TYPE Review PUBLISHED 01 November 2022 DOI 10.3389/fnagi.2022.1034684 Neurofilaments contribution in OPEN ACCESS clinic: state of the art EDITED BY Miriam Sklerov, Constance Delaby 1,2 , Olivier Bousiges 3 , Damien Bouvier 4 , University of North Carolina at Chapel Hill, United States Catherine Fillée 5 , Anthony Fourier 6 , Etienne Mondésert 1 , REVIEWED BY Nicolas Nezry 7 , Souheil Omar 8 , Isabelle Quadrio 6 , Chiara Fenoglio, University of Milan, Italy Benoit Rucheton 9 , Susanna Schraen-Maschke 7 , Francesca Gilli, Dartmouth College, United States Vincent van Pesch 10 , Stéphanie Vicca 11 , Sylvain Lehmann 1 * *CORRESPONDENCE and Aurelie Bedel 12 Sylvain Lehmann 1 Université de Montpellier, IRMB, INM, INSERM, CHU de Montpellier, Laboratoire s-lehmann@chu-montpellier.fr Biochimie-Protéomique clinique, Montpellier, France, 2 Sant Pau Memory Unit, Hospital de la Santa SPECIALTY SECTION Creu i Sant Pau—Biomedical Research Institute Sant Pau—Universitat Autònoma de Barcelona, This article was submitted to Barcelona, Spain, 3 Laboratoire de biochimie et biologie moléculaire (LBBM)—Pôle de biologie Cellular and Molecular Mechanisms of Hôpital de Hautepierre—CHU de Strasbourg, CNRS, laboratoire ICube UMR 7357 et FMTS Brain-aging, (Fédération de Médecine Translationnelle de Strasbourg), équipe IMIS, Strasbourg, France, 4 Service a section of the journal de Biochimie et Génétique Moléculaire, CHU de Clermont-Ferrand, Clermont-Ferrand, France, 5 Frontiers in Aging Neuroscience Cliniques universitaires Saint-Luc UCLouvain, Service de Biochimie Médicale, Brussels, Belgium, 6 Biochimie et Biologie Moléculaire—LBMMS, Unité de diagnostic des pathologies dégénératives, RECEIVED 01September 2022 Centre de Biologie et Pathologie Est, Groupement Hospitalier Est, Lyon, France, 7 Univ. Lille, Inserm, ACCEPTED 10 October 2022 CHU Lille, UMR-S-U1172, LiCEND, Lille Neuroscience & Cognition, LabEx DISTALZ, Lille, France, PUBLISHED 01 November 2022 8 Laboratoire de biologie médicale de l’Institut de Neurologie de Tunis, Tunis, Tunisia, 9 Laboratoire CITATION de Biologie, Institut Bergonié, Bordeaux, France, 10 Cliniques universitaires Saint-Luc UCLouvain, Delaby C, Bousiges O, Bouvier D, Service de Neurologie, Brussels, Belgium, 11 Hôpital Necker-Enfants malades, Paris, Laboratoire de Fillée C, Fourier A, Mondésert E, Biochimie générale, DMU BioPhyGen, AP-HP.Centre—Université de Paris, Paris, France, 12 Service de Nezry N, Omar S, Quadrio I, Biochimie, CHU Pellegrin, Bordeaux, France Rucheton B, Schraen-Maschke S, van Pesch V, Vicca S, Lehmann S and Bedel A (2022) Neurofilaments Neurological biomarkers are particularly valuable to clinicians as they can contribution in clinic: state of the art. be used for diagnosis, prognosis, or response to treatment. This field of Front. Aging Neurosci. 14:1034684. doi: 10.3389/fnagi.2022.1034684 neurology has evolved considerably in recent years with the improvement COPYRIGHT of analytical methods, allowing the detection of biomarkers not only in © 2022 Delaby, Bousiges, Bouvier, cerebrospinal fluid (CSF) but also in less invasive fluids like blood. These Fillée, Fourier, Mondésert, Nezry, Omar, advances greatly facilitate the repeated quantification of biomarkers, including Quadrio, Rucheton, Schraen-Maschke, van Pesch, Vicca, Lehmann and Bedel. at asymptomatic stages of the disease. Among the various informative This is an open-access article biomarkers of neurological disorders, neurofilaments (NfL) have proven to be distributed under the terms of the Creative Commons Attribution License of particular interest in many contexts, such as neurodegenerative diseases, (CC BY). The use, distribution or traumatic brain injury, multiple sclerosis, stroke, and cancer. Here we discuss reproduction in other forums is these different pathologies and the potential value of NfL assay in the permitted, provided the original author(s) and the copyright owner(s) management of these patients, both for diagnosis and prognosis. We also are credited and that the original describe the added value of NfL compared to other biomarkers currently used publication in this journal is cited, in accordance with accepted academic to monitor the diseases described in this review. practice. No use, distribution or reproduction is permitted which does KEYWORDS not comply with these terms. neurofilament, biomarkers, neurological and neurodegenerative diseases, cut-off, biological fluids Abbreviations: AD, Alzheimer’s disease; AUC, area under the curve; CBD, corticobasal degeneration; CJD, Creutzfeldt Jakob disease; CNS, central nervous system; CSF, cerebrospinal fluid; FTD, fronto-temporal dementia; Gd+ , gadolinium lesions; GRN, progranulin; HTT, hungtintin; ICANS, Immune Cell Associated Neurotoxicity Syndrome; LBD, Lewy body disease; MCI, mild cognitive impairment; MS, multiple sclerosis; MSA, multisystem atrophy; NfL, neurofilament light; OCB, oligoclonal bande in the CSF; PD, Parkinson’s disease; PPD, primary psychiatric disorders; PS, Parkinson’s syndromes; PSP, progressive supranuclear palsy; ROC, receiver operating characteristic; TBI, traumatic brain injury. Frontiers in Aging Neuroscience 01 frontiersin.org Delaby et al. 10.3389/fnagi.2022.1034684 Introduction NfL and neurodegenerative diseases Neurofilaments (Nf) belong to the family of intermediate Alzheimer’s disease filaments and their localization is exclusively neuronal. Nf differ from other types of intermediate filaments by the complexity Background and state of the art of their structure and the composition of their subunits. Three subunits of Nf can be identified (and differentiated Alzheimer’s disease (AD) is the most prevalent cognitive on SDS gel according to their molecular weight): NfH neurodegenerative disease with approximately 900,000 people (heavy chain, 200 kDa), NfM (medium chain, 160 kDa), affected in France and more than 225,000 new cases per and NfL (light chain, 68 kDa; Yuan et al., 2017; Figure 1). year (Helmer et al., 2006). From a pathophysiological point Each protein subunit consists of a globular head, an alpha of view, AD is characterized by neurodegeneration due to helix portion and a C-terminal domain of variable length, the development of neurofibrillary tangles (NFTs, formed by thus determining the molecular weight of each of these aggregates of hyperphosphorylated tau protein) and amyloid subunits (Yuan et al., 2006, 2012). Nf are involved in plaques [formed by agglomerates of amyloid peptides Aβ the radial growth of the axon during neuron development produced by the cleavage of amyloid precursor protein (APP)]. and in the maintenance of its structure and diameter, The diagnosis of AD is based on clinical examination, interview which are necessary for the transmission of nerve impulses. with the patient and relatives, neuropsychological tests (e.g., Nf are also involved in the organization and docking of mini-mental state examination, MMSE), imaging (structural the different components of the axon to the microtubule Magnetic Resonance Imaging, MRI, FDG-PET, amyloid PET), network. and finally lumbar puncture with the quantification of Because of their enrichment in axons and their release CSF biomarkers Aβ42, Aβ40 (together with the ratio of into blood and cerebrospinal fluid (CSF) during neuronal Aβ42/Aβ40), total tau (t-tau), and phosphorylated tau on damage, the measurement of NfL in these biological fluids as threonine 181 (p-tau). These CSF biomarkers perform well potential biomarkers of axonal damage, axonal loss and neuronal in the diagnosis of AD at the dementia stage (Hansson death raises many hopes in terms of diagnosis and prognosis et al., 2006) but also allow diagnosis of the disease at (Figure 2). early stage (MCI, mild cognitive impairment; Jack et al., NfL thus appears as one of the biomarkers of 2013). neurodegeneration, non-specific, but indirectly involved The number of publications related to the determination of in the pathogenesis of many neurological diseases. These NfL in AD is very high, both in CSF and in blood (546 referenced biomarkers are therefore being actively investigated and several in Pubmed on 01/06/2022). As for almost all neurodegenerative assays for their quantification are currently available: the first diseases, NfL is increased