Amyotrophic lateral sclerosis: Correlations between fluid biomarkers of NfL, TDP-43, and tau, and clinical characteristics.

OBJECTIVES: We previously reported the diagnostic and prognostic performance of neurofilament light chain (NfL), TAR DNA-binding protein 43 (TDP-43), and total tau (t-tau) in cerebrospinal fluid (CSF) and plasma as amyotrophic lateral sclerosis (ALS) biomarkers. The present study aimed to elucidate associations between clinical characteristics and the markers as well as mutual associations of the markers in ALS patients using the same dataset.
METHODS: NfL, TDP-43, and t-tau levels in CSF and plasma in 75 ALS patients were analyzed. The associations between those markers and clinical details were investigated by uni- and multivariate analyses. Correlations between the markers were analyzed univariately.
RESULTS: In multivariate analysis of CSF proteins, the disease progression rate (DPR) was positively correlated with NfL (β: 0.51, p = 0.007) and t-tau (β: 0.37, p = 0.03). Plasma NfL was correlated with age (β: 0.53, p = 0.005) and diagnostic grade (β: -0.42, p = 0.02) in multivariate analysis. Plasma TDP-43 was correlated negatively with split hand index (β: -0.48, p = 0.04) and positively with % vital capacity (β: 0.64, p = 0.03) in multivariate analysis. Regarding mutual biomarker analysis, a negative correlation between CSF-NfL and TDP-43 was identified (r: -0.36, p = 0.002).
CONCLUSIONS: Elevated NfL and t-tau levels in CSF may be biomarkers to predict rapid DPR from onset to sample collection. The negative relationship between CSF NfL and TDP-43 suggests that elevation of CSF TDP-43 in ALS is not a simple consequence of its release into CSF during neurodegeneration. The negative correlation between plasma TDP-43 and split hand index may support the pathophysiological association between plasma TDP-43 and ALS.

Introduction

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder specifically affecting motor neurons, characterized by progressive muscle weakness and atrophy, resulting in respiratory failure and death. It is untreatable at present, but an effective therapy is strongly desired [1]. In order to develop novel treatments, objective and responsive biomarkers are essential for successfully carrying out clinical trials. Various potential biomarkers of ALS have been reported, and recently neurofilament light chain (NfL), which is associated with neuroaxonal damage and loss [2], is considered the most promising biomarker of ALS progression [3-6]. The level of TAR DNA-binding protein 43 (TDP-43) was also reported to be high in the cerebrospinal fluid (CSF) and plasma of ALS patients compared with controls [7,8], but the inconsistency of the TDP-43 immunoassay used to measure the level of this protein has been problematic. CSF total-tau (t-tau) is expected to serve as a responsive biomarker for ALS diagnosis and progression, despite a lack of consistent results [9,10]. The diagnostic and prognostic value of such biomarkers has been well-investigated at individual levels; however, associations between biomarkers and clinical characteristics, as well as the mutual associations of those biomarkers, are not fully understood.

We previously examined the diagnostic and prognostic performance of NfL, TDP-43, and t-tau in CSF and plasma as biomarkers of ALS in two individual case-control cohorts, revealing increased levels of CSF NfL, plasma NfL, and CSF TDP-43 in ALS compared with control groups and shorter survival associated with higher levels of CSF and plasma NfL [11]. We herein conducted additional analysis of the same cohorts of ALS patients and biomarker dataset with the aim of comprehensively elucidating the association with clinical findings and mutual associations of CSF and plasma NfL, TDP-43, and t-tau levels in ALS patients, because such clinico-biomarker and mutual-biomarker associations were insufficiently investigated in the initial study.

