Prion-specific and surrogate CSF biomarkers in Creutzfeldt-Jakob disease: diagnostic accuracy in relation to molecular subtypes and analysis of neuropathological correlates of p-tau and Aβ42 levels

Human prion diseases, also known as transmissible spongiform encephalopathies (TSE), are rapidly progressive neurodegenerative disorders caused by prion protein misfolding [57]. With an annual incidence of ~2 cases per million persons sporadic Creutzfeldt-Jakob disease (sCJD) is by far the most common form (85–90% of cases) [53], followed by genetic CJD (gCJD) and fatal familial insomnia (10–15% of cases), which are linked to point or insertion mutations in the prion protein gene (PRNP) [14]. Rare acquired forms of CJD caused by human to human or animal to human transmission of infectious prions [74], and atypical disease variants with a slower progression, such as Gerstmann-Sträussler-Scheinker syndrome (GSS), prion protein-cerebral amyloid angiopathy (PrP-CAA) and variably protease-sensitive prionopathy (VPSPr) complete the phenotypic spectrum of human prion diseases of the CNS [25, 81].

CJD is increasingly recognised in the differential diagnosis of rapidly progressive neurological syndromes, highlighting the need for reliable tools to provide an early clinical diagnosis [23, 33, 61, 62]. The ageing population, the improved awareness of CJD as a heterogeneous disorder covering a wider phenotypic spectrum than previously recognised [53], and the identification of potentially treatable disorders that manifest as rapidly progressive dementia (RPD) [8, 16, 24] have all contributed to this scenario.

Current diagnostic criteria for sCJD [80] were mainly developed for epidemiological purposes and do not take into account the extent of clinical heterogeneity shown by sCJD patients. This is a major cause of the difficulties in the clinical diagnosis and differentiation of prion diseases against other neurological disorders. Currently, at least six major subtypes of sCJD are recognised, which are largely determined by the genotype at the polymorphic codon 129 (encoding methionine, M or valine, V) in PRNP, and by the type (type 1 or type 2) of PrP^Sc accumulating in the brain [50, 52]. The number and variety of clinical symptoms (especially at onset), the rate of disease progression, and the underlying regional brain pathology vary significantly among sCJD subtypes; this affects the relative accuracy of the proposed diagnostic criteria.

CSF protein assays combined with special MRI techniques such as FLAIR and DWI currently represent the most useful in vivo markers for sCJD [13, 78]. Specifically, several brain-derived CSF proteins serving as surrogate markers for neuronal damage have been considered, alone or in combination, for their utility in supporting the clinical diagnosis of probable CJD. Among them, CSF protein assays for 14‑3-3, t-tau and p-tau (for the calculation of the t-tau/p-tau ratio) have contributed the most promising and significant results regarding sensitivity and specificity in distinguishing CJD from other RPDs, although with a significant heterogeneity both in terms of number of studies and agreement regarding the relative performance of each assay [6, 9, 12, 17, 18, 22, 30, 44, 48, 60, 63, 67, 68, 72, 79]. More recently, the development of the RT-QuIC assay [5], an in vitro fluorimetric assay which is able to indirectly detect very low amounts of prions, based on the capacity of PrP^Sc to induce recombinant (rec-) PrP conversion and aggregation, has provided a very promising tool to improve the early diagnosis of human prion diseases in a noteworthy manner given its high degree of sensitivity (82–97%) and specificity (99–100%) [5, 19, 41, 45, 46, 55, 64].

The evaluation of biomarker accuracy for an early clinical diagnosis should be ideally conducted on a clinically based cohort of patients with RPD in which the clinical suspicion of prion disease was raised. Nevertheless, only a few studies considered this approach [6, 9, 17, 48, 62, 71, 80]. Furthermore, only a portion of previous studies specifically analysed the effect of the disease subtype on the sensitivity and specificity of the available biomarker assays [15, 28, 30, 35, 38, 48, 63, 77]. Finally, knowledge about the reliability of measuring CSF proteins such as p-tau and Aβ42 to predict the underlying neuropathology is based on a limited number of studies that often used post-mortem ventricular CSF or brain biopsies to maintain the time lapse between CSF and brain assessment at minimum [11, 65, 69, 70]. In this respect, brains affected by CJD provide the unique opportunity to correlate the CSF findings with the post-mortem neuropathology within a short time interval.

