Serum pNF-H Research Articles

Serum phosphorylated neurofilament heavy-chain levels reflect phenotypic heterogeneity and are an independent predictor of survival in motor neuron disease.

To investigate the prognostic role and the major determinants of serum phosphorylated neurofilament heavy -chain (pNfH) concentration across a large cohort of motor neuron disease (MND) phenotypes. Enzyme-linked immunosorbent assay (ELISA) was used to measure serum pNfH concentration in 219 MND patients consecutively enrolled in our tertiary MND clinic. A multifactorial analysis was carried out to investigate the major clinical determinants of serum pNfH. Kaplan–Meier survival curves and Cox regression analysis were performed to explore the prognostic value of serum pNfH. Serum pNfH levels were not homogenous among MND phenotypes; higher concentrations in pyramidal, bulbar, and classic phenotypes were observed. C9orf72-MND exhibited higher pNfH concentrations compared to non-C9orf72 MND. Multiple linear regression analysis revealed mean MEP/cMAP and disease progression rate as the two major predictors of serum pNfH levels (R2 = 0.188; p ≤ 0.001). Kaplan–Meier curves showed a significant difference of survival among MND subgroups when divided into quartiles based on pNfH concentrations, log-rank X2 = 53.0, p ≤ 0.0001. Our study evidenced that higher serum pNfH concentration is a negative independent prognostic factor for survival. In Cox multivariate model, pNfH concentration showed the highest hazard ratio compared to the other factors influencing survival included in the analysis. pNfH differs among the MND phenotypes and is an independent prognostic factor for survival. This study provides supporting evidence of the role of pNfH as useful prognostic biomarker for MND patients. Neurofilament measurements should be considered in the future prognostic models and in clinical trials for biomarker-based stratification, and to evaluate treatment response.Electronic supplementary materialThe online version of this article (10.1007/s00415-020-09838-9) contains supplementary material, which is available to authorized users.

Neurofilament Proteins as Prognostic Biomarkers in Neurological Disorders.

Neurofilaments: light, medium, and heavy (abbreviated as NF-L, NF-M, and NF-H, respectively), which belong to Type IV intermediate filament family (IF), are neuron-specific cytoskeletal components. Neurofilaments are axonal structural components and integral components of synapses, which are important for neuronal electric signal transmissions along the axons and post-translational modification. Abnormal assembly of neurofilaments is found in several human neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), infantile spinal muscular atrophy (SMA), and hereditary sensory-motor neuropathy (HSMN). In addition, those pathological neurofilament accumulations are known in α-synuclein in Parkinson's disease (PD), Aβ and tau in Alzheimer's disease (AD), polyglutamine in CAG trinucleotide repeat disorders, superoxide dismutase 1 (SOD1), TAR DNA-binding protein 43 (TDP43), neuronal FUS proteins, optineurin (OPTN), ubiquilin 2 (UBQLN2), and dipeptide repeat protein (DRP) in amyotrophic lateral sclerosis (ALS). When axon damage occurs in central nervous disorders, neurofilament proteins are released and delivered into cerebrospinal fluid (CSF), which are then circulated into blood. New quantitative analyses and assay techniques are well-developed for the detection of neurofilament proteins, particularly NF-L and the phosphorylated NF-H (pNF-H) in CSF and serum. This review discusses the potential of using peripheral blood NF quantities and evaluating the severity of damage in the nervous system. Intermediate filaments could be promising biomarkers for evaluating disease progression in different nervous system disorders.

The Association Between Phosphorylated Neurofilament Heavy Chain (pNF-H) and Small Fiber Neuropathy (SFN) in Patients with Impaired Glucose Tolerance.

