Neurofilaments in blood and CSF for diagnosis and prediction of onset in Creutzfeldt-Jakob disease

While cerebrospinal fluid (CSF) biomarkers for Creutzfeldt-Jakob disease (CJD) are established and partly included in the diagnostic criteria, no blood biomarkers are available. Here, we assessed the utility of serum neurofilament light chain (NF-L) and tau protein in comparison to CSF markers (NF-L and phosphorylated NF heavy chain (pNF-H), tau, S100B, 14-3-3) and prion conversion assay (real-time quaking induced conversion (RT-QuIC)) for sporadic and genetic CJD. Importantly, a Gerstmann-Sträussler-Scheinker mutation carrier in the asymptomatic phase and at disease onset was included as well. Both NF-L and tau were markedly increased in CJD serum, reaching similar or even better performance as in CSF (sensitivity and specificity for serum NF-L 100% and 85.5%, and for serum tau 84.6% and 96.2%, respectively). Serum S100B showed high sensitivity as well (84.2%), but lower specificity (63%). CSF neurofilaments were increased before symptom onset, while prion seeding assay was negative. Just before a clinical diagnosis could be made, all CSF markers and NF-L in the serum were increased and CSF prion conversion assay was positive. The data suggest that neurofilaments are sensitive and specific blood markers for the diagnosis of genetic and sporadic CJD and might represent promising tools to predict disease onset.

In the present study we therefore addressed the questions whether we can measure these biomarkers with newly established assays in blood, of which diagnostic value they are, and how early in the disease process these markers appear.

Results
A summary of the marker concentrations, cut-off levels, and diagnostic performance in terms of sensitivity and specificity as well as the positive and negative predictive values for the discrimination between CJD and controls is given in Table 1.

Surrogate marker concentrations in serum.
Boxplots of marker levels in serum of the diagnostic groups are shown in Fig. 1A-C. NF-L serum concentrations were higher in both sCJD and gCJD compared to DCo or Co. At 44.7 pg/ml cut-off 100% sensitivity and 85.5% specificity was reached for discrimination between CJD and controls.
Serum tau was significantly different in sCJD, but not gCJD, and controls. At 2.2 pg/ml cut-off, CJD was recognized with 84.6% sensitivity and 96.2% specificity.    Table 1. Demographic characteristics of the patient cohort, marker concentrations in serum and CSF, and diagnostic performance. Co, non-demented control; DCo, demented control; f, female; m, male; pos., positive; neg., negative; n.d., not determined; n.a., not applicable; PPV, positive predictive value; NPV. negative predictive value. a The age is given as median with interquartile range. b The markers are given as mean with standard deviation. c Codon 129 polymorphism was determined for 28 sporadic CJD cases. d For 5 out of 7 CJD analyzed for CSF prion seeding activity the codon 129 polymorphism was known. e For the patients with M/V individual marker concentrations are given; CSF parameters missing for one patient. f This patient also had an A117A polymorphism. g Calculated for discrimination between all CJD versus all control measures; given with 95% confidence intervals in brackets.
At the presymptomatic stage serum tau and S100B were similar to control levels, whereas NF-L tended to be elevated, and at symptom onset serum markers were clearly increased (green and red circles respectively in Fig. 1A-C).
ROC analysis of serum marker concentrations of all CJD versus all controls yielded comparable estimates for the AUC for NF-L and tau (Fig. 1G).

