 0.0020) (Table I) (Supplementary
Figure 1A). ROC analysis showed an area under the
curve (AUC) of 0.7704 (Figure 1). A cut-off value
of 554 pg/ml for pNFH generated sensitivity of
75% and specificity of 60% were recorded. pNFH
levels correlated negatively with disease duration
and positively with rate of functional decline
[(40 - ALSFRS)/disease duration (months)]
(Supplementary Figure 1B, C). Higher pNFH
levels were observed in patients with short disease
duration (51 year) (Supplementary Figure 1D).
VEGF levels were lower in patients than in
disease controls (median 6.7 pg/ml vs. 10.9 pg/ml,
p ¼ 0.0356) (Table I) (Supplementary Figure 2).
ROC analysis showed AUC 0.6870 (Figure 1).
pNFH/VEGF ratio in ALS patients was higher
than in controls (median 224.3 and 54.6, respect-
ively) (p50.0001) (Supplementary Figure 3A).
ROC analysis showed AUC 0.8481 (Figure 1).
A cut-off value of 75.02 for pNFH/VEGF ratio
increased the sensitivity (83.3%) and the specificity
(80.0%). Significant correlations between pNFH/
VEGF ratio and disease duration (negative), rate of
functional decline or ALSFRS-R (positive) were
found (Supplementary Figure 3B–D). Furthermore,
ALS patients with short disease duration displayed

Correspondence: J. Costa, ITQB NOVA, Avenida República, 2780-157 Oeiras, Portugal. E-mail: jcosta@itqb.unl.pt

(Received 22 December 2015; revised 2 June 2016; accepted 21 June 2016)

ISSN 2167-8421 print/ISSN 2167-9223 online ß 2016 World Federation of Neurology on behalf of the Research Group on Motor Neuron Diseases
DOI: 10.1080/21678421.2016.1212894
 2

Table I. Demographic and clinical features of the patients included.

Gender Disease duration FVC Onset site Total protein

(M/F) Age (years) (years) ALS/FRS (% predicted value) (bulbar/spinal) (mg/mL) pNFH [VEGF] (pg/mL)
Controls 7/8 64.1 (55.3–67.5) – – – – 0.48 (0.39–0.58) 417.4 (243.5–843.9)* [10.9 (7.7–13.0)]#
M. Gonçalves et al.

ALS 26/10 56.0 (49.1–63.2) 0.87 (0.58–1.92) 36 (34–38) 104 (90–117)a 4/31 0.48 (0.37–0.58) 1739 (490.5–3750)* [6.7 (1.3–10.5)]#

Notes:
1. The control group was formed by patients with the final diagnosis of chronic inflammatory demyelinating neuropathy, hereditary demyelinating polyneuropathy, diabetic neuropathy, axonal polyneuropathy,
brachial plexitis, myelitis, atypical headache, normal pressure hydrocephalus, undetermined spinal cord lesion, multiple sclerosis and multiple system atrophy.
2. Demographic and clinical information presented as median and between brackets the interquartile range IQR (25% percentile–75% percentile).
3. Testing for normality was performed using the D’Agostino & Pearson omnibus normality test. The non-parametric Mann–Whitney test was applied to determine statistical differences between two groups.
p values 50.05 were considered as statistically significant.
4. *p ¼ 0.0020; #p ¼ 0.0356.
a
Values calculated for a subgroup of 30 patients with measured FVC.

Figure 3E).

Discussion
comparing controls and ALS patients.

lower level of VEGF in ALS CSF.

To our knowledge pNFH and VEGF are not
ment with other reports (1–3). Concerning diag-
nostic specificity and sensitivity our values are
progression and disease duration, which is in agree-
We have observed that pNFH levels were increased

diagnostic value of combining both biomarkers,

interdependent; however, we have explored the
slightly lower than those reported elsewhere (2),

analysed and number of subjects. On the other

which might be due to the different populations

hand, in agreement with others (6) we observed a

in the CSF and correlated with the rate of disease
higher levels of pNFH/VEGF ratio (Supplementary
Figure 1. ROC curves for pNFH, VEGF and pNFH/VEGF

both abnormal in ALS (1,2,6). We found that this
index has a potentially higher diagnostic accuracy
than the single evaluation of pNFH. Higher CSF
pNFH/VEGF ratio was associated with a more
aggressive disease course and respiratory failure,
probably explained by the combination of rapid
motor neurons death and deregulation in the syn-
thesis of VEGF in ALS.
Our results underscore the potential of CSF
pNFH as a diagnostic and prognostic biomarker in
ALS and combined analysis of pNFH/VEGF could
provide higher diagnostic yield. This should be
investigated in a larger population of patients.
Supplementary material for this article is avail-
able via the supplementary tab on the article’s online
page at http://dx.doi.org/10.1080/21678421.2016.
1212894.
Acknowledgements
We wish to thank the participants in this study. This
work was supported by EU JPND project SOPHIA,
Fundação para a Ciência e a Tecnologia (FCT)
[JPND/0003/2011], Portugal; Euronanomed 2
ERA-NET project GlioEx, FCT [ENMed/0001/
2013]; iNOVA4Health, UI FCT/Ministério da
Educação e Ciência [UID/Multi/04462/2013].
Declaration of interest
The authors report no conflicts of interest.
Supplementary material available online
pNFH and VEGF as CSF biomarkers for ALS 3
References
1. Costa J, de Carvalho M. Emerging molecular biomarker
targets for amyotrophic lateral sclerosis. Clin Chim Acta.
2016;455:7–14.
2. Steinacker P, Feneberg E, Weishaupt J, Brettschneider J,
Tumani H, Andersen PM, et al. Neurofilaments in the
diagnosis of motor neuron diseases: a prospective study on
455 patients. J Neurol Neurosurg Psychiatry. 2016;87:12–
20.
3. Boylan KB, Glass JD, Crook JE, Yang C, Thomas CS,
Desaro P, et al. Phosphorylated neurofilament heavy subunit
(pNF-H) in peripheral blood and CSF as a potential
prognostic biomarker in amyotrophic lateral sclerosis.
J Neurol Neurosurg Psychiatry. 2013;84:467–72.
4. Ganesalingam J, An J, Bowser R, Andersen PM, Shaw CE.
pNfH is a promising biomarker for ALS. Amyotroph Lateral
Scler Frontotemporal Degener. 2013;14:146–9.
5. Oosthuyse B, Moons L, Storkebaum E, Beck H, Nuyens D,
Brusselmans K, et al. Deletion of the hypoxia-response element
in the vascular endothelial growth factor promoter causes
motor neuron degeneration. Nat Genet. 2001;28:131–8.
6. Devos D, Moreau C, Lassalle P, Perez T, De Seze J,
Brunaud-Danel V, et al. Low levels of the vascular endothe-
lial growth factor in CSF from early ALS patients.
Neurology. 2004;62:2127–9.
7. Teunissen CE, Petzold A, Bennett JL, Berven FS, Brundin
L, Comabella M, et al. A consensus protocol for the
standardization of cerebrospinal fluid collection and bio-
banking. Neurology. 2009;73:1914–22.
8. Goncalves M, Tillack L, de Carvalho M, Pinto S, Conradt
HS, Costa J. Phosphoneurofilament heavy chain and N-
glycomics from the cerebrospinal fluid in amyotrophic lateral
sclerosis. Clin Chim Acta. 2015;438:342–9.
9. Carilho R, de Carvalho M, Swash M, Pinto S, Pinto A,
Costa J. Vascular endothelial growth factor and amyotrophic
lateral sclerosis: the interplay with exercise and non-invasive
ventilation. Muscle Nerve. 2014;49:545–50.