IntroductionSmall fiber neuropathy (SFN)—the early stage of diabetic peripheral neuropathy (DPN)—progresses gradually and is difficult to diagnose using neurophysiological tests. To facilitate the early diagnosis of SFN, biomarkers for SFN must be identified. The purpose of this study was to investigate the characteristics of SFN in prediabetic patients and the relationship between pNF-H and SFN.Methods44 IGT patients (inpatients and outpatients) were selected at random. 33 healthy subjects served as controls. Data on clinical characteristics and laboratory parameters were collected. Quantitative sensory testing (QST), electromyography (EMG), and Sudoscan were performed, and pNF-H was measured by ELISA.Results24 of the 44 patients with impaired glucose tolerance (IGT) were diagnosed with SFN according to the modified Toronto Diabetic Neuropathy Expert Group consensus criteria. The thermal sensory thresholds of the IGT-SFN group were significantly different from those of the CTRL group (p < 0.05), except for the heat pain threshold. The sensory nerve action potential (SNAP) of the sural nerve was 12.39 in the IGT-SFN group, which was significantly lower than those in the other groups. No significant difference in nerve conduction velocity (NCV) was observed among the three groups. The electrochemical skin conductance (ESC) in the IGT-SFN group was 69.78 ± 14.03uS, which was significantly lower than that in the CTRL group. The pNF-H in the IGT-SFN group was 170.6 (140.0, 223.6) pg/ml, which was significantly higher than those in the CTRL and IGT-non-SFN groups (76.55 and 64.7 pg/ml, respectively). Multivariate regression analysis demonstrated that pNF-H and 2h plasma glucose were independently correlated with SFN; the ORs (95% CI) were 1.429 (1.315, 1.924) and 2.375 (1.157, 4.837), respectively.ConclusionsSerum pNF-H may be associated with SFN in IGT patients, and serum pNF-H could therefore serve as a sensitive biomarker for the detection of SFN.

Dysfunction of the blood-brain barrier in postoperative delirium patients, referring to the axonal damage biomarker phosphorylated neurofilament heavy subunit.

BackgroundDelirium is the most common postoperative complication of the central nervous system (CNS) that can trigger long-term cognitive impairment. Its underlying mechanism is not fully understood, but the dysfunction of the blood-brain barrier (BBB) has been implicated. The serum levels of the axonal damage biomarker, phosphorylated neurofilament heavy subunit (pNF-H) increase in moderate to severe delirium patients, indicating that postoperative delirium can induce irreversible CNS damage. Here, we investigated the relationship among postoperative delirium, CNS damage and BBB dysfunction, using pNF-H as reference.MethodsBlood samples were collected from 117 patients within 3 postoperative days. These patients were clinically diagnosed with postoperative delirium using the Confusion Assessment Method for the Intensive Care Unit. We measured intercellular adhesion molecule-1, platelet and endothelial cell adhesion molecule-1, vascular cell adhesion molecule-1, E-selectin, and P-selectin as biomarkers for BBB disruption, pro-inflammatory cytokines (tumor necrosis factor-alpha, interleukin-1 beta, interleukin-6), and pNF-H. We conducted logistic regression analysis including all participants to identify independent biomarkers contributing to serum pNF-H detection. Next, by multiple regression analysis with a stepwise method we sought to determine which biomarkers influence serum pNF-H levels, in pNF-H positive patients.ResultsOf the 117 subjects, 41 were clinically diagnosed with postoperative delirium, and 30 were positive for serum pNF-H. Sensitivity and specificity of serum pNF-H detection in the patients with postoperative delirium were 56% and 90%, respectively. P-selectin was the only independent variable to associate with pNF-H detection (P < 0.0001) in all 117 patients. In pNF-H positive patients, only PECAM-1 was associated with serum pNF-H levels (P = 0.02).ConclusionsSerum pNF-H could be an objective delirium biomarker, superior to conventional tools in clinical settings. In reference to pNF-H, P-selectin may be involved in the development of delirium-related CNS damage and PECAM-1 may contribute to the progression of delirium- related CNS damage.

Serum neurofilament heavy chains as early marker of motor neuron degeneration

ObjectiveTo determine whether serum phosphorylated neurofilament heavy chain (pNfH) levels are elevated before patients were diagnosed with sporadic or familial ALS, and what the prognostic value of these prediagnostic pNfH levels is.MethodspNfH was measured via ELISA in leftovers of serum drawn for routine purposes before the time of diagnosis. These prediagnostic samples were retrieved from the biobank of the University Hospitals Leuven for 95 patients who in follow‐up received a diagnosis of ALS. Additionally, 35 patients with mild cognitive impairment (MCI) and 85 healthy controls (HC) were included in this retrospective study.ResultsThe median disease duration (range) from onset to prediagnostic sampling and from onset to diagnosis was 6.5 (−71.9–36.1) and 9.9 (2.0–40.7) months, respectively. Fifty‐eight percent of the prediagnostic samples had serum pNfH levels above the 95th percentile of pNfH levels measured in HC. Serum pNfH levels (median (range)) were elevated up to 18 months before the diagnosis of ALS (91 pg/mL (6–342 pg/mL)) in comparison with HC (30 pg/mL (6–146 pg/mL); P = 0.05), and increased during the prediagnostic stage, which was not observed in patients with MCI. Furthermore, prediagnostic pNfH levels were a univariate predictor of survival in ALS (hazard ratio (95% CI): 2.16 (1.20–3.87); P = 0.01).InterpretationOur findings demonstrate that serum pNfH is elevated well before the time of diagnosis in mainly sporadic ALS patients. These results encourage to prospectively explore if pNfH has an added value to shorten the diagnostic delay in ALS.