Materials and methods

Patients and data collection

The study cohort consisted of 75 ALS patients visiting our institute between September 2009 and May 2018, who were from the discovery cohort enrolled from September 2009 to March 2014 (n = 29) and validation cohort enrolled from April 2014 to May 2018 (n = 46) included in our previous study [11]. The participating ALS patients were diagnosed with suspected, possible, probable, or definite ALS based on El Escorial criteria [12]. All ALS patients in the suspected or possible grade were converted to probable or definite ALS within the follow-up period. All participants underwent CSF sampling at the time of diagnosis. At the time of CSF collection, clinical data including age, sex, upper motor neuron (UMN) score, Medical Research Council (MRC) sum score, % slow vital capacity (%VC), ALS Functional Rating Scale revised (ALSFRS-R) [13], disease progression rate (DPR), and split hand index were collected. The UMN score comprised a sum of pathologically brisk reflexes that included bilateral assessment of the biceps, supinator, triceps, finger, knee, and ankle reflexes, plantar responses, and facial and jaw jerks, with a maximum score of 16 [14]. The MRC sum score, which is a sensitive indicator of the degree of muscle weakness as a lower motor neuron symptom, was the total score of manual muscle testing on bilateral arm abduction, elbow flexion, wrist extension, hip flexion, knee extension, and ankle dorsiflexion, and the score ranged from 0 to 60 [15]. The DPR was calculated as follows: (48 − ALSFRS-R score at the time of sampling)/months elapsed between disease onset and sampling [16]. Disease onset was defined as the month when patients become aware of their symptoms caused by ALS. Based on the nerve conduction study, split hand index was calculated by dividing the product of the compound muscle action potential (CMAP) amplitude recorded over the first dorsal interosseous and abductor pollicis brevis by the CMAP amplitude recorded over the abductor digiti minimi on the affected side [17] (Note: a reduced split hand index indicates preferential atrophy in the first dorsal interosseous and abductor pollicis brevis, which is typically observed in ALS).

All study subjects provided written informed consent before participation and the study protocol was approved by the University Ethics Committee (ERB-G-12). Informed consent from patients was obtained when possible and also from the nearest relative.

Sample collection and measurement of NfL, TDP-43, and t-tau

CSF samples were collected in polypropylene vials by lumbar puncture, and the samples were cleared by centrifugation at 400 x g for 10 min at 4°C immediately after collection. Plasma samples were obtained via venous puncture and collected in EDTA-containing tubes. After collection, plasma was separated by centrifugation for 10 min at 3,000 rpm and placed in polypropylene vials. CSF and plasma samples obtained from the enrolled subjects were immediately stored at -80°C until analysis. In the analysis, CSF and plasma NfL, TDP-43, and t-tau concentrations were measured with Simoa NF-light assay, TDP-43 assay, and Human Total Tau assay kits, respectively, on a Simoa HD-1 Analyzer (Quanterix, Lexington, MA, USA) according to protocols provided by the manufacturer. All samples were analyzed in duplicate on one occasion independently in the two cohorts (discovery and validation cohorts). Because of inter-assay variation between the two cohorts, we corrected the values based on those of internal controls [11]. For this sub-analysis in the present study, we used the same levels of those combined values of each biomarker in the ALS group of our previous paper. In the original dataset, we conducted a case-control study involving two independent cohorts (validation and discovery cohorts). To correct for inter-assay variation, we adjusted the values of the validation cohort based on the correction formula: raw values x correction factors. In the current study, we used the corrected biomarker levels for the analysis of the two cohorts. The correction factors were determined as the mean value ratios between the discovery and validation assays based on four internal controls for each biomarker (See the previous study [11]).

Statistics

Associations between biomarker values and clinical characteristics were analyzed by uni- and multivariate linear regression models using each biomarker value as a dependent variable, and age, sex, diagnosis, UMN score, MRC score, %VC, ALSFRS-R score, DPR, and split hand index as independent variables. The independent variables in multivariate analysis were chosen based on the importance in the association with the prognosis of ALS, not by p- values of univariate analysis or step-wise method, unless otherwise stated. Multivariate linear regression was performed after the exclusion of patients whose data were missing. The F test was used to assess how each multivariate linear regression model fitted the data. When dependent variables or residuals in multiple linear regression were not normally distributed (P<0.05 in the Shapiro-​Wilk test), we reanalyzed those after natural logarithm transformation of dependent variables. The absence of multicollinearities among these variables was confirmed when the value of the variance inflation factor was less than ten. The association between biomarkers was analyzed by Spearman’s rank correlation tests. P< 0.05 was considered significant in all analyses. JMP software 14.0 (SAS Institute, Cary, North Carolina, USA) was used for all statistical analyses.