in blood and CSF of AD patients one is based on an ELISA immunoassay (marketed by Uman (Gaetani et al., 2019; Mattsson et al., 2019; Delaby et al., Diagnostic, Umeå, Sweden), the second one is based on Simoa 2020). Thus, AD patients can be differentiated from healthy (single molecule array) technology and is implemented on controls with very good accuracy, by measuring NfL in both the Quanterixr (Billerica, MA, USA) SimoaTM device, and the CSF (Lista et al., 2017) and the blood (Forgrave et al., the third one is an immunoassay, based on a microfluidic 2019; Mattsson et al., 2019). However, several studies reported approach, which can be implemented on the EllaTM device absence of direct correlation between NfL concentrations in from the Protein Simpler company. These three approaches CSF or plasma and amyloid pathology (Aβ+ and Aβ−) as use the same detection antibodies. A fourth approach, based assessed by amyloid PET (Dhiman et al., 2020; Verberk et al., on electro-chemiluminescence, can be implemented on the 2020). This suggests that changes in NfL concentration are Mesoscale Discovery (MSD) devices (Rockville, MD, USA). independent of amyloid pathology in AD, whereas they are However, the cut-offs for NfL measurement remain to be correlated with neurodegeneration and tauopathy (Dhiman homogenized, or even defined, depending on the method, the et al., 2020). clinical contexts and the physiological parameters influencing the concentrations such as age, body mass index or renal function (see Table 1). The objective of this article is to Diagnostic and prognostic values overview the interest of NfL in various neurodegenerative diseases and in other contexts of neurological impairment. This almost systematic increase of NfL in neurodegenerative We present the prognostic or diagnostic implications of diseases limits the diagnostic interest of this biomarker in AD. measuring this biomarker in biological fluids. We also However, it is noteworthy that the increase in NfL levels (both discuss the place and informative value of NfL in relation in blood and CSF) of AD patients remains more moderate than to other biomarkers commonly used to monitor the described in most other neurodegenerative diseases (Gaetani et al., 2019). pathologies. NfL may be differentially involved in the pathological processes Frontiers in Aging Neuroscience 02 frontiersin.org Delaby et al. 10.3389/fnagi.2022.1034684 FIGURE 1 Structure and organization of neurofilaments (Nf), adapted from Gaetani et al. (2019). FIGURE 2 NfL release after axonal damages. NfL are detectable in CSF and blood. of these disorders, in particular, neuronal death may be greater Changes in blood NfL appear to precede the first clinical in some of them than in AD (which is chronic), thus generating manifestations of AD by about 16 years, as shown in longitudinal higher levels of sNfL. In addition, pathologies associated with studies of AD mutation carriers (Preische et al., 2019): NfL motor neuron death, such as ALS, might also exhibit higher could thus be used to monitor subjects with genetic risk levels of NfL than others. factors for AD. Indeed, serum NfL has been shown to correlate Thus, for frontal forms of AD (e.g., primary progressive with the estimated number of years before symptoms appear logopenic aphasia), the measurement of NfL could be of in carriers of autosomal dominant AD mutations (Sánchez- interest in the differential diagnosis with other primary Valle et al., 2018) or in Down syndrome (Fortea et al., 2018, progressive aphasias and, more generally, with frontotemporal 2020). lobar degeneration (FTLD), where NfL levels are higher Elevated CSF NfL is also associated with faster brain atrophy (Pijnenburg et al., 2015; Disanto et al., 2016; Steinacker and cognitive decline in AD patients followed up longitudinally et al., 2017a; Paterson et al., 2018; Lleó A et al., 2019). (Zetterberg et al., 2016; Osborn et al., 2019; Dhiman et al., The elevated levels of NfL in prion diseases would also 2020). Therefore, elevated CSF NfL in the early clinical stages allow a differential diagnosis between prions and AD of AD may predict accelerated cognitive decline and conversion (Steinacker et al., 2016; Thompson et al., 2018). to dementia in AD (Zetterberg et al., 2016; Lim et al., 2021). Frontiers in Aging Neuroscience 03 frontiersin.org Delaby et al. 10.3389/fnagi.2022.1034684 TABLE 1 Diagnostic/prognostic blood NfL’s threshold in neurological pathological context, according to various technological approaches. Clinical contexts sNfL cut-off Technological Reference value (pg/ml) approach used for cut-off definition Neurodegenerative Diseases Alzheimer’s Disease (AD) AD vs. control subjects 37.0 Simoa Ashton et al. (2021) and Smirnov et al. (2022) 19.3 Ella Oeckl et al. (2022) 26.6 MSD Gaiottino et al. (2013) AD vs. FTD 42.7 Ella Oeckl et al. (2022) Parkinson’s syndrome (PS) PS vs. control subjects 21.0 Simoa Zhang et al. (2022) PD vs. atypical forms of parkinsonism (MSA, PSP) 14.8 Simoa Marques et al. (2019) PD vs. atypical forms of parkinsonism (MSA, PSP, CBD) 17.2 Simoa Quadalti et al. (2021) PD vs. PSP/CBD 16.6 Simoa Quadalti et al. (2021) PD vs. MSA 17.2 Simoa Quadalti et al. (2021) Atypical forms of parkinsonism vs. control subjects 13.6 Simoa Marques et al. (2019) Fronto-temporal dementia Fronto-temporal dementia vs. PPD 19.5–33.0 Simoa Forgrave et al. (2019) Amyotrophic lateral sclerosis (ALS) ALS vs. control subjects 500 Elisa Shi et al. (2022) ALS vs. non-ALS patients 32.7 Simoa Vacchiano et al. (2021) Multiple sclerosis (MS) OCB/Gd + occurence 31.0 Simoa Bittner et al. (2020) Prognosis clinical disease activity 1.5 (z-score) Simoa Benkert et al. (2022) Neurological Damages Traumatic brain injury (TBI) TBI diagnosis 20.0 Simoa Thelin et al. (2017) Prediction of intracranial lesions 8.38 Simoa Kahouadji et al. (2022) Stroke Stroke prognosis 46.12 Simoa Wang Z. et al. (2021) Oncology Response to cancer therapy 28.0–60.0 Simoa Schoeberl et al. (2022) Brain metastasis diagnosis >22.0 Simoa Kim et al. (2021) Chemotherapy toxicity >36.0 Simoa Huehnchen et al. (2022) ICANS occurence 75.0 Simoa Schoeberl et al. (2022) AD, Alzheimer’s disease; PS, Parkinson’s syndromes; PD, Parkinson’s disease; FTD, fronto-temporal dementia; MSA, multisystem atrophy; PSP, progressive supranuclear palsy; CBD, corticobasal degeneration; PPD, primary psychiatric disorders; TBI, traumatic brain injury; MS, multiple sclerosis; ICANS, Immune Cell Associated Neurotoxicity Syndrome; OCB, oligoclonal bande in the CSF; Gd + , gadolinium lesions. Thus, NfL assay could serve as a prognostic marker of worsening region immunohistochemistry techniques; Smirnov et al., 2022). cognitive function in AD. Using Ella technique, thresholds of 19.3 pg/ml and 42.7 pg/ml were reported to differentiate AD from controls and AD from Position of NfL compared to other biomarkers FTD, respectively (Oeckl et al., 2022). When using the MSD and cut-off value technique, a threshold of 26.6 pg/ml was found to discriminate AD from controls (Gaiottino et al., 2013). These cut-offs are NfL measurement in CSF does not seem to provide further not comparable since the techniques are not standardized, but information to the amyloid biomarkers, t-tau and p-tau, already all studies agree in finding areas under the curve in the range used to predict conversion to dementia in AD (Fortea et al., of 0.7, which is insufficient for diagnostic use. But these blood 2018). thresholds might however be of interest for a first screening Measurement of NfL in blood could be useful as a first line, step. In addition, age-dependent cut-offs should increase the i.e., screening test for AD and other neurodegenerative diseases. performance of the test. Finally, the combination of this test This would indeed be a straightforward implement due to the with blood measurements of amyloid and tau could also be less invasive nature of blood collection compared to lumbar interesting. puncture. Thresholds for the use of the blood NfL for diagnosis have been proposed in some studies, although the areas under the Parkinson’s syndromes and curve never reach 0.8. Two large studies found similar cut-off synucleopathies values with the Simoa technique, around 37 pg/ml: the first study being