Neurofilaments in pre-symptomatic ALS and the impact of genotype

Objective. To evaluate serum and cerebrospinal fluid (CSF) levels of phosphorylated neurofilament heavy (pNfH), and to compare these to levels of neurofilament light (NfL), as biomarkers of pre-symptomatic ALS. Design. The study population includes 34 controls, 79 individuals at-risk for ALS, 22 ALS patients, and 14 phenoconverters. At-risk individuals are enrolled through Pre-Symptomatic Familial ALS (Pre-fALS), a longitudinal natural history and biomarker study of individuals who are carriers of any ALS-associated gene mutation, but who demonstrate no clinical evidence of disease at the time of enrollment. pNfH and NfL in serum and CSF were quantified using established enzyme-linked immunosorbent assays. Results. There is a longitudinal increase in serum pNfH in advance of the emergence of clinically manifest ALS. A similar pattern is observed for NfL, but with the absolute levels also frequently exceeding a normative threshold. Although CSF data are more sparse, similar patterns are observed for both neurofilaments, with absolute levels exceeding a normative threshold prior to phenoconversion. In serum, these changes are observed in the 6–12 months prior to disease among SOD1 A4V mutation carriers, and as far back as 2 and 3.5 years, respectively, in individuals with a FUS c.521del6 mutation and a C9ORF72 hexanucleotide repeat expansion. Conclusions. Serum and CSF pNfH increase prior to phenoconversion. In CSF, the temporal course of these changes is similar to NfL. In serum, however, pNfH is less sensitive to pre-symptomatic disease than NfL. The duration of pre-symptomatic disease, as defined by changes in neurofilaments, may vary depending on underlying genotype.

Analysis of neurofilament concentration in healthy adult horses and utility in the diagnosis of equine protozoal myeloencephalitis and equine motor neuron disease

Neurofilaments (NFs) are structural proteins of neurons that are released in significant quantities in the cerebrospinal fluid and blood as a result of neuronal degeneration or axonal damage. Therefore, NFs have potential as biomarkers for neurologic disorders. Neural degeneration increases with age and has the potential to confound the utility of NFs as biomarkers in the diagnosis of neurologic disorders. We investigated this relationship in horses with and without neurological diagnosis. While controlling for horse type (draft, pleasure, and racing), we evaluated the relationship between serum heavy-chain phosphorylated neurofilaments (pNF-H) and age, sex, and serum vitamin E concentrations. Serum pNF-H concentrations increased by 0.002 ng/ml for each year increase in age. There were significant differences in the serum pNF-H concentration among the type of activity performed by the horse. The highest serum pNF-H concentration was found in horses performing heavy work activity (racehorse) and with lower serum pNF-H concentration found among light (pleasure riding) and moderate (draft) activity. There was no significant association between the pNF-H concentration and sex or vitamin E concentration. Serum pNF-H concentration was elevated among horses afflicted with EMND and EPM when compared with control horses without evidence of neurologic disorders. Accordingly, serum pNF-H concentration can serve as a useful biomarker to complement the existing diagnostic work-up of horses suspected of having EPM or EMND.

Correlations between serum and CSF pNfH levels in ALS, FTD and controls: a comparison of three analytical approaches.

Background Phosphorylated neurofilament heavy (pNfH), a neuronal cytoskeleton protein, might provide a promising blood biomarker of neuronal damage in neurodegenerative diseases (NDDs). The best analytical approaches to measure pNfH levels and whether serum levels correlate with cerebrospinal fluid (CSF) levels in NDDs remain to be determined. Methods We here compared analytical sensitivity and reliability of three novel analytical approaches (homebrew Simoa, commercial Simoa and ELISA) for quantifying pNfH in both CSF and serum in samples of amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD) and control subjects. Results While all three assays showed highly correlated CSF measurements, Simoa assays also yielded high between-assay correlations for serum measurements (ϱ = 0.95). Serum levels also correlated strongly with CSF levels for Simoa-based measurements (both ϱ = 0.62). All three assays allowed distinguishing ALS from controls by increased CSF pNfH levels, and Simoa assays also by increased serum pNfH levels. pNfH levels were also increased in FTD. Conclusions pNfH concentrations in CSF and, if measured by Simoa assays, in blood might provide a sensitive and reliable biomarker of neuronal damage, with good between-assay correlations. Serum pNfH levels measured by Simoa assays closely reflect CSF levels, rendering serum pNfH an easily accessible blood biomarker of neuronal damage in NDDs.