In the present study, we aimed to investigate the utility of several CSF biomarkers (14-3-3, t-tau, p-tau, Aβ42 and rec-PrP seeded conversion by RT-QuIC) in the differential diagnosis of CJD from other neurological disorders, in a large non-selected clinical population suspected to be affected by a prion disease. Furthermore, we assessed the diagnostic accuracy (specificity and sensitivity) of each assay employed in the present study, also considering the molecular subtypes in sporadic cases and the mutation type in genetic CJD patients. Finally, in definite CJD cases we correlated the CSF findings concerning p-tau and Aβ42 with the type and amount of tau and Aβ pathology in the brain.

The present study reports the results of a comprehensive analysis of all major available CSF biomarkers for the differential diagnosis of CJD in a large unselected clinical population reflecting clinical practice, taking also into account the effect of the disease subtype on diagnostic accuracy and the influence of tau and Aβ brain pathology on p-tau and Aβ42 CSF levels.

Our results confirm the very high specificity of RT-QuIC for the diagnosis of sCJD in a large series of CJD and non-CJD CSF samples. However, they also provide, for the first time, convincing evidence for a lower sensitivity of the assay in atypical subtypes of the disease characterised by PrP^Sc type 2 (e.g.,VV2, MV2K, and MM2C). The negative results we repeatedly obtained with the CSF samples from a patient affected by VPSPr and one from a GSS patient carrying the D202N mutation [P. Parchi, personal communication] are also consistent with this finding. Thus, as with CSF protein surrogate markers, the in vitro conversion reaction exploited by RT-QuiC is significantly affected by the neurobiological heterogeneity of human prions.

Recent studies using a different recombinant PrP substrate or olfactory mucosa brushings instead of CSF as an alternative seed reported a 96–97% sensitivity of the RT-QuIC assay for the diagnosis of sCJD [45, 46]. However, although very promising, these novel modified RT-QuIC assays have, to date, only been applied to sCJD cases largely represented by typical CJD patients affected by the MM(V)1 type. Furthermore, at variance with the methodology used in the present study [42], the modified assays have not been, as yet, validated in the inter-laboratory setting.

The reasons behind the variability in seeding activity among CSF samples from CJD patients remain, unfortunately, partially unclear. Indeed, besides the above-mentioned correlation with the disease subtype, we failed to reveal other common features among the CSF samples giving a negative RT-QuIC result. In particular, there was no significant correlation with age at onset, disease duration at the time of lumbar puncture, or with the results of both conventional protein assays and brain MRI. Future studies should further address this issue and establish, in the first instance, whether seeding activity correlates or not with PrP^Sc levels. Nevertheless, our observation of a higher conversion activity of the samples from gCJD patients carrying the E200K-129M haplotype (and PrP^Sc type 1), in comparison to those of the sCJD MM(V)1 group, which is confirmatory of the results of a previous study [19], supports the view of a direct role of PrP^Sc in seeding rec-PrP conversion and aggregation.

Regarding the more conventional CSF surrogate diagnostic markers, such as proteins t-tau and 14-3-3, our data demonstrate that t-tau is (moderately) superior to 14-3-3 in the diagnosis of sCJD. More specifically, t-tau yields a lower number of false positive results, especially in cases suffering from inflammatory-related conditions and subacute dementias; moreover, it has a higher sensitivity than 14-3-3 for the sCJD MV2K type. These results, which are in line with those reported by Hamlin et al. [30], together with the notion that the t-tau assay has technical advantages with respect to the standard western blot 14-3-3 assay, suggest a change in the current recommendations to prioritise t-tau analysis over 14-3-3. Interestingly, t-tau appears to be less accurate than 14-3-3 only in the differential diagnosis with AD; however, in cases where the differential diagnosis with AD is an issue, biomarker analysis can also take advantage of Aβ42 and p-tau and possibly total-PrP dosage, allowing for the calculation of t-tau/p-tau and other ratios based on different combinations of these four biomarkers, which have been reported to significantly improve diagnostic accuracy in such cases [1, 21, 67]. Outside this specific clinical scenario (CJD vs rapidly progressive AD), however, our data do not support the additional diagnostic value of t-tau/p-tau ratio with respect to t-tau alone. This lower diagnostic performance of t-tau/p-tau ratio compared to what is generally reported in the literature [9, 12, 21, 60, 67] likely reflects differences in the characteristics of the examined patient populations. Indeed, while previous studies mainly analysed the diagnostic accuracy of the t-tau/p-tau ratio in the differential diagnosis between typical CJD and AD or between CJD and large clinically unselected populations of patients with dementia, our study focused on patients referred for the suspicion of CJD and included a relatively high number of CJD cases of less common subtypes such as VV2, MV2K and MM2C, showing a significantly lower t-tau/p-tau ratio than the typical CJD MM(V)1.