based on the comparison of amyloid+ vs. amyloid− Background and state of the art subjects (defined on the basis of Aβ1–42 assays in CSF or amyloid PET imaging, N = 805; Ashton et al., 2021) and the second being Parkinson’s disease (PD) is the second most common based on a pathology cohort (N = 312) comparing Braak 0_II neurodegenerative disease in France (after AD) and the first vs. Thal 4–5 and Braak V–VI subjects (Braak being the scale leading cause of motor disorders. In France, 150,000 people measuring DNF pathology and Thal amyloid pathology by brain are affected and 25,000 new cases are diagnosed every Frontiers in Aging Neuroscience 04 frontiersin.org Delaby et al. 10.3389/fnagi.2022.1034684 year (Elbaz et al., 2016). PD is due to the progressive loss 2020). In PSP, higher baseline NfL values in CSF and blood of dopaminergic neurons of the nigro-striatal pathway in appear to be associated with accelerated worsening of motor association with the formation of Lewy bodies corresponding and cognitive symptoms (Rojas et al., 2016). Furthermore, to alpha-synuclein aggregation (Elbaz et al., 2016). Other some patients with HOI evolve to synucleinopathies with motor neurodegenerative pathologies present alpha-synuclein or cognitive impairment such as PD, LBD or MSA and the aggregates, such as Lewy body disease (LBD), Parkinson’s level of NfL in the CSF could help predict this conversion dementia, multisystem atrophy (MSA) or idiopathic orthostatic (Singer et al., 2021). hypotension (IOH). All these pathologies are part of the so-called synucleinopathies. The differential diagnosis between these synucleinopathies is complex and is compounded Position of NfL compared to other biomarkers by diseases with atypical Parkinsonian syndromes, such and cut-off value as progressive supranuclear palsy (PSP) or corticobasal degeneration (CBD), making the differential diagnosis NfL assessed alone appears to have modest performance sometimes challenging. To date, no specific biomarkers in predicting conversion from normal cognition to MCI or can be used for such differential diagnosis. parkinsonian dementia individually, suggesting that NfL should The determination of NfL, both in CSF and in blood, has be integrated into a multi-marker panel to improve prediction been intensively studied in the context of PD (156 results in of clinical conversion to dementia. Some studies propose to Pubmed as of 01/06/2022). Interestingly, NfL levels do not seem incorporate, for example, Aβ1–42 assay in CSF for this purpose to increase in the CSF of PD patients and several publications (Bäckström et al., 2015). Some articles have attempted to have reported similar levels to those of healthy controls (Gaetani determine a threshold value using ROC curve to support the et al., 2019). differential diagnosis of parkinsonian syndromes. Interestingly, all these studies achieved NfL quantification using the Simoa technique. Thus, a cut-off value of NfL at 21 pg/ml in plasma Diagnostic and prognostic values would allow a satisfactory discrimination of MSA patients and healthy subjects, with a sensitivity of 81% and a specificity While NfL levels are not increased in either blood or CSF of of 93% (AUC = 0.912; Zhang et al., 2022). Similarly, NfL parkinsonian patients, it is noteworthy that they rise in atypical cut-off value in serum of 14.8 pg/ml (assessed by the Simoa forms of parkinsonian syndromes such as PSP, MSA, and CBD approach) allows a clear discrimination (AUC = 0.91) between in both CSF (Bäckström et al., 2015; Herbert et al., 2015) and PD patients and those with atypical forms of parkinsonism (MSA blood (Hansson et al., 2017; Lin et al., 2018; Marques et al., and PSP), yielding a sensitivity of 86% and a specificity of 85% 2019; Ashton et al., 2021). This discrepancy could be due in (Marques et al., 2019). Thus, among patients whose serum NfL part to milder and less extensive axonal degeneration in PD concentration is above the cut-off value, the probability of having than in these atypical forms of parkinsonian syndromes. NfL an atypical parkinsonism syndrome is 76% (positive predictive levels are indeed related to severity, motor neurons scores, and value), and patients whose serum NfL amount is below this stratification of PD. cut-off value have a 92% probability of having PD (negative Of note, while LBD patients (or those with parkinsonian predictive value). In the same study, a cut-off value of 13.6 ng/L dementia) also have significant elevated levels of NfL in CSF was used to differentiate patients with atypical parkinsonism compared to healthy and PD patients, they have lower levels from control subjects with a sensitivity of 93% and a specificity than PSP, MSA, and CBD patients (Hall et al., 2012). On the of 71% (AUC = 0.88; Marques et al., 2019). One study further other hand, NfL levels in CSF are not very specific and do detailed the differences in threshold values (assessed by Simoa) not differentiate LBD and AD patients (de Jong et al., 2007). according to parkinsonian syndromes (Quadalti et al., 2021): The assessment of NfL in CSF or blood could thus be useful for example, to differentiate PD from MSA, the best cut-off for the differential diagnosis of PD vs. atypical parkinsonian value for plasma NfL is at 17.2 pg/ml for a sensitivity of 90.3% syndromes. and specificity of 96.4% (AUC = 0.972), and to differentiate a The prognostic value of NfL has been evaluated in PD group of PD patients from a group of PSP/DCB patients, the and PSP, but no data are available on its prognostic value in optimal cut-off value is 16.6 pg/ml with a sensitivity of 88.7% MSA and CBD. In PD, baseline CSF NfL values are associated and specificity of 87.8% (AUC = 0.936). When grouping atypical with mean change per year in Dementia rating scale scores parkinsonian syndromes (MSA, PSP, DCB) vs. PD, the cut-off (Olsson et al., 2019; Aamodt et al., 2021). The determination value of plasma NfL is 17.2 pg/ml for a sensitivity of 90.3% of NfL in CSF (Bäckström et al., 2015) and blood (Aamodt and a specificity of 91.7% (Quadalti et al., 2021). These results et al., 2021) thus predicts the risk of conversion to parkinsonian highlight the interest of NfL measurement in clinical setting, dementia in the following years, with not only cognitive but however, prospective validation and real-life clinical use are still also motor impairment (Lerche et al., 2020; Mollenhauer et al., needed to confirm such value. Frontiers in Aging Neuroscience 05 frontiersin.org Delaby et al. 10.3389/fnagi.2022.1034684 Fronto-temporal dementia (FTD) In a second study, high serum NfL concentrations were also associated with shorter patient survival and these concentrations Background and state of the art were correlated with cortical atrophy of the prefrontal, temporal and parietal brain regions (Benussi et al., 2020). Frontotemporal dementia (FTD) is a heterogeneous group Interestingly, serum NfL concentrations showed a high accuracy of neurodegenerative diseases characterized by behavioral in discriminating between FTD and healthy controls (area under disorders and deficits in executive and language functions. It the curve (AUC): 0.86, p < 0.001; Benussi et al., 2020). In is the third most common cause of neurocognitive impairment subjects with genetic mutations in the C9ORF72, GRN or MAPT after AD and LBD. In clinical practice, the challenge is to genes (pre-symptomatic subjects), increased serum NfL has been differentiate FTD patients with various primary psychiatric described during the conversion phase corresponding to the disorders (PPD), because of the overlap of some behavioral onset of symptoms (Wilke et al., 2022). Finally, as in ALS, symptoms with FTD. To date, there is no validated biomarker to serum NfL determination seems to be particularly relevant for distinguish PPD from FTD but NfL, as a non-specific biomarker monitoring future therapies in FTD (Toft et al., 2020; Saracino of neuronal death, appears to be promising to fill this gap in et al., 2021). diagnosis. Several studies have investigated NfL levels in FTD subjects: Position of NfL compared to other biomarkers a total of 19 publications reporting NfL results in CSF and cut-off value (Goossens et al., 2018) and seven in serum were identified. Among