Serum Phosphorylated Neurofilament Heavy Subunit-H, a Potential Predictive Biomarker for Postoperative Cognitive Dysfunction in Elderly Subjects Undergoing Hip Joint Arthroplasty

BackgroundPostoperative cognitive dysfunction (POCD), which refers to a cognitive impairment subsequent to surgical procedures, is a common complication in the elderly subjects. This study aimed to investigate potential risk factors for POCD in elderly subjects undergoing hip joint arthroplasty. MethodsConsecutive elderly osteoarthritis patients who were scheduled to undergo hip arthroplasty under epidural anesthesia were enrolled into this single-center, prospective observational study. Serum phosphorylated neurofilament heavy subunit-H (pNF-H) was measured by the enzyme-linked immunosorbent assay method. A level of >70.5 pg/mL was accepted as pNF-H positivity. Neuropsychological assessment at baseline (1 day before the surgery) and postoperative day 7 was conducted. POCD was defined according to the calculated Z scores. Risk factors for POCD were evaluated by univariate and multivariate logistic regression analyses. ResultsIn final, 287 patients were enrolled, and 55 had suffered POCD within postoperative 7 days with an incidence of 19.2%. The final multiple logistic regression analysis revealed a higher pNF-H positivity was the only independent risk factor for POCD (odds ratio: 2.03, 95% confidence interval: 1.21-3.29, P = .012). ConclusionsOur results revealed an increased preoperative serum pNF-H expression was an independent risk factor for POCD development in elderly subjects undergoing hip joint arthroplasty, suggesting the close association between anatomical damage in central nervous system and POCD.

Time course and prognostic value of serum GFAP, pNFH, and S100β concentrations in dogs with complete spinal cord injury because of intervertebral disc extrusion.

BackgroundA noninvasive biomarker is needed to predict recovery from severe spinal cord injury (SCI) because of thoracolumbar intervertebral disc extrusion (TL‐IVDE). Proteins released from neural and glial cells can be detected in the blood and show promise as prognostic tools, but their concentration is influenced by time after injury.Hypothesis/ObjectivesSerum concentrations of glial fibrillary acidic protein (GFAP), phosphorylated neurofilament heavy chain (pNFH), and S100β will follow different time courses; measurement of combinations of these proteins will predict outcome.AnimalsThirty‐one dogs with TL‐IVDE causing paralysis with no pain perception.MethodsProspective study. Serum samples were taken at presentation and intervals over 56 days and banked at −80°C. Glial fibrillary acidic protein, pNFH, and S100β concentrations were measured using ELISA tests and plotted against time from onset of nonambulatory status. Outcome was established at 6 months. The association between biomarker concentration and outcome was examined using logistic regression, receiver operator characteristics curve analysis, and model development.ResultsThirty‐one dogs participated, 3/31 (10%) developed progressive myelomalacia and 19/31 (62%) recovered ambulation. Glial fibrillary acidic protein and S100β concentrations rose for the first 1 to 3 days, and were undetectable by 14 and 28 days, respectively. Phosphorylated neurofilament heavy chain concentrations peaked at 14 days and were detectable at 56 days. Glial fibrillary acidic protein concentrations in the first 72 hours after onset of nonambulatory status predicted recovery with an accuracy of 76.7%‐89% depending on sample timing.Conclusions and Clinical ImportanceSerum GFAP concentrations can be used to predict outcome in clinically complete SCI. A rapid inexpensive bedside test is needed.

Neurofilament changes in serum and cerebrospinal fluid after acute ischemic stroke