Results

Patient characteristics

The demographic characteristics of 75 patients with ALS are summarized in Table 1. For detailed clinical data from each patient, see S1 Table. Case numbers 1–29 were in the discovery cohort of our previous report and case numbers 30–75 were in the validation cohort [11]. This cohort included four familial ALS cases in which relatives with ALS or motor neuron disease were found within the second degree by consanguinity: cases 3 (younger brother), 8 (maternal grandmother), 41 (younger brother), and 57 (mother) (S1 Table). In those familial cases, no genetic abnormality was identified.

Table 1

Clinical and biological data of patients with amyotrophic lateral sclerosis.

Characteristic
Age at sample collection (years)	72 [63–77]
Sex (Male:Female) (n)	47:28
*Clinical diagnosis at sample collection (Definite:Probable:Possible:Suspected) (n)	25:28:15:7
ALSFRS-R score (n = 75)	41 [35 – 44]
Disease progression rate (n = 75)	0.47 [0.16–1.00]
Upper motor neuron score (n = 75)	8 [4 – 11]
MRC sum score (n = 75)	52 [47–57]
% vital capacity (%) (n = 69)	87 [73–103]
Split hand index (n = 62)	5.0 [2.0–10.0]
Neurofilament light chain in cerebrospinal fluid (pg/mL) (n = 70)	7965 [4261–13433]
TAR DNA-binding protein 43 in cerebrospinal fluid (pg/mL) (n = 70)	63 [60–68]
Total-tau in cerebrospinal fluid (pg/mL) (n = 69)	12 [11 – 16]
Neurofilament light chain in plasma (pg/mL) (n = 49)	109 [68–160]
TAR DNA-binding protein 43 in plasma (pg/mL) (n = 49)	391 [215–761]
Total-tau in plasma (pg/mL) (n = 48)	0.63 [0.40–0.86]

Data are given as median [interquartile range].

ALSFRS-R, Amyotrophic Lateral Sclerosis Functional Rating Scale revised score; MRC, Medical Research Council.

* Patients were diagnosed based on El Escorial criteria.

Association between levels of biomarkers and clinical data

Univariate and multivariate analyses are shown in Tables 2–4. Levels of NfL, TDP-43, and t-tau in plasma were analyzed after natural logarithm transformation because they were not normally distributed.

Table 2

A: The association between NfL in CSF and clinical characteristics of ALS patients (n = 70).

B: The association between NfL in plasma and clinical characteristics of ALS patients (n = 49).

	Univariate analysis				Multivariate analysis (N = 53, R2 = 0.40, P = 0.005)
	B value	SE	β value	P-value	B value	SE	β value	P-value
Age	27	69	0.05	0.70	-87	75	-0.17	0.25
Sex*	-1468	1568	-0.11	0.35	-368	1689	-0.03	0.83
Diagnosis**	-4682	1554	-0.34	0.004	-3123	1864	-0.25	0.10
Upper motor neuron score	454	154	0.34	0.005	172	181	0.14	0.35
MRC score	-87	92	-0.11	0.35	-247	149	-0.26	0.11
% vital capacity	-21	35	-0.07	0.56	67	49	0.22	0.18
ALSFRS-R score	-182	111	-0.2	0.11	261.00	241	0.25	0.28
Disease progression rate	4458	1097	0.44	0.0001	5418	1908	0.51	0.007
Split hand index	-303	163	-0.24	0.07	-75.00	154	-0.06	0.63
* Male = 0; Female = 1.
**Definite/Probable = 0; Possible/Suspected = 1.
NfL, neurofilament light chain; CSF, cerebrospinal fluid; ALS, amyotrophic lateral sclerosis.
MRC, Medical Research Council; ALSFRS-R score, ALS Functional Rating Scale revised score.
Bold characters indicate a significant difference with a p-value less than 0.05.
The data were analyzed after being log transformed.

* Male = 0; Female = 1.

**Definite/Probable = 0; Possible/Suspected = 1.

NfL, neurofilament light chain; ALS, amyotrophic lateral sclerosis; MRC, Medical Research Council; ALSFRS-R score, ALS Functional Rating Scale revised score.

Bold characters indicate a significant difference with a p-value less than 0.05.

Table 4

A: The association between t-tau in CSF and clinical characteristics of ALS patients (n = 69).

B: The association between t-tau in plasma and clinical characteristics of ALS patients (n = 48).