these studies, three described NfL levels in patients Low blood progranulin level is a validated biomarker used to with a definite diagnosis of FTD on post-mortem pathology predict the presence of GRN mutations for hereditary FTD (van analysis and 14 of them described this marker for an FTD Swieten and Heutink, 2008). Biomarkers are however lacking for population including also familial forms (Steinacker et al., the other etiologies and analysis of AD biomarkers in CSF (t-tau, 2018). p-tau, amyloid peptides) remains recommended to exclude this pathology as it is the main differential diagnosis for degenerative dementia (Ducharme et al., 2020). On the other hand, no difference in t-tau and p-tau concentrations was found between Diagnostic and prognostic values DFT and control groups (Abu-Rumeileh et al., 2018) and no difference in the concentration of other Nf subunits (NfM and The existing studies describe increased NfL concentrations NfH) is observed between FTD patients and PPD (Escal et al., in both matrices (CSF and serum) in FTD groups compared to 2022). PPD and control groups (Forgrave et al., 2019), with sensitivity To date, no consensual NfL threshold value has been and specificity values above 80%. The highest concentrations validated for the differential diagnosis between FTD and of NfL are observed in FTD associated with Amyotrophic PPD. However, the studies mentioning cut-offs and using Lateral Sclerosis (ALS, or Charcot disease; Pijnenburg et al., the UmanDiagnostics ELISA kit range from 1,063 pg/ml to 2015). Thus, according to recently published international 1,877 pg/ml in the CSF (Forgrave et al., 2019) and for the Simoa recommendations (Ducharme et al., 2020), NfL measurement technique from 19.5 pg/ml to 33 pg/ml in blood (Forgrave et al., in CSF or blood could be used in practice for the differential 2019). As the assay is not standardized, it is therefore necessary diagnosis between FTD and SPD, as long as validated thresholds to assess and validate this threshold in each laboratory according are available. to the technique used. This is not so easy, as it requires samples Cut-off values found in studies comparing FTD vs. controls with a probable diagnosis, which is quite rare in this type of or FTD vs. PPD are similar (Davy et al., 2021), which suggests uncommon neurocognitive disorder. that NfL amounts are comparable in patients with PPD and healthy subjects; this is confirmed by studies comparing PPD and controls (Eratne et al., 2020). Studies investigating the prognostic performance of NfL Other neurodegenerative in the context of FTD described a 5-year survival estimate proteinopathies respectively at 36% in FTD patients with a high NfL concentration in the CSF (>3,675 pg/ml) and at 73% in patients Creutzfeld-Jakob disease (CJD), the most common form whose NfL level was lower than 1,989 pg/ml (both measures of prion disease, is responsible for extremely rapid cognitive determined by ELISA; Meeter et al., 2018). CSF NfL achieved an decline. Its diagnosis is based on clinical criteria, EEG and the AUC of 0.87 (95% CI 0.81–0.92, p < 0.001), with a sensitivity of detection of the 14.3.3 protein in the CSF. Alternatively, a very 79% and specificity of 89% (cutoff ≥1,613 pg/ml) to discriminate large increase in t-tau concentration, with a high t-tau/p-tau patients from controls (Meeter et al., 2018). ratio are strong arguments for the diagnosis. Recently, studies Frontiers in Aging Neuroscience 06 frontiersin.org Delaby et al. 10.3389/fnagi.2022.1034684 have shown the interest of NfL measurement in CSF (Abu- Diagnostic and prognostic values Rumeileh and Parchi, 2021) and in blood (Schmitz et al., 2022) to help in the early diagnosis of the disease and to Increased blood and/or CSF NfL levels have been reported differentiate it from other causes of dementia, in particular from in ALS patients (Xu et al., 2016; Verde et al., 2019), reflecting progressive forms of AD. Of note, CSF NfL, either alone or axonal damage of motor neurons during disease progression. in combination with other biomarkers, yielded a performance The mean increase of NfL is significantly greater in ALS patients similar to t-tau in the distinction of prion disease from other compared to patients with slowly progressing amyotrophy neurodegenerative diseases (AUC 0.926 vs. 0.939) and showed (Gaiani et al., 2017). even a higher diagnostic value than t-tau in the specific More recently, the interest of blood NfL in predicting the comparisons between atypical prion disease and other rapidly course of the disease has become apparent due to the correlation progressive neurodegenerative dementias (AUC 0.839 vs. 0.722; of this biomarker with the severity of clinical signs (Kojima Abu-Rumeileh and Parchi, 2021). As for the blood, NfL shows et al., 2021) and/or the course of the disease (Thouvenot lower values compared to blood t-tau for the discrimination et al., 2020). Monitoring NfL blood levels could also allow the between CJD and non-prion rapidly progressive dementias detection of pre-symptomatic forms of ALS in subjects at risk (AUC 0.497–0.724 and AUC 0.722–0.837, respectively; Abu- (Benatar et al., 2018). Finally, although no cure for ALS is yet Rumeileh and Parchi, 2021). available, NfL blood levels have been suggested as a monitoring Huntington’s disease is a rare, inherited disorder of the marker for potential future therapies (Benatar et al., 2018; CNS. It is manifested by motor, cognitive, and psychiatric Witzel et al., 2021). disorders. The mutated huntingtin (HTT) protein becomes abnormal and toxic to the neurons of the striatum when the Position of NfL compared to other biomarkers number of CAG repeats is greater than 35. Mean concentrations and cut-off value of NfL in plasma at baseline were significantly higher in HTT mutation carriers than in controls [3.63 (SD 0.54) log pg/ml vs. Diagnostic performance of blood NfL for ALS is superior 2.68 (0.52) log pg/ml, p < 0.0001] and the difference increased to other biomarkers of axonal degeneration [such as S100B from one disease stage to the next, thus correlating with the and progranulin (Steinacker et al., 2017b) or neuroinflammation severity of symptoms (Byrne et al., 2017, 2018; Rodrigues et al., (Brodovitch et al., 2021)]. This can be partly explained by the 2020). Interestingly, the elevation of NfL precedes the clinic in severity of the disease, but also by the fact that the degeneration children with the mutation (Byrne et al., 2018). Concentrations affects large myelinated axons that have a strong expression of of NfL in CSF and plasma were correlated in mutation carriers NfL. The combination of NfL and C-reactive protein in blood (r = 0.868, p < 0.0001; Byrne et al., 2017). (De Schaepdryver et al., 2020) or ferroptosis markers (Devos et al., 2019) have been proposed for the prognostic evaluation of ALS. Determination of TDP-43 and t-tau proteins levels in CSF has also been described in this context (Kojima et al., 2021). Amyotrophic lateral sclerosis A recent study described optimal cut-off values to discriminate between ALS and controls at 500 pg/ml (a sensitivity of 88.5% Background and state of the art and specificity of 83.3%), using ELISA assay (Shi et al., 2022). When evaluating the ROC curves in discriminating patients with Amyotrophic lateral sclerosis (ALS) is a neurodegenerative ALS and subjects with an alternative ALS-mimicking disease, disease of central and peripheral motor neurons, resulting sNfL (measured through Simoa) yields a diagnostic value of in rapidly progressive amyotrophy and a greatly reduced 0.873 ± 0.036 (sensitivity 84.7%, specificity 83.3%), for a cut-off life expectancy. Diagnosis can be challenging due to the 32.7 pg/ml (Vacchiano et al., 2021). heterogeneity of clinical presentations and the criterion of evolutivity. Therefore, it is common for patients initially identified as having ALS to have their diagnosis reconsidered as a slowly evolving motor neuron disease. In recent years, the NfL Multiple sclerosis assay has emerged as a promising biomarker for the diagnosis and prognosis of ALS. Background and state of the art Published studies first focused on the determination of NfL in CSF (Zetterberg et al., 2007), then in blood (Gaiottino et al., Multiple sclerosis (MS) is a chronic inflammatory disease of 2013) but also on drying blood or plasma spots (Lombardi et al., the central nervous system (CNS), which can cause significant 2020). To our knowledge, 36 articles illustrating the interest physical and cognitive disability and is responsible for a of NfL blood determination in the context of ALS have been significant deterioration in quality of life. It is the most common published since 2013. neurological disease affecting young adults, three women for one Frontiers in Aging Neuroscience 07 frontiersin.org Delaby et al. 10.3389/fnagi.2022.1034684 man, with onset occurring mainly between the ages of 20 and NfL levels also seem to correlate with markers of B cell activity 40. It is a multifactorial disease linked to genetic susceptibility in the CSF, such as the CD80 + marker or the CD20 + /CD14 + factors in association with environmental risk factors and ratio (Engel et al., 2019; Uher et al., 2021). epigenetic factors leading to immune dysfunction (Reich et al., In the short term, elevation of NfL blood levels above the 2018). Eighty-five percent of multiple sclerosis evolves in the 80th percentile of measured samples correlates with worsening form of relapses alternating with phases of remission (so- disability within 1 year, as measured by the Expanded Disability called relapsing-remitting MS or RRMS). After 10–15 years Status Scale (EDSS) score. This elevation is also predictive of of disease, RRMS may progress, especially without treatment, the occurrence of relapses and progression of brain atrophy in to a phase where disability progresses (so-called secondarily the short term (Disanto et al., 2017; Barro et al., 2018; Calabresi progressive MS or SPMS). Fifteen percent of patients present a et al., 2021a; Thebault et al., 2022). In the long term, elevated progressive form from the start (so-called primary progressive blood NfL levels seem to be predictive of increased brain atrophy MS or PPMS). Relapses are related to CNS invasion by at 5 or even 10 years (Kuhle et al., 2017; Barro et al., 2018; pro-inflammatory peripheral immune cells causing multifocal Chitnis et al., 2018; Cantó et al., 2019; Jakimovski et al., 2019; demyelinating lesions and secondary axonal degeneration. Later Srpova et al., 2021). Contrast enhancing and new/enlarging phases of the disease are associated with diffuse microglial lesions were independently associated with increased serum activation and the formation of ectopic lymphoid meningeal neurofilament light chain (17.8% and 4.9% increase per lesion follicles (Cree et al., 2021). Although great progress in the respectively; P < 0.001; Barro et al., 2018). The association accuracy of diagnostic criteria and immunotherapies for RR with disability progression, however, is inconsistent across forms of MS has been achieved, no blood or CSF biomarkers studies (Manouchehrinia et al., 2020a). One hypothesis that to monitor disease activity or prognosis or finally to monitor may explain the weaker long-term predictive value is that response to treatment are available to date. NfL could be a part of the increase in NfL blood levels related to chronic useful biomarker in these indications, and more than 300 studies neurodegenerative processes (associated with the progression of related to the measurement of NfL in MS have been published in disability in the long term) could be masked by neuro-axonal CSF and since 2016 in serum (n = 178) or plasma (n = 49). damage/injury due to the acute inflammatory activity of MS. Blood NfL as a marker of treatment response Diagnostic and prognostic values Numerous studies have reported decreased blood NfL levels Measurement of NfL is not relevant for the differential following initiation of MS immunotherapies (Pop and Viuleţ, diagnosis of neuroinflammatory CNS pathology. However, 1985; Disanto et al., 2017; Novakova et al., 2017; Varhaug various parameters related to the neuro-axonal damage induced et al., 2018; Cantó et al., 2019; Bittner et al., 2020; Calabresi by the inflammatory activity of MS are correlated with increased et al., 2021a,b), so that this assay is currently included in NfL levels in the CSF and in the blood: presence of a relapse most pharmaceutical clinical trials as a secondary measure (Barro et al., 2018), presence and number of gadolinium- of treatment effectiveness. The decrease appears to be greater enhancing lesions on gadolinium injection on MRI (Gd+), upon initiation of high efficacy therapy (Delcoigne et al., 2020), indicating “active” lesions (Novakova et al., 2017; Varhaug compared to moderate or basic therapy (p < 0.05). This indicates et al., 2018), increase in the number of new lesions on MRI that longitudinal sNfL changes rather than absolute sNfL values (Bittner et al., 2020), and the brain volume (Barro et al., 2018). at a given time point might be indicative of disease activity and Interestingly, sNfL levels were described to be higher in RRMS treatment stratification (Bittner et al., 2020). Longitudinal and than in clinically isolated syndrome patients (p = 0.001), thus individual measurement of NfL blood levels to assess the effect suggesting sNfL might serve as a biomarker from very early of initiated treatment on MS inflammatory activity is therefore stages of MS (Bittner et al., 2020). Importantly, the prediction promising. Early detection of a suboptimal response to treatment accuracy of OCB (presence of oligoclonal bands in the CSF) could indeed help to individualize therapeutic decisions, and and/or Gd+ (sensitivity: 72%, specificity: 76%, accuracy: 79%) some treatment strategies are already proposed on the basis of were increased by including the 90th percentile of sNfL in measured NfL values (Bittner et al., 2021; Thebault et al., 2021). addition to the above two variables (sensitivity: 73%; specificity: 79%, accuracy: 84%). These findings pointed towards a potential value of especially high sNfL levels (>31 pg/ml) at time of Position of NfL compared to other biomarkers first demyelinating event as indicators of ongoing chronic CNS and cut-off value neuroinflammation and may be considered for inclusion in a future refinement of the McDonald criteria (Bittner et al., 2020). Astrocytic biomarkers [such as GFAP and chitinase-3-like In the context of relapse occurrence or so-called “active” lesions, protein 1 (CHI3L1)] are currently being studied to discriminate this increase may persist for a few weeks to a few months. Blood RRMS from SPMS patients (Selner, 1988; Huss et al., 2020). Frontiers in Aging Neuroscience 08 frontiersin.org Delaby et al. 10.3389/fnagi.2022.1034684 However their determination is not a substitute for other that may include progressive neurodegeneration, cognitive markers currently used in clinical routine (such as determination impairment, and dementia. Head injuries must be carefully of the presence of CSF-specific IgG oligoclonal bands or diagnosed to foresee, anticipate, and treat the medical and/or visualization of lesion progression on MRI). social after-effects they cause. Blood and CSF biomarkers of Comparison of ELISA and electrochemiluminescence neurodegeneration, particularly NfL, could help in the diagnosis techniques showed better sensitivity of the SimoaTM platform and prognosis assessment. for the determination of blood NfL in this context (Kuhle et al., 2016) and comparison between Simoa and Ella technologies Diagnostic and prognostic values showed similar results (Gauthier et al., 2021). NfL levels increase with age (Gauthier et al., 2021) and The number of publications regarding the interest of NfL fluctuate with body mass index and total blood volume in TBI is growing rapidly (391 articles in PubMed as of (Manouchehrinia et al., 2020b), but are not influenced by gender 01/06/2022). An increase in NfL levels in the CSF and blood is (Harp et al., 2022). Renal function only influences NfL levels found during TBI, reflecting the axonal damage suffered (Khalil when the glomerular filtration rate is below 60 ml/min/1.73 m2 et al., 2018). Following TBI, whether severe or mild in athletes, (Benkert et al., 2022). A meta-analysis in MS and independent NfL is detectable from the first hour and continues in a linear cohort studies of young patients (between 18 and 