BackgroundNeurofilament light (NFL) is a well-validated biomarker for neuronal injury and neurodegeneration. Increased cerebrospinal fluid (CSF) levels have been shown after stroke, as well as in patients with a broad range of neurodegenerative and neuroinflammatory diseases. Neurofilament heavy (NFH) belongs to the same family of structural proteins but it is less extensively studied. The potential of phosphorylated NFH (pNFH) as a stroke biomarker and for the prediction of clinical outcome is unknown. In this study, we aimed to examine the temporal pattern of NFL and pNFH concentrations in serum and CSF after acute ischemic stroke. Materials and MethodsA quantitative Enzyme-Linked ImmunoSorbent Assay (ELISA) for pNFH was developed and tested on CSF and serum samples. NFL and pNFH were analysed in serum and CSF of acute ischemic stroke patients, who were followed over time (Day 0–1, Day 2–3, Day 7–9, three weeks, and 3–5 months after stroke). ResultsNFL and pNFH concentrations in serum and CSF increased after stroke, peaked during the 3rd week, and then decreased back to almost baseline levels at 3–5 months. CSF-NFL and serum-NFL correlated to the outcome measured by Barthel Index after 3–5 months, whilst no such association was seen for pNFH. DiscussionThese findings suggest that NFL and pNFH in both CSF and serum reflect the temporal pattern of the post ischemic axonal injury and that this process does not seem to progress after 3–5 months. ConclusionNFL and pNFH in CSF and serum are promising biomarkers for axonal injury following stroke. Further studies in larger populations are needed to fully understand the progression of the neuronal damage after acute ischemic stroke and to evaluate if these biomarkers can provide additive information and how they relate to outcome.

Cerebrospinal Fluid Neurofilaments May Discriminate Upper Motor Neuron Syndromes: A Pilot Study

Background: Patients presenting with upper motor neuron (UMN) signs may widely diverge in prognosis, ranging from amyotrophic lateral sclerosis (ALS) to primary lateral sclerosis (PLS) and hereditary spastic paraplegia (hSP). Neurofilaments are emerging as potential diagnostic and prognostic biomarkers for ALS, but the diagnosis of UMN syndromes still relies mostly on clinical long-term observation and on familiarity or genetic confirmation. Objectives: To test whether phosphorylated neurofilament heavy chain (pNfH) may discriminate different UMN syndromes at diagnosis and to test their prognostic role among these diseases. Methods: We measured the cerebrospinal fluid (CSF) and serum pNfH of 30 patients presenting with UMN signs and diagnosed with ALS, hSP, and PLS, plus 9 healthy controls (HC). Results: ALS patients had higher levels of pNfH in CSF and serum compared to HC (p < 0.001 and p < 0.001 in CSF and serum, respectively) and PLS (p = 0.015 and p = 0.038) and hSP (p = 0.003 and p = 0.001) patients. PLS and hSP patients had similar CSF and serum pNfH concentrations, but a higher CSF pNfH concentration, compared to HC (p = 0.002 and p = 0.003 for PLS and hSP, respectively). Receiver operating characteristic curves for discriminating ALS from PLS and hSP showed an area under the curve of 0.79 for CSF and 0.81 for serum. In multivariable survival analysis including relevant clinical factors CSF pNfH represented the strongest variable predicting survival (HR 40.43; 95% CI 3.49–467.79, p = 0.003) independently of clinical group. Conclusions: Despite some statistical instability of the results due to limitations in sample size, our study supports the role of CSF pNfH as a prognostic biomarker for motor neuron diseases presenting with UMN signs. A potential power to discriminate between ALS and other UMN syndromes at presentation, and between all of the examined MND and HC, has been detected for both CSF and serum pNfH.

A Novel Brain Injury Biomarker Correlates with Cyanosis in Infants with Congenital Heart Disease.

Cyanotic heart lesions are a complex subset of congenital heart disease (CHD) in which patients are desaturated until surgical repair or palliation. We hypothesized that a direct relationship would exist between degree of desaturation and presence of systemic inflammation and brain injury in unrepaired patients less than 1year of age. The pre-operative desaturation with augmented systemic inflammation would predict a more complex post-operative course. Fifty patients with CHD were enrolled in this study and classified as cyanotic (O2 ≤ 90%) or acyanotic (O2 > 90%) based on SpO2. Serum inflammatory mediators measured included interleukins (IL)-6, IL-8, IL-12p70, IL-10, IL-1β, tumor necrosis factor (TNF)-α, interferon (INF)-γ; macrophage inhibitory factor (MIF) and a novel brain biomarker, phosphorylated neurofilament heavy subunit (pNF-H). Twenty-two cyanotic and 28 acyanotic subjects were enrolled with SpO2 of 78 ± 18% and 98 ± 2% (p < 0.001), respectively, and mean age of 72days (range 2-303) and 102days (range 1-274), respectively. Cyanotic vs acyanotic subjects had elevated serum IL-6 (6.6 ± 7.6 vs 2.9 ± 2.9pg/ml, p = 0.019) and pNF-H (222 ± 637 vs 57 ± 121pg/ml, p = 0.046), and both biomarkers correlated with degree of desaturation (Spearman rank-order correlation ρ = - 0.30, p = 0.037 and ρ = - 0.29 p = 0.049, respectively). Post-operative inotrope scores at 24h and duration of mechanical ventilation correlated inversely with pre-operative oxygen saturation (ρ = - 0.380, p = 0.014 and ρ = - 0.362, p = 0.020, respectively). The degree of pre-operative desaturation correlated with a more complicated post-operative course supporting the need for advanced peri-operative therapy in this population.