Our results further underline the profound implications that the phenotypic heterogeneity of the disease has on diagnostic criteria and the overall clinical approach to a patient suspected of having CJD. Indeed, as also clearly revealed by our study, the sensitivity of both surrogate protein markers and RT-QuIC varies significantly according to the CJD type. Given that knowledge about the patient’s PRNP codon 129 genotype, one of the two major molecular determinants of the disease type, can be obtained from a blood sample, it is crucial to include codon 129 genotyping in the diagnostic work-up of patients with suspected CJD. This information, combined with those obtained from the patient’s clinical history and neurological examination, which is usually able to discriminate between a typical (MM(V)1) or atypical (all the others) disease subtype are crucial for the correct interpretation of the results of CSF assays. The most striking example of the importance of codon 129 genotyping for the clinical diagnosis of CJD concerns the VV2 subtype, the second most common sCJD type [53]. Given the relatively uniform and consistent clinical phenotype early in the disease course in these subjects, and the 100% sensitivity of standard CSF assays, the finding of VV at codon 129 in such a clinical scenario is highly confirmatory of the diagnosis of sCJD, even in cases with a negative RT-QuIC result. In contrast, the finding of a negative 14-3-3 test and/or t-tau levels below 1250 pg/ml in a codon 129 VV patient will exclude the diagnosis of sCJD VV2 with a 100% NPV.

One of the major issues in the field of diagnostic biomarkers concerns their correlation with the underlying brain pathology. Besides the analyses of the effect of CJD subtypes on all biomarkers analysed, in this study we have correlated neuropathological and CSF findings for both p-tau and Aβ. Our findings of increased CSF p-tau concentrations in sCJD types VV2 and MV2K, (both caused by CJD strain V2), is of particular significance in this respect. Indeed, although the CSF tau profile in CJD patients usually combines excessively high t-tau with relatively low p-tau concentrations [21, 57, 67, 68], we and other authors have clearly shown that CSF p-tau levels can be significantly raised in prion disease too, owing, once again, to the heterogeneity of the disease. In previous studies, variant CJD subjects showed raised CSF p-tau in most cases [29], and elevated levels of CSF p-tau have also been detected in VPSPr and GSS [56], as also shown in individual cases in the present study. Interestingly, a secondary tauopathy characterised by brain deposition of abnormal p-tau aggregates has been demonstrated in all these atypical phenotypes of prion disease by post-mortem studies [26, 27, 31, 58], although the data on VPSPr are very limited. Thus, our CSF findings raise the question of whether a tauopathy in response to PrP^Sc deposition also develops in classic CJD, especially in the VV2 or MV2K subtypes. Initial studies were either negative [27 or led to the conclusion that only prion diseases associated with PrP-amyloid plaques develop p-tau deposits, whereas those characterised by synaptic PrP deposits do not [66]. More recently, Reiniger et al. [59] demonstrated that a tiny punctate (also described as rod-, dot-, or stub-shaped), PrP-specific, p-tau immunoreactivity, positively correlating with PrP^Sc burden in the neocortex, is consistently found in sCJD brains, irrespective of the molecular subtypes and the AD-related associated pathology. Consistently, in a recent study by Kovacs et al. [36] these small neuritic profiles were regarded as the most frequent type of tau immunoreactivity in CJD brains. Pursuing the findings of these studies we demonstrated here, for the first time, that in classic CJD the prion-specific secondary tauopathy especially affects the sCJD subtypes linked to the V2 strain (e.g., VV2 and MV2K) and, most significantly, that in such cases the tauopathy is severe enough to determine a significant increase in p-tau levels in the CSF. Furthermore, we reported the detection of prion specific p-tau dots in VPSPr, a finding we have confirmed in a second case, for whom we had no CSF [P. Parchi personal communication]. Small neuritic p-tau positive profiles have been, to date, only documented in the frontal cortex of a single case of VPSPr [4]. Overall, our results expand on but also help, to some extent, to understand some of the previous findings. Indeed, the fact that both VV2 and MV2K are sCJD types characterised by plaque-like PrP deposits or even PrP-amyloidosis (MV2K) and are associated, on average, with a higher PrP^Sc burden than those of the MM(V)1 type [49] somehow fits with the data of both Sikorska et al. [66] and Reiniger et al. [59]. Moreover, our observation that the PrP-related tau deposits were rarely detected in the cases with severe neurodegeneration also explains the previously described lack of correlation between tau deposits and disease duration [59].