50 years fashion with a maximum at Day 12 in the CSF and blood of age) did not show any association between CSF NfL level (Zetterberg et al., 2013; Zetterberg and Blennow, 2016; Shahim and age, in contrast to healthy subjects or those with other et al., 2017; McDonald et al., 2021). neurodegenerative diseases. This suggests that in younger The 24-h assessment of this biomarker could also be of patients, inflammatory activity may mask the effect of age on prognostic interest. The NfL increase seems to be correlated measured NfL levels (Bridel et al., 2019; Bittner et al., 2020; with the severity of the damage found on the brain scan and Manouchehrinia et al., 2020a; Rosso et al., 2021; Uher et al., with the severity of the neurological sequelae at 1 year, as well 2021). Nevertheless, a recent study of more than 10,000 blood as with the survival of the patients (Shahim et al., 2016, 2018; samples confirms that NfL level varies with age, with an Graham et al., 2021). Measurement at 24 h allows prediction of increase of 2.5% per year, but that this increase is greater the long-term evolution of patients and high values are observed after 50 years of age (Benkert et al., 2022). This study enabled in case of unfavorable evolution. In athletes, NfL blood levels the determination and validation in independent cohorts of are correlated with the number of concussions and their impact cut-off values expressed as percentile/Z-scores that quantify the (Verduyn et al., 2021). A recent study in rugby players showed deviation of serum NfL level from the control group, adjusted that those who have suffered more than three concussions for age and body mass index: in this study, a sNfL Z score in a year maintained high NfL levels after the off-season, above 1.5 was associated with an increased risk of future clinical indicating chronic suffering. Chronically elevated levels of this or MRI disease activity in all people with multiple sclerosis biomarker have also been associated with the development of (odds ratio 3.15, 95% CI 2.35–4.23; p < 0.0001) and in people frontotemporal cognitive deficits and damage to the blood-brain considered stable with no evidence of disease activity (2.66, barrier (Verduyn et al., 2021). Thus, a persistent increase in 1.08–6.55; p = 0.034; Benkert et al., 2022). A freely available web NfL could indicate the presence of progressive post-traumatic application allows calculation of the adjusted percentile/Z-score neurodegeneration. These results have yet to be confirmed by from an individual NfL value1 . larger studies but suggest that NfL blood determination may be useful to identify patients at risk of developing chronic traumatic encephalopathy and to adapt their follow-up. NfL and neurological damages Position of NfL compared to other biomarkers Traumatic brain injuries and cut-off value Background and state of the art The monitoring of serum biomarkers, such as S100B protein in TBI patients is of great interest for rapid and Traumatic brain injury (TBI) is a major public health inexpensive diagnosis and prognostic evaluation. Other blood problem (more than 500 per day in France). About 80% of biomarkers are being studied and the NfL assay could be a cases of TBI are classified as mild head injury (also called complementary tool to enhance its diagnostic performance. concussions). The morbidity associated with head trauma is Indeed, the cellular origin and the kinetics of these biomarkers considerable. Nearly 50% of victims have residual disabilities are different (Figure 3): the determination of S100B must be performed within 3 h after the TBI [because of its half-life 1 https://shiny.dkfbasel.ch/baselnflreference of 2 h (Jackson et al., 2000)], and the serum determination Frontiers in Aging Neuroscience 09 frontiersin.org Delaby et al. 10.3389/fnagi.2022.1034684 of NfL (whose half-life is longer and whose levels remain Considering the extensive literature in this area, NfL is likely high several days after the TBI) would thus present a real to be of greater interest for prognostic assessment. However, no informative added value in case of delayed patient management cut-off value has yet been determined to predict the secondary (Blomquist et al., 1997; Shahim et al., 2016; Thelin et al., occurrence of cognitive disorders. 2017). A mean diagnostic serum threshold of 20 pg/ml is found in several studies in this context (Thelin et al., 2017). Other biomarkers of interest in this context are emerging, Stroke such as neuron-specific enolase (NSE), glial fibrillary acidic protein (GFAP), t-tau, and ubiquitin C-terminal hydrolase L1 Background and state of the art (UCH-L1; Figure 3). Further studies comparing the diagnostic and prognostic performance of these markers are needed for Stroke is the worldwide leading cause of death and long-term their clinical use, alone or in combination, as well as for the morbidity. NfL may be of interest as a predictive biomarker advancement of CE and IVD labeling, which are essential for for the outcome of ischemic stroke, and for the long-term their use in clinical care. In the field of mild TBI (MTBI) and consequences. Indeed, only one third of patients recover with the interest of blood biomarkers in the reduction of unnecessary minimal or no deficit, whereas the majority remain moderately brain scans prescriptions, a recent study compared the diagnostic or severely disabled for life. As life expectancy and population performances of S100B and NfL in the early management (blood ages increase, stroke management has become a societal issue samples within 3 h post-trauma) of 179 MTBI patients referred and efforts are underway to identify appropriate prognostic to an emergency department. S100B predicted intracranial indicators for optimizing patient management. lesions with a sensitivity of 100% and a 36% specificity. The NfL measurement did not enhance the predictive value. At a Diagnostic and prognostic values threshold of 8.38 ng/L, NfL predicted intracranial lesions with a 28% specificity. On the other hand, in the same population, CSF and blood NfL levels are both increased after stroke, NfL proved to be a high effective marker for the detection of involving both small and large vessels, and several studies patients with degenerative neurological pathologies, as shown by have investigated the relationship between this increase and the data collected in this study (area under the ROC curve of the prognosis of ischemic stroke (Gattringer et al., 2017; Pujol- 0.87 compared with only 0.57 for the S100B protein; Kahouadji Calderón et al., 2019; Peters et al., 2020). A recent meta-analysis et al., 2022). confirmed existence of a correlation between serum NfL level FIGURE 3 Cellular origin (A) and kinetics (B) of NfL increase following traumatic brain injury (TBI) compared with other biomarkers: S100B, UCH-L1, Tau, NSE, and GFAP. Frontiers in Aging Neuroscience 10 frontiersin.org Delaby et al. 10.3389/fnagi.2022.1034684 and the volume of the cerebral infarct area (Liu et al., 2020), different from NfL with a much faster rise and fall. The with a pool adjusted odds ratios (ORs) from multivariate time between stroke and the determination of each of these regression models, = 1.71 (95% CI: 1.17–4.29), representing biomarkers should be evaluated for optimal use (Pujol-Calderón that the patients with higher sNfL had a 1.71 times higher et al., 2022). Although the place of blood NfL remains to be risk of poor functional outcome during follow-up (compared confirmed, it will undoubtedly improve, in combination with with lower sNfL patients; Liu et al., 2020). Kinetic assays the available tools (biomarkers, clinical, and imaging scores), reveal that the highest concentration would be reached 1 week a better assessment of the prognosis of stroke patients. After after stroke (Tiedt et al., 2018; Gendron et al., 2020). Patients an acute stroke, prophylaxis is implemented to prevent or with the highest levels between 1 and 7 days after stroke reduce subsequent events, and a blood biomarker that captures were 1.7 times more likely to have sequelae within 3 months subclinical events would help both monitor and determine than those with low NfL levels. A large prospective study the best treatment for this purpose (Campbell et al., 2019). (n = 1,694 patients) recently confirmed that NfL levels at 48 h Thus, blood levels of NfL are related to the risk of developing were an independent risk factor for cognitive sequelae within stroke in the years after the acute event and low NfL could 3 months after stroke (Wang Z. et al., 2021), p < 0.001. Levels be a real-time biomarker of the effectiveness of prophylactic also correlated with the Rankin score used to measure disability treatment (Uphaus et al., 2019). secondary to stroke in the acute phase. Other studies show There is no validated consensus cut-off value for the that initial NfL levels predict longer-term patient outcomes, assessment of stroke prognosis by blood NfL. Only one study particularly cognitive decline at 1 year (Wang J.