Serum-Based Phospho-Neurofilament-Heavy Protein as Theranostic Biomarker in Three Models of Traumatic Brain Injury: An Operation Brain Trauma Therapy Study.

Glial fibrillary acidic protein (GFAP) and ubiquitin C-terminal hydrolase (UCH-L1), markers of glial and neuronal cell body injury, respectively, have been previously selected by the Operation Brain Trauma Therapy (OBTT) pre-clinical therapy and biomarker screening consortium as drug development tools. However, traumatic axonal injury (TAI) also represents a major consequence and determinant of adverse outcomes after traumatic brain injury (TBI). Thus, biomarkers capable of assessing TAI are much needed. Neurofilaments (NFs) are found exclusively in axons. Here, we evaluated phospho-neurofilament-H (pNF-H) protein as a possible new TAI marker in serum and cerebrospinal fluid (CSF) across three rat TBI models in studies carried out by the OBTT consortium, namely, controlled cortical impact (CCI), parasagittal fluid percussion (FPI), and penetrating ballistics-like brain injury (PBBI). We indeed found that CSF and serum pNF-H levels are robustly elevated by 24 h post-injury in all three models. Further, in previous studies by OBTT, levetiracetam showed the most promising benefits, whereas nicotinamide showed limited benefit only at high dose (500 mg/kg). Thus, serum samples from the same repository collected by OBTT were evaluated. Treatment with 54 mg/kg intravenously of levetiracetam in the CCI model and 170 mg/kg in the PBBI model significantly attenuated pNF-H levels at 24 h post-injury as compared to respective vehicle groups. In contrast, nicotinamide (50 or 500 mg/kg) showed no reduction of pNF-H levels in CCI or PBBI models. Our current study suggests that pNF-H is a useful theranostic blood-based biomarker for TAI across different rodent TBI models. In addition, our data support levetiracetam as the most promising TBI drug candidate screened by OBTT to date.

Abstract P6-12-26: The axonal damage marker, serum phosphorylated neurofilament heavy subunit, as a potential marker of chemotherapy-induced neuropathy

Abstract Background: Chemotherapy-induced neurologic disorders such as peripheral neuropathy and cognitive disturbance are clinically significant problems for cancer survivors, but their objective assessment methods have not been established. We previously reported in a cross-sectional study that the serum phosphorylated neurofilament heavy subunit (pNF-H), a biomarker of axonal damage, was increased in breast cancer patients treated with chemotherapy. The aim of this study is to temporally assess the neurological adverse events and evaluate the association of serum pNF-H level with cognitive functions and neuropathy following sequential chemotherapy. Methods: Thirty-five breast cancer patients who received neoadjuvant or adjuvant chemotherapy were enrolled prospectively. They underwent brain MRI and cognitive function tests including Controlled Oral Word Association (COWA), Trail Making Test (TMT), and Hopkins Verbal Learning Test-Revised (HVLT-R) before chemotherapy (baseline), one month after completing sequential chemotherapy (post-phase) and more than six months after completing chemotherapy (late-phase). Serum pNF-H levels and questionnaires reporting peripheral neuropathy were measured at the three phases, and every 3 weeks during chemotherapy. Brain MRI volumetry was calculated by the automatic analysis software, BAAD® (Brain Anatomical Analysis using Dartel). The correlations among cognitive functions, brain volume, peripheral neuropathy and serum pNF-H levels were statistically analyzed. Results: Patients' median age was 48 years (range 24-71). A decrease of more than 10% in cognitive function test (COWA) scores was seen in 10 cases (31%) at post-phase. A brain volume loss of more than 10% was seen in 5 cases (15%) at post-phase. The correlation between brain volume change and cognitive disturbance was not significant (p=0.45) and both changes were improved at late-phase. A peripheral neuropathy grade above CTCAE grade 2 was seen in 19 cases (54%). The neuropathy was significantly more severe in anthracycline followed by taxane regimen than taxane followed by anthracycline during chemotherapy (p=0.016), although this difference was not seen at the late-phase (p=0.08). An elevated serum pNF-H level at baseline was seen in only one case, and this case demonstrated the cognitive disturbance, brain volume loss, and peripheral neuropathy following chemotherapy. During chemotherapy, pNF-H was elevated in 24 patients (69%), with especially higher levels noted during the taxane regimen compared to the anthracycline regimen (p=0.019). In the cases treated with anthracycline followed by taxane, the taxane-phase elevation was especially significant (p=0.014). The maximum pNF-H level during taxane therapy was significantly correlated with peripheral neuropathy grade (p=0.002). At late-phase, the significant reduction of pNF-H level was seen in all cases. Conclusions: Change of cognitive function, brain volume and peripheral neuropathy was observed following chemotherapy in breast cancer patients. This study suggests that the serum axonal damage marker, pNF-H, may reflect chemotherapy-induced neuropathy. Citation Format: Kida K, Sumitani M, Ogata T, Kotake R, Natori A, Hashimoto J, Shimokawa T, Yamauchi H, Yamauchi T. The axonal damage marker, serum phosphorylated neurofilament heavy subunit, as a potential marker of chemotherapy-induced neuropathy [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P6-12-26.

