Diagnostic Accuracy of a Combined Analysis of Cerebrospinal Fluid t-PrP,t-tau,p-tau,and Aβ 42 in the Differential Diagnosis of Creutzfeldt-Jakob Disease from Alzheimer’s Disease with Emphasis on Atypical Disease Variants

INTRODUCTION

Creutzfeldt-Jakob disease (CJD) and Alzheimer’s disease (AD) are neurodegenerative disorders with overlapping clinical and laboratory features. CJD includes six major clinical-pathological subtypes that are largely determined by the genotype at the methionine (M)/valine (V) polymorphic codon 129 of the PRNP gene and the type (1 or 2) of pathological prion protein accumulating in the brain (namely MM1, MM2, MV1, MV2, VV1, and VV2) [1]. While the most common MM/MV1 subtype usually manifest with a subacute clinical course, atypical sporadic CJD variants such as MV 2K, MM 2C, and VV1 show a significantly slower clinical course, which may mimic AD and other neurodegenerative dementias [2]. On the other hand, although AD typically presents with a slowly progressive and well defined profile of cognitive decline, there also are well recognized variants of the disease with a rapid clinical progression and/or focal neurological signs manifesting relatively early in the disease course [3–6]. Cerebrospinal fluid (CSF) biomarker analysis is increasingly used in the diagnostic work-up of patients with neurodegenerative dementia. In particular, the search for elevated, above threshold, levels of protein 14-3-3 and/or t-tau is recommended by diagnostic criteria for CJD [7, 8], whereas a reduced concentration of amyloid-β peptide 1–42 (Aβ₄₂) in combination with increased levels of both total (t-tau) and/or phosphorylated tau (p-tau) represents a well-established neurochemical profile in support of the clinical diagnosis of AD [9, 10]. However, the lack of full sensitivity and specificity of these biomarkers still represents a significant limitation. In particular, approximately 10% of AD patients have t-tau levels compatible with CJD and/or show a positive 14-3-3 assay [5, 11–19], whereas a similar proportion of CJD cases, especially those of the MV2K, MM2C, and MM2T subtypes, is associated with a negative 14-3-3 test and/or with low t-tau levels [2, 19–21]. Finally, reduced Aβ₄₂ CSF levels have also been found in CJD [22–27]. To improve the differential diagnosis between AD and CJD based on CSF biomarkers, some studies analyzed the performance of the combined analyses of multiple protein markers including t-tau and p-tau, but also total prion protein (t-PrP) [28–30]. Most significantly, the addition of t-PrP dosage and the calculation of the t-tau/(p-tau×t-PrP) ratio, also designated as “CJ factor”, has allowed, to date, the most accurate diagnosis in AD with clinical overlap with CJD [29]. However, these results have not been widely validated and there have been some divergent results between studies in terms oft-PrP CSF levels. Furthermore, the accuracy of the proposed CSF biomarker combination in the atypical variants of CJD has not been investigated as yet. In this study, we evaluated the diagnostic performance of CSF t-PrP levels either alone or in combination with other protein biomarkers, including Aβ₄₂ in typical and atypical variants of both CJD and AD.

MATERIALS AND METHODS

RESULTS

DISCUSSION

In this study we investigated for the first time the accuracy of CSF protein t-PrP, t-tau, 14-3-3, p-tau, and Aβ₄₂ assays in the differential diagnosis between CJD and AD in a patient series including a high proportion of clinically atypical cases of both diseases. Furthermore, at variance with most previous studies which have mainly focused on t-tau, p-tau, and, to a lesser extent t-PrP, we also considered the added diagnostic value of Aβ₄₂.

At variance with classic biomarkers such as t-tau, 14-3-3, p-tau, and Aβ_42, there is still limited knowledge about t-PrP in the CSF in both controls and patients with neurodegenerative diseases. To date, few studies have measured CSF PrP levels in CJD, AD, and controls. Our finding of a statistically significant decrease in CSF t-PrP levels in CJD patients is consistent with those of other authors [29, 37–39]. Thus, as CSF Aβ₄₂ levels inversely correlate with amyloid burden in AD, t-PrP levels likely reflect the extent of PrP^c conversion into abnormal PrP (PrP^Sc) and the burden of PrP^Sc deposition in CJD. The latter may also explain our finding of an even higher reduction of t-PrP levels in atypical CJD cases. Indeed, atypical CJD variants such as MV 2K and MM 2C are usually associated with a relatively high amount of PrP^Sc accumulation involving major areas of the brain such as the cerebral cortex, basal ganglia, and thalamus.