-H. et al., proposes a threshold value of 46 pg/ml, determined by the Simoa 2021) and mortality (Gendron et al., 2020), regardless of approach, to identify patients who will remain with cognitive whether the stroke was ischemic or hemorrhagic. The optimal sequelae at 3 months [sensitivity of 71% and specificity of 81.5% cut-off value of the sNfL concentration was 46.12 pg/ml, (AUC 0.79; Wang Z. et al., 2021)]. This value should be adjusted which yielded a sensitivity of 71.0% and a specificity of 81.5%, according to the assay technique used, the patient’s history with the area under the curve (AUC) at 0.785 (95% CI (neurodegenerative diseases) and age. 0.762–0.808, p < 0.001; Wang Z. et al., 2021). These results demonstrate the value of early NfL determination as a prognostic biomarker. CSF NfL levels are also increased in subarachnoid Neurological damages in oncology hemorrhage due to aneurysm rupture (Nylén et al., 2006; Gendron et al., 2020). As in stroke, the levels correlate with Background and state of the art a poor prognosis, and in particular with 3-month mortality (Gendron et al., 2020; Hviid et al., 2020). The area under Neurological disorders in cancerology are numerous and the receiver operating characteristic curve (ROC AUC) for varied in terms of both their etiology and the symptoms discrimination of day-30 mortality was significant on admission observed. They can be due to the primary cerebral localization of [AUC = 0.83, 95% confidence interval (CI): 0.56–1.0] and a tumor (glioblastoma, oligodendroglioma, medulloblastoma...), increased on 24-h follow-up (AUC = 0.93, 95% CI: 0.84–1.0; to the cerebral or medullary localization of metastases of an Hviid et al., 2020). extra-cerebral cancer (lung, breast, melanoma, digestive cancer, NfL levels may remain elevated for up to 6 months lymphoma...), to a carcinomatous meningitis (inflammation after stroke. The sustained high levels could be explained by meningeal invasion by cancerous cells) or to autoimmune by secondary Wallerian degeneration affecting motor neurons reactions triggered (paraneoplastic neurological syndrome). The and by the poststroke inflammatory and immune response. treatment of these cancers is also responsible for neurological High 6-month NfL levels correlate with the presence of complications, and this neurotoxicity is in most cases dose- secondary lesions and quantitative measure of secondary dependent. Neurological damage may affect the central nervous neurodegeneration detected by MRI (Tiedt et al., 2018; Peng system (CNS), the peripheral nervous system (PNS) or et al., 2021). both simultaneously. Early diagnosis of these disorders can often reduce symptoms and prevent permanent neurological deficits. Imaging tests such as MRI or electromyography Position of NfL compared to other biomarkers (EMG) are used to identify these disorders, but there are and cut-off value no blood biomarkers (except for anti-neuronal antibodies) that can be useful for the diagnosis of these pathologies. Other biomarkers are studied in the stroke context: GFAP Moreover, the currently available markers do not allow the and Tau levels increase after stroke and are correlated with evaluation of lesion progression and response to treatment. lesion volume and the NIHSS clinical score used for diagnosis Various studies have explored the use of NfL in these and severity assessment. The kinetics of these biomarkers are indications. Frontiers in Aging Neuroscience 11 frontiersin.org Delaby et al. 10.3389/fnagi.2022.1034684 Diagnostic and prognostic values The increase correlated with the development and severity of polyneuropathy (Huehnchen et al., 2022; Karteri et al., 2022). Primary CNS tumors and brain metastases are major causes However, no association between increased blood NfL and of morbidity and mortality. The earlier the diagnosis, the better cognitive impairment could be demonstrated in this context the management of the patients and the survival rates, which are (Argyriou et al., 2022). anyway low in these cases. Several publications have thus focused Emerging therapies such as antibody immunotherapy on the use of NfL as a diagnostic and prognostic blood marker. and CAR-T cells may also induce neurotoxicity, particularly High blood levels of NfL in brain metastases (Hepner et al., 2019; autoimmune encephalitis, and the Immune Cell Associated Kim et al., 2021; Lin et al., 2022) was reported, these levels being Neurotoxicity Syndrome (ICANS), 1 week after treatment. correlated with the number and size of metastases (Lin et al., In the case of post-immunotherapy autoimmune encephalitis, 2022). A decrease in levels of NfL was also detected following blood and CSF NfL rises and this result might be useful for treatment (Kim et al., 2021). tolerance monitoring (Piepgras et al., 2021). A very recent Interestingly, Winther-Larsen et al. demonstrated an increase study reports that patients with ICANS after CAR-T therapy in NfL in the blood (35 vs. 16 pg/ml, p = 0.001) early, even before had elevated NfL levels (Schoeberl et al., 2022) and increased the diagnosis of brain metastases (median 3 months before; levels correlated with severity of the symptoms [(ICANS grade Winther-Larsen et al., 2020). sNfL discriminated these patients 0–1: 28.4 pg/ml (IQR, 19.2–49.7 pg/ml); ICANS grade 2–4: with an area under the curve of 0.77 (0.66–0.89; Winther-Larsen 60.0 pg/ml (IQR, 31.7–109.0 pg/ml); p < 0.01; Schoeberl et al., 2020). An increase in NfL could be measured median et al., 2022)]. More surprisingly, patients who developed this 3 months (range: 1–5) before the brain metastasis diagnosis. neurotoxicity had already elevated pre-transplant levels of NfL A very high level of NfL at the time of diagnosis seemed to (60 pg/ml vs. 28 pg/ml in the group that developed no or little be correlated with a lower survival, with an inferior survival neurotoxicity, as assessed by Simoa technology; Schoeberl et al., [hazard ratio: 2.10 (95% confidence interval: 1.11–3.98; Winther- 2022). Larsen et al., 2020)]. Of note, these findings were recently These initial results are promising in terms of the usefulness confirmed in a cohort of lung cancer patients (Lin et al., of NfL in oncology but remain to be validated on larger and 2022). longer-term cohorts for neurotoxicity. NfL determination may also have prognostic value in primary CNS tumors. Thus, Hepner et al. showed in a cohort of glioma patients that blood levels of NfL varied closely with tumor activity (Hepner et al., 2019), and that patients with progressive Position of NfL compared to other biomarkers disease had on average 10-fold higher levels than those with and cut-off value stable disease. For metastases diagnosis, NfL threshold values may be defined according to age: for example, NfL levels >22 pg/ml Response to cancer therapy (measured by the Simoa approach) in cancer patients aged 51–60 years may predict the presence of brain metastases (Kim Iatrogenic neurological complications can appear as early et al., 2021). Other biomarkers are also being evaluated, in as the first days of treatment or even years later. Some particular the GFAP protein which would, according to Darlix alkylating agents are well known to be responsible for adverse et al. (2021) outperform NfL as a diagnostic and prognostic effects with neurological manifestations (e.g., neuropathy factor for brain metastases in breast cancer patients. Further and platinum salts). The increase in treatment lines and studies will determine