Comparison of elevated phosphorylated neurofilament heavy chains in serum and cerebrospinal fluid of patients with amyotrophic lateral sclerosis

ObjectivePhosphorylated neurofilament heavy chain (pNfH) levels are elevated in cerebrospinal fluid (CSF) of patients with amyotrophic lateral sclerosis (ALS). Instead of CSF, we explored blood as an alternative source to...

Phosphorylated neurofilament H (pNF-H) as a potential diagnostic marker for neurological disorders in horses

The current study aimed at the investigating the potential use of phosphorylated neurofilament H (pNF-H) as a diagnostic biomarker for neurologic disorders in the horse. Paired serum and cerebrospinal fluid (CSF) samples (n=88) and serum only (n=30) were obtained from horses diagnosed with neurologic disorders and clinically healthy horses as control. The neurologic horses consisted of equine protozoal myeloencephalitis (EPM) (38 cases) and cervical vertebral malformation (CVM) (23 cases). Levels of pNF-H were determined using an ELISA. The correlation between CSF and serum concentrations of pNF-H was evaluated using Spearman's Rank test and the significance of the difference among the groups was assessed using a nonparametric test. Horses had higher pNF-H levels in the CSF than serum. Horses afflicted with EPM had significantly higher serum pNF-H levels in comparison to controls or CVM cases. The correlation between CSF and serum pNF-H levels was poor in both the whole study population and among subgroups of horses included in the study. There was significant association between the likelihood of EPM and the concentrations of pNF-H in either the serum or CSF. These data suggest that pNF-H could be detected in serum and CSF samples from neurologic and control horses. This study demonstrated that pNF-H levels in serum and CSF have the potential to provide objective information to help in the early diagnosis of horses afflicted with neurologic disorders.

Brain injury with systemic inflammation in newborns with congenital heart disease undergoing heart surgery.

The potential role of systemic inflammation on brain injury in newborns with congenital heart disease (CHD) was assessed by measuring levels of central nervous system (CNS)-derived proteins in serum prior to and following cardiac surgery. A total of 23 newborns (gestational age, 39±1 weeks) with a diagnosis of CHD that required cardiac surgery with cardiopulmonary bypass (CPB) were enrolled in the current study. Serum samples were collected immediately prior to surgery and 2, 24 and 48 h following CPB, and serum levels of phosphorylated neurofilament-heavy subunit (pNF-H), neuron-specific enolase (NSE) and S100B were analyzed. Systemic inflammation was assessed by measuring serum concentrations of complement C5a and complement sC5b9, and the following cytokines: Interleukin (IL)-1β, IL-6, IL-8, IL-10, IL12p70, interferon γ and tumor necrosis factor (TNF)-α. Analysis of cord blood from normal term deliveries (n=26) provided surrogate normative values for newborns. pNF-H and S100B were 2.4- to 2.8-fold higher (P<0.0001) in patient sera than in cord blood prior to surgery and remained elevated following CPB. Pre-surgical serum pNF-H and S100B levels directly correlated with interleukin (IL)-12p70 (ρ=0.442, P<0.05). pNF-H was inversely correlated with arterial pO2 prior to surgery (ρ=−0.493, P=0.01) and directly correlated with arterial pCO2 post-CPB (ρ=0.426, P<0.05), suggesting that tissue hypoxia and inflammation contribute to blood brain barrier (BBB) dysfunction and neuronal injury. Serum IL12p70, IL-6, IL-8, IL-10 and TNF-α levels were significantly higher in patients than in normal cord blood and levels of these cytokines increased following CPB (P<0.001). Activation of complement was observed in all patients prior to surgery, and serum C5a and sC5b9 remained elevated up to 48 h post-surgery. Furthermore, they were correlated (P<0.05) with low arterial pO2, high pCO2 and elevated arterial pressure in the postoperative period. Length of mechanical ventilation was associated directly with post-surgery serum IL-12p70 and IL-8 concentrations (P<0.05). Elevated serum concentrations of pNF-H and S100B in neonates with CHD suggest BBB dysfunction and CNS injury, with concurrent hypoxemia and an activated inflammatory response potentiating this effect.