	Univariate analysis				Multivariate analysis (n = 53, R2 = 0.47, P = 0.0006)
	B value	SE	β value	P-value	B value	SE	β value	P-value
Age	0.14	0.05	0.32	0.007	0.06	0.06	0.14	0.29
Sex*	4.43	1.09	0.44	0.0001	4.60	1.29	0.46	0.0009
Diagnosis**	0.07	1.28	0.007	0.95	0.29	1.43	0.03	0.84
Upper motor neuron score	-0.04	0.13	-0.04	0.77	-0.13	0.11	-0.12	0.38
MRC score	-0.01	0.07	-0.02	0.84	0.02	0.11	0.02	0.89
% vital capacity	0.01	0.03	0.03	0.83	0.06	0.04	0.25	0.10
ALSFRS-R score	-0.01	0.09	-0.01	0.93	-0.02	0.18	-0.03	0.91
Disease progression rate	1.70	0.92	0.22	0.07	3.23	1.46	0.37	0.03
Split hand index	0.08	0.13	0.09	0.52	0.13	0.12	0.13	0.29
* Male = 0; Female = 1.
**Definite/Probable = 0; Possible/Suspected = 1.
t-tau, total Tau; CSF, cerebrospinal fluid; ALS, amyotrophic lateral sclerosis; MRC, Medical Research Council; ALSFRS-R score, ALS Functional Rating Scale revised score.
Bold characters indicate a significant difference with a p-value less than 0.05.

The data were analyzed after being log transformed.

* Male = 0; Female = 1.

**Definite/Probable = 0; Possible/Suspected = 1.

t-tau, total Tau; ALS, amyotrophic lateral sclerosis; MRC, Medical Research Council; ALSFRS-R score, ALS Functional Rating Scale revised score.

Bold characters indicate a significant difference with a p-value less than 0.05.

As shown in Table 2A and 2B, NfL in CSF was significantly correlated with the diagnostic grade (β = -0.34, p = 0.004), UMN score (β = 0.34, p = 0.005), and DPR (β = 0.44, p = 0.0001) in univariate analysis. A significant correlation in multivariate analysis was observed only between CSF NfL and DPR (β = 0.51, p = 0.007). NfL in plasma showed significant correlations with age (β = 0.45, p = 0.001), diagnostic grade (β = -0.39, p = 0.006), %VC (β = -0.42, p = 0.004), and DPR (β = 0.29, p = 0.04) in univariate analysis. NfL in plasma was correlated with age (β = 0.53, p = 0.005) and diagnostic grade (β = -0.42, p = 0.02) in multivariate analysis. Regarding the goodness of fit in the multivariate linear regression model, the model significantly fitted the data on NfL in both CSF and plasma.

Regarding CSF TDP-43, there was no significant correlation on univariate analysis. The level in CSF was significantly negatively correlated with DPR in multivariate analysis (β = -0.47, p = 0.03). (Note, this weak correlation cannot be generalized because of the small R2 value and lack of significance of model-fitting in the F test.) (Table 3A) TDP-43 in plasma was significantly positively correlated with % vital capacity (β = 0.34, p = 0.03) and ALSFRS-R score (β = 0.33, p = 0.02) in univariate analysis. The significant correlation between TDP-43 in plasma and %VC was preserved in multivariate analysis (β = 0.64, p = 0.03). Multivariate analysis revealed another significant negative correlation between TDP-43 in plasma and split hand index (β = -0.61, p = 0.008), as shown in Table 3B. Although the goodness of fit in the multivariate regression model for plasma TDP-43 did not reach significance in the F test, the R2 value of the model (= 0.42) was not small. Moreover, when we employed a backward step-wise method for modeling, a significant model using age, diagnosis, MRC score, ALSFRS-R, %vital capacity, and split hand index, was obtained (R2 value = 0.36; p-value in F test = 0.042). The significant correlation of levels of plasma TDP-43 with %VC and split hand index was preserved in this model (β = -0.64, p = 0.021 and β = -0.56, p = 0.006, respectively). Based on those results from forced entry and backward step-wise methods in multiple regression, we concluded that the correlations between plasma TDP-43 and %VC as well as between plasma TDP-43 and split hand index show statistical reliability.

Table 3

A: The association between TDP-43 in CSF and clinical characteristics of ALS patients (n = 70).

B: The association between TDP-43 in plasma and clinical characteristics of ALS patients (n = 49).