whether the combination of the two the development of new therapeutic modalities increase the biomarkers is worthwhile, but studies have already described risk of neurotoxicity. It can be challenging to make the increased levels of sNfL and GFAP in patients with CNS diagnosis, but it is essential to get it done as soon as tumors with disease in progression vs. CNS with stable disease possible. Several studies have shown that the concentration (p = 0.03 and p = 0.01, respectively; Hepner et al., 2019). of NfL correlates with the development and severity of Regarding chemotherapy toxicity, an increase in serum NfL toxicity resulting from various treatments. Thus, patients concentration (assessed by Simoa) of more than 36 pg/ml treated with paclitaxel, known to induce peripheral neurological (Huehnchen et al., 2022) or an elevated level at 3 or 4 weeks manifestations, show an increase in blood NfL concentration (>50 pg/ml and >85 pg/ml) is associated with high risk of 4 weeks after chemotherapy (compared to controls), resulting developing polyneuropathy (Huehnchen et al., 2022; Karteri in an 86% sensitivity and 87% specificity (Huehnchen et al., et al., 2022). In addition, high level of blood NfL could predict 2022). An increase of sNFL of +36 pg/ml from baseline was the occurrence of ICANS in case of CAR-T cell treatment (a associated with a predicted CIPN probability of more than 0.5 threshold value of 75 pg/ml determined by the Simoa approach (Huehnchen et al., 2022). has been proposed; Schoeberl et al., 2022). Frontiers in Aging Neuroscience 12 frontiersin.org Delaby et al. 10.3389/fnagi.2022.1034684 Other NfL applications Conclusion It is difficult to be exhaustive on the use of NfL as Development of ultrasensitive immunoassays has made it this marker is in full expansion. For example, Nf could possible to detect biomarkers of neuronal damage in easily be of interest in other peripheral neuropathies, such as collected blood samples, even though the concentrations are TTR amyloidosis (Ticau et al., 2021) and AL amyloidosis lower than in CSF. This opens a promising new avenue for (Louwsma et al., 2021). Growing literature also highlights the development of neurological biomarkers. Neurofilaments the predictive value of NfL concentrations in the intensive (Nf) are proteins selectively expressed in the cytoskeleton care setting, as it may be used to assess the risk of of neurons, the increase of which is a marker of neuronal neurological events following either resuscitation (Fisse et al., damage. NfLs are highly specific to neurons but increase in 2021; Page et al., 2022) or cardiac arrest. Thus, high levels many clinical settings and other candidates for neurological of blood NfL 48 h after cardiac arrest (>500 pg/ml) were damage are currently under development. Indeed, neuronal described to predict neurological complications related to damage is common and can be observed both during cerebral ischemia/hypoxia with high sensitivity (100%, 95%CI neurodegenerative diseases (such as AD, PD, FTD) but also 70.0–100%) and specificity (91.7%, 95%CI 62.5–100%; Adler in other neurological contexts (such as head injury, stroke or et al., 2022; Hoiland et al., 2022) and were correlated cancer). In this review, we have provided an overview of these with EEG abnormalities, p < 0.001 (Grindegård et al., different contexts, showing that the determination of NfL in 2022). biological fluids has a wide range of potential uses, sometimes Recent research has focused on the determination of NfL for differential diagnosis, but more often for prognosis or values in children, and norms could be proposed in the pediatric monitoring of the therapeutic response of many neurological population (Nitz et al., 2021). Increased NfL is described to be diseases (Figure 4). We discuss the place and informative added correlated with the development of motor neurological disorders value of NfL compared to other commonly used biomarkers or retinopathy in premature infants (Goeral et al., 2021; Sjöbom for monitoring neurodegenerative or no-neurodegenerative et al., 2021). The value of NfL in CSF and blood also appears pathologies. to be associated with the severity of inherited diseases with The NfL assay may be particularly relevant for the neurological impairment, such as SMA (Johannsen et al., 2021; diagnosis of ALS or PD dementia for example. It would Nitz et al., 2021), cerebral adrenoleukodystrophy (Wang et al., also be useful for the differential diagnosis between FTD 2022) or mitochondrial diseases (Sofou et al., 2019). Finally, NfL and psychiatric disorders, between different Parkinsonian could be relevant for monitoring treatment response (Ru et al., syndromes, or between Alzheimer’s disease and Prion disease, 2019). for which the levels of circulating NfL are very different. FIGURE 4 Physiological and pathological factors modifying the NfL levels in CSF and blood (non-exhaustive). A rise in NfL is not specific for a specific disease factor and may be caused by both neurodegenerative diseases or a head impact during sports for example. Frontiers in Aging Neuroscience 13 frontiersin.org Delaby et al. 10.3389/fnagi.2022.1034684 It is important to note that the level of NfL is a good a must for the clinical activity of the neurologist (Giovannoni, marker of severity: its level is indeed correlated with a poor 2018; Lambertsen et al., 2020). prognosis in many neurodegenerative diseases (ALS, Parkinson’s disease syndromes, MS, Huntington’s,...) and predicts the cognitive decline following repeated head trauma and stroke. Author contributions The signs and symptoms on which the clinical diagnosis is based sometimes appear late in the course of the disease All authors contributed to the writing of the different parts compared to the time of onset; the dynamics of blood NfL of the article. SL, AB, and CD assembled the final version. All in the preclinical phases of neurological diseases and the authors contributed to the article and approved the submitted possibility of detecting blood levels of NfL in an ultrasensitive version. manner could be used to identify diseases at an earlier stage. Finally, NfL can also be used to monitor the response to therapies and the neurotoxicity that such treatments may Funding cause. Nonetheless, it is important to keep in mind that NfL No funding was required for this study which was carried is a marker of neuroaxonal destruction independent of the out within the “Neurofilaments” working group of the French mechanism causing the neuronal damage. The specific diagnosis Society of Clinical Biology (SFBC). will require consideration of a great deal of information, including the patient’s history, physical examination, imaging, and other laboratory tests. It seems essential to define reference Conflict of interest ranges of normality for this biomarker, depending on the biological sample considered (CSF or blood), the analytical The authors declare that the research was conducted in the test performed, age and BMI for each medical indication absence of any commercial or financial relationships that could in order to correctly interpret NfL results according to be construed as a potential conflict of interest. the context. Indeed, many factors other than the primary neurological disease, including age, BMI, cardiovascular risk factors, unrecognized head injury, etc., may be confounding Publisher’s note factors that influence blood NfL and should therefore be taken into account when evaluating this biomarker in All claims expressed in this article are solely those of the patients. authors and do not necessarily represent those of their affiliated The homogenization of the thresholds of this biomarker organizations, or those of the publisher, the editors and the according to the assay methods would allow a rapid development reviewers. Any product that may be evaluated in this article, or of its use in clinical routine for an optimized management of the claim that may be made by its manufacturer, is not guaranteed patients. The extent of its use means that NfL could soon become or endorsed by the publisher. References Aamodt, W. W., Waligorska, T., Shen, J., Tropea, T. F., Siderowf, A., Ashton, N. 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