Surrogate marker concentrations in CSF.
Boxplots of the marker levels in CSF samples of the diagnostic groups are shown in Fig. 1D-F.
The levels of NF-L and pNF-H in CSF samples were strongly increased in CJD compared to both control groups. CSF tau concentrations were significantly increased in CJD as well, however, due to low levels in an E200K and the insert 4 × 24 carrier, the difference between gCJD and DCo was not significant. Displayed are the results for CJD patients (indicated separately for sCJD and gCJD) and from demented (DCo) and non-demented controls (Co). The concentrations of analytes determined in serum and CSF from a gCJD patient at presymptomatic and early symptomatic disease stage are indicated by green and red circles, respectively. Boxes give median values with interquartile ranges, whiskers indicate the concentration range. Three and two asterisks indicate statistically significant differences of p < 0.0001 and p < 0.01, respectively. The diagnostic performance of markers is illustrated by ROC curves for serum (G) and CSF (H). Except for S100B, of which levels were determined in DCo only, ROC analyses were conducted considering all CJD measures (i.e. sCJD and gCJD) versus all controls (i.e. DCo and Co). The results of RT-QuIC are shown in (I). Displayed are the mean fluorescence signals with SD detected in the CSF of sCJD patients (n = 6), gCJD patients (n = 4), and controls (n = 6). Separately shown are the results for 3 replicate measurements of the gCJD mutation carrier in the asymptomatic disease stage (orange) and at disease onset (red).
Scientific RepoRts | 6:38737 | DOI: 10.1038/srep38737 At the presymptomatic disease stage, CSF tau was normal and NF-L and especially pNF-H were already elevated above the 75% quartile of controls. At disease onset all CSF markers were increased. See green and red circles in Fig. 1D-F, respectively.
In the ROC analyses pNF-H, tau, and NF-L showed similar performance (Fig. 1H). Fig. 1I. In 10/14 CJD CSF samples the RT-QuIC assay was positive (71.4% sensitivity). False negative results were obtained for one sample from a patient with M/M codon 129 polymorphism and an NF-L serum just above the cut-off level, and for 3 gCJD samples from patients with E200 K and E196 K mutations. These 3 patients were positive for CSF 14-3-3 and above the cut-off for all other markers, with the exception that the E200 K mutation carrier had a tau CSF level clearly below the diagnostic threshold. CSF samples from 6 non demented control cases failed in the RT-QuIC assay (100% specificity). After a prolonged amplification time, in 2 out of 6 control CSF samples a signal emerged after 48 h and 96 h, respectively, resulting in a signal less than 10% of the signal obtained with CJD samples after 5 days of measurements. In the CSF from the presymptomatic gCJD patient no seeding activity could be detected. At the onset of first symptoms the signal was positive with minimally delayed starting point of about 3 h and a maximum similar to that determined in the other positive CJD samples.

Discussion
Today, improvement of CJD diagnosis is attempted in two ways: (1) There is a search for surrogate markers in the serum of CJD patients, and (2) Methods are developed to amplify the minute amounts of abnormal PrP present in body fluids for a specific diagnosis.
In the present study we show that the known biomarkers NF-L and tau measured in serum samples parallel the increase found in CSF, yielding similar statistical significance for differentiation. NF-L, which also is elevated in motor neuron disease [13][14][15] , is found at even higher levels in CJD patients. pNF-H which was found to be increased also in amyotrophic laterla sclerosis (ALS) plasma by in house assays 16,17 , yielded excellent diagnostic performance in CSF in our cohort. Additionally, the analysis of the serum from a presymptomatic gCJD patient provides first evidence for pNF-H to be elevated already before symptoms appear and clinical diagnosis can be made.
If serum NF-L qualifies as marker for broad differential diagnoses has to be examined in future studies including especially patients with rapidly progressive neurodegenerative dementia. As recent data point to a generally strong correlation between CSF and blood levels of NF-L and proteopathic lesions 18 , high diagnostic power for the discrimination of CJD and at least neurodegenerative diseases is likely to be expected.
Serum tau can now be detected more sensitively and is shown for the first time to be increased not only in sCJD 19 but also in gCJD. Differences between gCJD and controls, however, were less marked compared to NF-L and pNF-H.
The time point at which the biomarkers increase in CSF and also in serum is a mostly open question. It can be hypothesized that the analyzed markers are increased early in CJD as they indicate neuroaxonal degeneration. We found the presymptomatic stage of one CJD mutation carrier characterized by normal serum and CSF tau and serum S100B. Neurofilaments, especially pNF-H in the CSF, already show a trend for increased levels and might therefore represent candidate markers for the onset of the disease as it was shown for asymptomatic ALS gene carrier 15 . At symptom onset of the gCJD patient, all markers are clearly increased in serum and CSF to the range of the CJD cohort.
Similar results were obtained with the specific approach, the RT-QuIC, which has been established for CJD diagnostics from CSF 12,20,21 and urine 22 , however, failed to be successful in serum until now. Seeding activity could not be detected in the CSF from the presymptomatic phase, but in parallel to the appearance of first CJD symptoms.
Taken together, we show that serum NF-L and serum tau can be used in the diagnosis of both sCJD and gCJD and provide preliminary evidence that neurofilament levels tend to be increased presymptomatically.

Methods
Patients. This study was conducted according to the principles expressed in the Declaration of Helsinki.
Collection and analysis of samples were approved by the Ethics Committees of the Medical Faculties of the University Göttingen (approval number 100305) and Ulm (approval number 20/10). All patients or their next relatives in case of severe dementia gave written informed consent to their participation in the study.
In total, our retrospective study included 103 patients that were seen in the general outpatient clinic and the outpatient memory clinic of the Department of Neurology in Ulm and the surveillance unit for transmissible spongiform encephalopathies in the Department of Neurology in Göttingen.
All methods were performed in accordance with the relevant guidelines and regulations. CSF was obtained by lumbar puncture, centrifuged, aliquoted and stored within 2-48 h at − 80 °C until analysis. Serum was processed likewise and stored within 2 h at − 80 °C.
For demographic characteristics of the diagnostic groups see Table 1.