	Univariate analysis				Multivariate analysis (n = 53, R2 = 0.15, P = 0.59)
	B value	SE	β value	P-value	B value	SE	β value	P-value
Age	0.05	0.13	0.05	0.69	-0.01	0.18	-0.01	0.98
Sex*	3.46	2.84	0.15	0.23	7.09	4.05	0.29	0.09
Diagnosis**	0.46	3.01	0.02	0.88	-0.09	4.48	-0.003	0.98
Upper motor neuron score	0.01	0.30	0.004	0.97	0.02	0.44	0.01	0.96
MRC score	0.01	0.17	0.009	0.94	0.35	0.36	0.18	0.34
% vital capacity	-0.02	0.06	-0.04	0.74	0.06	0.12	0.09	0.64
ALSFRS-R score	-0.11	0.20	-0.07	0.57	-0.96	0.58	-0.46	0.10
Disease progression rate	-2.00	2.21	-0.11	0.37	-10.2	4.58	-0.47	0.03
Split hand index	0.05	0.33	0.02	0.89	-0.01	0.37	-0.004	0.98
* Male = 0; Female = 1.
**Definite/Probable = 0; Possible/Suspected = 1.
TDP-43, TAR DNA-binding protein 43; CSF, cerebrospinal fluid; ALS, amyotrophic lateral sclerosis; MRC, Medical Research Council; ALSFRS-R score, ALS Functional Rating Scale revised score.
Bold characters indicate a significant difference with a p-value less than 0.05.

The data were analyzed after being log transformed.

* Male = 0; Female = 1.

**Definite/Probable = 0; Possible/Suspected = 1.

TDP-43, TAR DNA-binding protein 43; ALS, amyotrophic lateral sclerosis; MRC, Medical Research Council; ALSFRS-R score, ALS Functional Rating Scale revised score.

Bold characters indicate a significant difference with a p-value less than 0.05.

On analyzing t-tau, there were significant correlations of t-tau in CSF with age (β = 0.32, p = 0.007) and sex (β = 0.44, p = 0.0001) in univariate analysis, and with sex (β = 0.46, p = 0.0009) and DPR (β = 0.37, p = 0.03) in multivariate analysis with a significant goodness of fit of the data (Table 4A). No association of t-tau in plasma with any clinical data was identified in either uni- or multivariate analysis (Table 4B).

Correlation between levels of each biomarker

As shown in Fig 1A, NfL levels in CSF were negatively correlated with TDP-43 levels in CSF (r = -0.36, p = 0.002). There were no correlations between t-tau and NfL levels or between t-tau and TDP-43 levels in CSF (Fig 1B (t-tau—NfL); r = 0.14, p = 0.26. Fig 1C (t-tau—TDP-43); r = 0.02, p = 0.87). Mutual associations of the plasma biomarkers are summarized in Fig 1D–1F. There were no correlations in any combinations of the plasma biomarkers (NfL—TDP-43; r = -0.14, p = 0.35. t-tau—NfL; r = 0.09, p = 0.53. t-tau—TDP-43; r = -0.04, p = 0.80) (Note: correlations between CSF and plasma levels of each biomarker were already described in our previous report [11]).

Fig 1

Correlation of inter-biomarker levels in CSF and plasma.

Levels of individual biomarkers in ALS patients are presented as logarithms. The rs and p-values generated by Spearman’s rank correlation tests are shown. The combinations of biomarkers in CSF are presented in A-C and those in plasma are in D-F. Correlations between the same markers in CSF and plasma were presented in our previous report [11]. A linear regression line is presented when there is a significant correlation. CSF, cerebrospinal fluid; NfL, neurofilament light; TDP-43, TAR DNA-binding protein 43; t-tau, total tau; ALS, amyotrophic lateral sclerosis.