Direct evidence of central nervous system axonal damage in patients with postoperative delirium: A preliminary study of pNF-H as a promising serum biomarker

IntroductionApproximately 50–80% patients experience postoperative delirium, an acute cognitive dysfunction associated with prolonged hospitalization, increased mortality, excess healthcare costs, and persistent cognitive impairment. Elucidation of the mechanism of delirium and associated diagnostic and therapeutic measures are urgently required. Here we investigated the role of phosphorylated neurofilament heavy subunit (pNF-H), a major structural protein in axons, as a predictive maker of postoperative delirium. Material and methodsTwenty-three patients who underwent surgery for abdominal cancer were screened for postoperative delirium, and they were assessed for its severity using the memorial delirium assessment scale (MDAS) at and 48h after delirium onset. Serum pNF-H levels were also measured at both time points. The patients were divided into two groups according to the presence or absence of pNF-H. Clinical variables were compared between groups using the Mann–Whitney U test, and the relationship between pNF-H levels and delirium severity was analyzed using the exponential curve fitting. ResultsFifteen of the 23 (65.2%) patients tested positive for pNF-H, and these patients exhibited significantly higher MDAS scores compared with the pNF-H-negative patients only at the onset of delirium. Although the MDAS score significantly improved over time in the positive group, pNF-H positivity persisted. There was a correlation between the maximum pNF-H level and maximum MDAS score (R2=0.31, p=0.013). ConclusionsMore severe postoperative delirium was directly related to higher serum pNF-H levels, suggesting the potential application of pNF-H as a quantitative biomarker of neural damage in postoperative delirium.

Cerebrospinal Fluid Levels of Phosphorylated Neurofilament Heavy as a Diagnostic Marker of Canine Degenerative Myelopathy.

BackgroundNo definitive, antemortem diagnostic test for canine degenerative myelopathy (DM) is available. Phosphorylated neurofilament heavy (pNF‐H) is a promising biomarker for nervous system diseases.Hypothesis/ObjectiveCerebrospinal fluid (CSF) and serum pNF‐H is a detectable biological marker for diagnosis of canine DM.AnimalsFifty‐three DM‐affected, 27 neurologically normal, 7 asymptomatic at‐risk, and 12 DM mimic dogs.MethodsArchived CSF and serum pNF‐H concentrations were determined by a commercially available ELISA. A receiver‐operating characteristic (ROC) curve was generated with CSF values.ResultsCompared with old control dogs, median CSF pNF‐H concentration was increased in all stages of DM; old dogs 5.1 ng/mL (interquartile range [IQR] 1.4–9.3) versus DM stage 1 23.9 ng/mL (IQR 20.8–29.6; P < .05) versus DM stage 2 36.8 ng/mL (IQR 22.9–51.2; P < .0001) versus DM stage 3 25.2 ng/mL (IQR 20.2–61.8; P < .001) versus DM stage 4 38.0 ng/mL (IQR 11.6–59.9; P < .01). Degenerative myelopathy stage 1 dogs had increased median CSF pNF‐H concentrations compared with asymptomatic, at‐risk dogs (3.4 ng/mL [IQR 1.5–10.9; P < .01]) and DM mimics (6.6 ng/mL [IQR 3.0–12.3; P < .01]). CSF pNF‐H concentration >20.25 ng/mL was 80.4% sensitive (confidence interval [CI] 66.09–90.64%) and 93.6% specific (CI 78.58–99.21%) for DM. Area under the ROC curve was 0.9467 (CI 0.92–0.9974). No differences in serum pNF‐H concentration were found between control and DM‐affected dogs.Conclusions and Clinical Importance pNF‐H concentration in CSF is a sensitive biomarker for diagnosis of DM. Although there was high specificity for DM in this cohort, further study should focus on a larger cohort of DM mimics, particularly other central and peripheral axonopathies.