Aβ42 concentration in the CSF of sCJD patients has been measured in a number of studies, but with inconsistent results [21, 34, 47, 71, 73, 75, 77]. Early studies, based on a limited number of cases, found significantly decreased Aβ42 levels in CSF of CJD patients with mean values comparable to those of AD patients and with no apparent correlation with either the APOE genotype or the number of Aβ-positive plaques in the brain [34, 47, 71]. More recently, however, Varges et al. [73] demonstrated a dose-dependent effect of APOE-4 on the decrease in Aβ42 in the CSF of sCJD patients. Furthermore, other preliminary data suggest that Aβ42 levels in the CSF are significantly higher in sCJD than in AD. In the present study we definitely show, in a large group of sCJD cases, that (1) the CSF levels of Aβ42 are significantly higher in sCJD than in AD and that (2) about 60% of sCJD cases have a concentration of Aβ42 in the CSF within the range of controls. Nevertheless, our analyses of Aβ load in neuropathologically verified patients clearly shows that the decrease in CSF Aβ42 in CJD patients, although influenced by the co-morbid AD pathology, may occur irrespective of the plaque load. Thus, the more likely explanation for the reduced Aβ42 levels in a subgroup of sCJD patients seems to be a combined effect of age-related co-morbidity, influenced by the APOE genotype, and the effect of neuronal loss which can be very significant in CJD even a few months after clinical onset. An alternative explanation, which has been put forward in previous studies [47, 75], indicates that in pathologic conditions such as CJD a fraction of Aβ42 cannot be detected by conventional ELISA methods as epitopes may be masked.

Our findings have also implications for the issue of CSF biomarkers reliability in AD. Indeed, at variance with most neurodegenerative disorders including AD, brains affected by CJD and other rapidly progressive dementias provide the unique opportunity to correlate the CSF findings with the post-mortem neuropathology within a short time interval. Although our data confirm an overall positive correlation between p-tau levels in the CSF and the extent of brain tauopathy and an inverse correlation between Aβ42 levels and β-amyloid load, it must be emphasized that the correlation for Aβ42 and β-amyloid is rather gross. Of most significance in this respect, also given the “negative” effect of CJD on Aβ42 concentration in the CSF, is our finding of Aβ42 levels within the control range in 68% of CJD brains with a co-occurring significant Aβ deposition in the brain (Thal phases I–III). If confirmed in brains with other pathologies, this finding would suggest that the Aβ42 level in the CSF is far from being an optimal marker of the early phases of Aβ deposition in the brain in pre-clinical AD. Given that our data are limited to Aβ42, it will be important to expand the correlative analysis to other Aβ peptides such as Aβ40. Indeed, evidence suggests a better diagnostic performance of the Aβ42/40 CSF concentration ratio compared to the Aβ42 concentration alone [39, 76]. Furthermore, although AD typically affects both cerebral hemispheres to a similar degree, the fact we have only examined one hemisphere might be considered as a potential limitation of our study.

At variance with Aβ42, according to our data, CSF p-tau level represents a more reliable marker of brain AD-related tau pathology since it fails to consistently discriminate between patients with or without neurofibrillary pathology only when p-tau deposition is relatively mild and limited to the transentorhinal/entorhinal cortices (Braak stages I–II).

In summary, our study provides confirmatory and novel evidence for a significantly improved value of CSF biomarkers for the clinical diagnosis of CJD. Although RT-QuIC is the most promising assay, given its high specificity, t-tau remains the most important surrogate marker. Our data support a change in the current diagnostic criteria for CJD, indicating both RT-QuIC and t-tau as primary laboratory investigations to be implemented in suspected cases of CJD, in combination, when possible, with codon 129 genotyping. In cases of a negative RT-QuIC result and elevated t-tau in a patient with rapidly progressive cognitive impairment, the differential diagnosis between CJD and atypical AD is also supported by the combined analyses of CSF p-tau, Aβ42 and possibly total-PrP [1, 21]. Finally, in cases with a negative RT-QuIC result the determination of CSF t-tau in combination with codon 129 genotyping remains of importance for the clinical diagnosis of sCJD VV2.

Despite the significant advances, the diagnostic value of both CSF biomarkers and cerebral MRI remain very low in some rare atypical variants of sporadic prion disease, such as sporadic fatal insomnia (MM2T) and VPSPr. Efforts should continue in the attempt to recognise such atypical forms through disease surveillance and the neuropathological examination of suspected cases. Future studies should also aim to develop a type-specific RT-QuIC or other assay to discriminate the molecular subtypes of sCJD cases in vitam to bring the epidemiological surveillance of sCJD to the next level.