Discussion

NfL, one of the neuron-specific cytoskeletal proteins, is highly expressed in large-caliber myelinated axons. NfL is released into extracellular spaces and levels increase in blood and CSF proportionally to the degree of axonal damage [18]. Mutations in the NfL gene (NEFL: OMIM162280) cause hereditary neuropathies, while a direct association between ALS and NEFL mutation has not been reported [19]. TDP-43 is a heterogeneous nuclear ribonucleoprotein mainly distributed in the nucleus under physiological conditions. In most cases of ALS, it is translocated to the cytoplasm where it changes structure and forms aggregates. A direct mechanistic link between TDP-43 and neurodegeneration of motor neurons is suggested by the identification of mutations in the gene coding TDP-43 (TARDBP: OMIM605078) in hereditary forms of ALS [20]. Tau protein is mainly expressed in neurons of the central nervous system where it exerts a role in stabilizing microtubules, key components of axonal transport and signal transduction. Mutations of the Tau gene (MAPT: OMIM157140) are responsible for a rare form of familial dementia called frontotemporal lobar degeneration-17 with tau pathology, while such mutations of tau have not been found in familial ALS. The increase of CSF t-tau is probably due to its leakage from damaged neurons into CSF; therefore, it reflects the severity of neuronal damage and degeneration [21].

In the present study, we carried out a comprehensive investigation of associations of multiple biomarkers in CSF and plasma with clinical findings and the mutual associations of those biomarkers.

Regarding the associations of NfL and TDP-43 in CSF with clinical findings, the following two results were obtained: First, NfL in CSF was positively associated solely with DPR in multivariate analysis. The results were consistent with previous findings [22-26]. Second, in contrast, TDP-43 in CSF was negatively associated with DPR in multivariate analysis, although the fitness of the multiple regression was insufficient. This contrasting association of NfL and TDP-43 in CSF with DPR of ALS from onset to sample collection may explain the negative correlation between NfL and TDP-43 in CSF, which was only significant in mutual correlations of biomarkers in the present study. It is logical that NfL, known as a strong indicator of axonal damage, was correlated with the rapid progression of ALS, while the result that TDP-43 was associated with slow progression of the disease was unexpected. This fact suggests that CSF TDP-43 elevation in ALS is not a simple consequence of its release into CSF during neurodegeneration. Although the significance of increased CSF TDP-43 in ALS is still unclear, this phenomenon might be a result of increased levels of TDP-43 expression in motor neurons in the disease. Intranuclear TDP-43 negatively regulates its own expression; therefore, intranuclear loss of function of the molecule resulting from its abnormal aggregation and redistribution can lead to an elevation of its own expression and cytosolic protein levels [27,28]. In fact, increased levels of TDP-43 mRNA were observed in the brains of patients affected by various forms of FTLD with TDP-43 pathology [27,29]. Such overexpression of TDP-43 has been reported to lead to more serious protein-aggregation and loss of intranuclear function of the molecule, causing a vicious cycle [28]. However, only in the early stage of the disease, such overexpression and overproduction of TDP-43 in ALS might work as a compensatory mechanism to maintain its disturbed intranuclear function. This hypothetical idea might not only explain the current result of the association between elevated CSF TDP-43 and slow DPR in the early stage (between onset to sampling), but also be consistent with our previous finding that CSF TDP-43 was not associated with survival after sampling [11].

The TDP-43 level in plasma showed a negative association with split hand index and a positive association with %VC in the current study. The direct relevance of the association between the pathophysiology of ALS and changes in plasma TDP-43 levels still remains controversial. Actually, TDP-43 levels in plasma were elevated not only in patients with ALS and inclusion body myositis but also in those with other inflammatory myopathies, suggesting that increased levels of plasma TDP-43 may be due to the release of non-pathological TDP-43 as a result of muscle damage or inflammatory processes to some extent [30]. On the other hand, elevated plasma TDP-43 levels have been reproducibly observed in patients with ALS and FTLD [8,11,31], leading us to consider its disease-specificity for ALS. Our result that the decrease in split hand index, which is recognized as a specific and early-diagnostic marker of ALS [32], was related to TDP-43 elevation in plasma independently of other non-specific indicators of muscle weakness such as MRC score supports the suggestion that raised TDP-43 levels in plasma reflect the ALS pathophysiology.

T-tau in CSF showed positive associations with DPR of ALS and female sex. This is consistent with the previous observation that t-tau levels in CSF are elevated in many patients with chronic neurodegenerative diseases as a non-specific indicator of neuronal damage and axonal degeneration [33]. The result was also in agreement with our previous report on the same dataset, showing that the correlation between CSF t-tau and poor survival after sample collection was significant only in one of the two cohorts despite a lack of consistency between the cohorts [11]. Considering the conflicting results on the association between CSF t-tau levels and disease progression in ALS [9,10,34], the prognostic potential of this biomarker may not be as powerful as that of CSF NfL. Regarding the association with sex, Rosen et al. reported higher t-tau levels in CSF from female patients with Alzheimer’s disease compared with male patients independent of the degree of cognitive impairment [35]. The sex difference in CSF t-tau has been insufficiently considered in the biomarker field of ALS [9,36]. The fact that t-tau in CSF increased in females should be paid attention to if t-tau is used as a prognostic biomarker of ALS.