Our results on t-PrP CSF levels in AD are at variance with both Dorey et al. [29] and Meyne et al. [40], who found either increased or decreased t-PrP levels in AD compared to controls. While the latter discrepancy has been attributed to the lack of specificity for the human prion protein of the assay used by Meyne et al. [40], the former may reflect differences in the selected patient populations. Whatever the case, further studies are needed to clarify the divergence, especially in view of the proposed putative role of PrP^C in AD pathogenesis [29].

Regarding the diagnostic value of t-PrP in the discrimination of CJD from AD, we reported an AUC of 0.825, a sensitivity of 73%, and a specificity of 75% using a cut-off value of 261 ng/ml, which is slightly worse than that reported by Dorey et al. (AUC = 0.886, 82.1% sensitivity and 82.4% specificity) using the same kit and a very similar cut-off value (263 ng/ml). The small discrepancy likely reflects their findings of increased t-PrP levels compared to normal subjects in a subgroup of ADpatients.

In this study, we also addressed the possible diagnostic role of CSF Aβ₄₂ and its derived ratios in the clinical distinction between CJD and AD. Van Everbroeck et al. [22] originally reported that Aβ₄₂ levels are decreased in CJD, a finding that has subsequently been confirmed by others [22–27]. More recently, however, some studies showed that Aβ₄₂ levels and Aβ₄₂/p-tau ratio are significantly higher in CJD than in AD patients [21, 41], thus indicating that the determination of Aβ₄₂ levels might contribute to the differential diagnosis between the two diseases. In our CJD population, Aβ₄₂ CSF levels were highly heterogeneous, with several cases of both typical and atypical CJD groups showing a lower than cut-off value. Whether this result simply reflects the burden of associated AD pathology, which has to be taken in account considering the mean age of death of CJD patients, or also depends on a pathogenic interaction between the two proteins, as it has recently been suggested [42, 43], remains to be seen.

Taken together, our results underline the added value of both Aβ₄₂ and t-PrP CSF assays in the differential diagnosis between CJD and AD. Indeed, the addition of t-PrP and Aβ₄₂ values to those of t-tau and p-tau in a novel ratio (t-tau×Aβ₄₂)/(p-tau×t-PrP) distinguishes CJD from AD patients with greater accuracy (0.995 accuracy, 98.1% sensitivity, and 97.7% specificity) than all previously-described ratios such as CJ factor, t-tau/p-tau, and Aβ₄₂/p-tau [21, 41]. Most importantly, the novel ratio also demonstrates superior diagnostic accuracy in the most difficult clinical scenario, which is the clinical distinction between atypical CJD and a/rpAD patients. Indeed, while CSF t-tau and 14-3-3 do not contribute at all to the differential diagnosis of such atypical variants (e.g., 61.1% and 25.9% sensitivity; 50% and 84.1% specificity), in the same situation the novel ratio yields 96.2% sensitivity and 95.5% specificity. We therefore strongly recommend extending the analyses to all four biomarkers in the presence of atypical clinical features and ambiguous results of standard CSF assays. Alternatively, particularly in a laboratory setting where the t-PrP assay is not available, Aβ₄₂×t-tau/p-tau and t-tau/p-tau ratios remain the best biomarkers for accuracy, sensitivity, and specificity in distinguishing CJD from AD.

We are aware that one potential limitation of our study is the discrepancy in the number of neuropathologically-verified cases between CJD and AD. Nevertheless, all AD affected subjects included in this study fulfilled the diagnostic criteria of probable AD with high evidence of the pathophysiological process of AD. Furthermore, in all a/rpAD cases, the final diagnosis was formulated at follow-up after at least 2 years of further clinical observation.

In summary, while individually none of the major CSF proteins that reflect the specific molecular pathology of AD (p-tau and Aβ₄₂) and CJD (PrP) or the associated neuronal damage (t-tau, 14-3-3) distinguish the two disorders with sufficient accuracy, various combinations of these markers significantly increase the diagnostic power. Among, them the (t-tau×Aβ₄₂)/(p-tau×t-PrP) ratio best distinguishes CJD from AD patients and is especially recommended in the diagnostic work-up of patients presenting with atypical clinical features that are compatible with both diseases.