In univariate analysis, NfL in plasma showed significant correlations with age, diagnostic grade, %VC, and DPR. Age and DPR were also associated with plasma NfL in multivariate analysis (Table 2B). Of these, the associations with DPR and diagnostic grade were consistent with previous reports [22,24,37], while the result of age was different from other reports, which identified no relationships among these parameters [24,38]. This discrepancies in the results may be explained by the confounding factor of physiological age-dependent elevation of NfL [39].

Major limitations were that this was a single-center study with a relatively small sample size, which may have resulted in a weak statistical power. A point to be aware of regarding the multiple regression analyses was the predictive power of the independent variables for each biomarker level. “DPR for CSF NfL”, “age and diagnostic grade for plasma NfL”, and “sex and DPR for CSF t-tau” had high beta values in the multiple linear regression models with significant goodness of fit of the data. Meanwhile, the linear regression model for CSF TDP-43 had a relatively low R2 value and did not significantly fit the data, although CSF TDP-43 was significantly correlated with a good prognosis. Given this fact, the predictive power of CSF TDP-43 for the progression of ALS was considered to remain uncertain. In addition, the longitudinal change of each biomarker could not be followed because CSF sampling was invasive and performed only once. The follow-up of biomarkers should be performed in a longitudinal study in order to clarify the significance of each biomarker in ALS progression.

Conclusions

NfL levels in CSF were positively associated with DPR during the time from onset to sample collection and negatively associated with CSF TDP-43. This negative relationship suggests that elevation of CSF TDP-43 in ALS is not a simple consequence of its release into CSF during neurodegeneration. T-tau in CSF showed a positive association with DPR and female sex independently. These findings may be important for utilizing t-tau in CSF as a progression biomarker of ALS. A negative association between TDP-43 in plasma and split hand index was identified. However, the present results need to be validated in a larger cohort.

Ethical publication statement

The authors confirm that they have read the journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines. The patients were evaluated at Kyoto Prefectural University of Medicine hospital under a protocol approved by the local ethics committee of the university. Written informed consent was obtained from the patients.

Clinical information and concentrations of biomarkers.

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27 Aug 2021

PONE-D-21-22395

Amyotrophic lateral sclerosis: Correlations between fluid biomarkers of NfL, TDP-43, tau, and clinical characteristics

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This research is interesting, but there might be some errors and main concerns:

In P3, There might be an error. “Plasma TDP-43 correlated negatively with split hand index......” was shown in “Results”, but in “Conclusions”, it was shown “The positive correlation between plasma TDP-43 and split hand index......”.

This study cohort consisted of 75 ALS patients, it might be better for the authors to indicate the patients are only sporadic or sporadic and familial cases.

In P6, The authors indicated that “The participating ALS patients were diagnosed as suspected, possible, probable, or definite ALS based on revised El Escorial criteria.” . But in revised El Escorial criteria, there are four diagnostic categories, clinically definite, clinically probable, clinically probable–laboratory-supported and clinically possible. The category of “clinically suspected ALS was deleted from the revised El Escorial Criteria for the Diagnosis of ALS.

The authors used “% vital capacity” as a clinical data, did it mean “% forced vital capacity (FVC)”? And, if ALS patients had bulbar dysfunction, decreased % FVC could not reflect the real situation of respiratory function. Even if all participants had normal bulbar function, it might be better to analyze the relation of abnormal % FVC with those fluid biomarkers.

“progression rate” was used in this study, it might be better to use “disease progression rate (DPR)”.

“split hand index” was used as a clinical data in this study, but the hand muscles innervated by median and ulnar nerves might not be involved in some patients in this cohort. And it was shown “the split hand index was calculated by dividing the product of he compound muscle action potential (CMAP) amplitude recorded over the first orsal interosseous and abductor pollicis brevis by the CMAP amplitude recorded over the abductor digiti minimi ” in page 7, but the authors did not indicate that nerve conduction study was made in which side, left or right, or the involved side. These will certainly influence the research results.

In discussion, it might be better to discuss the probable reasons of the most important research results in more detail.

Reviewer #2: This is an elegant study on a fundamental topic in ALS biomarker research. The authors are experts in the field. The results are interesting and well presented. I have only some minor issues:

1. Line 91: the authors state that the patients of the discovery and replication cohorts were "extracted" from the cohorts of a previous study. It is not clear whether only some of the patients evaluated in the previous study were taken, and, if so, which criteria were applied for this selection.

2. Lines 134-136 ("All samples were analyzed in duplicate on one occasion. For this sub-analysis, we used the levels of each biomarker in the ALS group of our previous paper"). I think that these sentences are not so clear. Does this mean that for these patients the levels of the biomarkers were not measured again after the work made for the previous publication? Please clarify.

3. Line 190: "genialized" --> "generalized"?

4. Lines 191-194 ("we would like to emphasize that CSF TDP-43 were negatively associated with the progression rate both in univariate and multivariate analyses regardless of significance, in contrast to those in CSF NfL and CSF tau"). I do not think that is correct to equalize a significant result (multivariate analysis) and a non-significant one. Perhaps the above-mentioned sentence could be eliminated.

5. Lines 353-354. Here it is written that the association between plasma TDP-43 and the split hand index is positive. However, I think that it is negative.

6. In table 1 I see that median t-Tau in CSF in ALS patients was 12 pg/mL. It seems a low value. Do the authors confirm this value?

Reviewer #3: The authors extended their analysis of CSF NfL and TDP-43 from a cohort of patients with ALS (published previously), by comparing measures to commonly used clinical measures of disease progression. Their methodology is clearly described and I have no concerns with their approach. I have no comments on their approach. This is sound. I have a few minor suggestions and some points of interest.

1. I initially suspected that the correlation between CSF NfL and TDP43 was driven by a few outliers. However after closer consideration (thank you for providing data points for individual patients) it appears that 5 of the participants present with very low CSF NfL levels relative to their TDP43 scores (patients 4, 9, 10, 17 and 23). Most of these individuals have very slow progression rates, except for #4, which has a very fast progression rate. Did the authors note any specific clinical features in these patients that may explain these low NfL scores. Could other clinical features be relevant? I note that all of these patients had a possible/probable diagnosis at study inclusion.

2. Did familial/sporadic status impact the results? Is the genetic status of patients known? If so, can the authors please comment.

3. I caution the authors against the use of “biomarker” for their current data results; factors are tested as possible prognostic indicators. This limitation is noted in the discussion, however the authors are asked to revise the term “biomarker” in their abstract.

4. Figure legend 1: Please correct the figure legend to state that correlations between plasma and CSF markers WERE completed in the previous manuscript. This should be followed by a reference to the report.

5. The manuscript is well-written and easy to follow, however I’ve noted a few minor grammatical mistakes; i.e. line 308 “missing the word “is” between CSF and elevated. The authors are encouraged to revise the document.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Frederik J Steyn

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

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5 Oct 2021

Responses to the reviewers’ comments

We appreciate the thoughtful comments from the reviewers that helped us to improve this manuscript. We have revised the manuscript with the point-by-point response as suggested, and believe that it has been significantly improved.

Submitted filename: Point-by-point responce20211002.docx

Click here for additional data file.

8 Nov 2021

Amyotrophic lateral sclerosis: Correlations between fluid biomarkers of NfL, TDP-43, and tau, and clinical characteristics

PONE-D-21-22395R1

Dear Dr. Kasai,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This research is interesting and provide some significant results. But the sample size is relatively small. And in the revised manuscript Ref. 12 should be “J Neurol Sci. 1994 Jul;124 Suppl:96-107”.

Reviewer #2: The authors have replied to my observations and according to me their answers and changes are satisfactory.

Reviewer #3: The authors have addressed all of my comments and the comments raised by the other reviewers; I have nothing more to add

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

12 Nov 2021

PONE-D-21-22395R1

Amyotrophic lateral sclerosis: Correlations between fluid biomarkers of NfL, TDP-43, and tau, and clinical characteristics

Dear Dr. Kasai:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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on behalf of

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