Head-to-head comparison of clinical performance of CSF phospho-tau T181 and T217 biomarkers for Alzheimer's disease diagnosis

Abstract

Introduction: Phosphorylated tau (p-tau) in cerebrospinal fluid (CSF) is an established Alzheimer's disease (AD) biomarker. Novel immunoassays targeting N-terminal and mid-region p-tau181 and p-tau217 fragments are available, but head-to-head comparison in clinical settings is lacking.

Methods: N-terminal-directed p-tau217 (N-p-tau217), N-terminal-directed p-tau181 (N-p-tau181), and standard mid-region p-tau181 (Mid-p-tau181) biomarkers in CSF were evaluated in three cohorts (n = 503) to assess diagnostic performance, concordance, and associations with amyloid beta (Aβ).

Results: CSF N-p-tau217 and N-p-tau181 had better concordance (88.2%) than either with Mid-p-tau181 (79.7%-82.7%). N-p-tau217 and N-p-tau181 were significantly increased in early mild cognitive impairment (MCI)-AD (A+T-N-) without changes in Mid-p-tau181 until AD-dementia. N-p-tau217 and N-p-tau181 identified Aβ pathophysiology (area under the curve [AUC] = 94.8%-97.1%) and distinguished MCI-AD from non-AD MCI (AUC = 82.6%-90.5%) signficantly better than Mid-p-tau181 (AUC = 91.2% and 70.6%, respectively). P-tau biomarkers equally differentiated AD from non-AD dementia (AUC = 99.1%-99.8%).

Discussion: N-p-tau217 and N-p-tau181 could improve diagnostic accuracy in prodromal-AD and clinical trial recruitment as both identify Aβ pathophysiology and differentiate early MCI-AD better than Mid-p-tau181.

Keywords: Alzheimer's disease; biomarker; cerebrospinal fluid; dementia; memory clinic; phosphorylated tau-181; phosphorylated tau-217; prodromal Alzheimer's.

FIGURE 1

Description and validation of the p‐tau assays studied. A, (i) Schematic illustration of full‐length tau‐441 with the different regions marked. (ii), Antibodies used in the N‐p‐tau217 assay. The capture antibody specifically recognizes tau phosphorylated at threonine‐217 while the detection antibody binds the N‐terminal 6‐QEFEVMEDHAGT‐18 epitope. (iii) For the N‐p‐tau181 assay, the capture antibody specifically recognizes phosphorylation at threonine‐181 and the detection antibody targets the N‐terminal 6‐QEFEVMEDHAGT‐18 epitope. (iv) The Mid‐p‐tau181 assay (commercially available from INNOTEST) uses a capture antibody that is specific to tau phosphorylated at threonine‐181 and a detection antibody directed at the 159‐PPGQK‐163 epitope. The Lumipulse Mid‐p‐tau181 assay uses a similar antibody combination as the INNOTEST assay but the identity and exact epitopes of these antibodies are not published. ⁴⁰ B, A schematic illustration of full‐length tau‐441 with the different regions and epitopes of the N‐p‐tau217 antibody pair marked (iii). (i) In human CSF immunoprecipitated with the p‐tau217 antibody, an endogenous peptide (amino acid 212‐224) was identified (blue line); this peptide was phosphorylated at threonine‐217 as indicated by the purple circle. In (ii), the range of tryptic peptides identified from glycogen synthase kinase (GSK)‐3β‐phosphorylated full‐length tau‐441 (the assay calibrator; SignalChem #TO8‐50FN), immunoprecipitated using the p‐tau217 antibody. Cleavage positions for trypsin are indicated with vertical lines and the identified peptides are indicated in black while sequence portions not detected are shown in gray. (iv) The range of tryptic peptides identified from human cerebrospinal fluid (CSF) immunoprecipitated using the detection antibody, Tau12. The detected peptide sequence was from the acetylated N‐terminus up to amino acid 254, including peptides phosphorylated at threonine‐217. C, The illustration shows a schematic of full‐length tau‐441 with the antibodies used in the N‐p‐tau181 assay shown in (iv). In human CSF immunoprecipitated with AT270, only endogenous peptides phosphorylated at threonine‐181 were identified (i). These peptides were in the amino acid range 155 to 196 (blue lines with phosphorylation site indicated by purple circles). (ii) The range of tryptic peptides identified from GSK‐3β‐phosphorylated full‐length tau‐441 (the assay calibrator; SignalChem # TO8‐50FN) immunoprecipitated using the AT270 antibody. (iii) The range of tryptic peptides identified from human CSF, both immunoprecipitated using the AT270 antibody. Cleavage positions for trypsin are indicated with vertical lines, the identified peptides are indicated in black while sequence portions not detected are gray. In (v) is shown the range of tryptic peptides identified from human CSF immunoprecipitated using Tau12. Here, the detected range was from the acetylated N‐terminus up to amino acid 254, including peptides phosphorylated at threonine‐181. D, Aliquots from two different CSF samples were analyzed untreated (neat) or immunodepleted with the capture and detection antibodies (IP'ed) used in the N‐p‐tau217 assay. More than 95% of the measurable N‐p‐tau217 levels were lost after immunodepletion. E, Immunodepletion of two different CSF samples with the N‐p‐tau181 assay antibodies led to the removal of more than 94% of the available N‐p‐tau181 signal in each sample

FIGURE 1

Description and validation of the p‐tau assays studied. A, (i) Schematic illustration of full‐length tau‐441 with the different regions marked. (ii), Antibodies used in the N‐p‐tau217 assay. The capture antibody specifically recognizes tau phosphorylated at threonine‐217 while the detection antibody binds the N‐terminal 6‐QEFEVMEDHAGT‐18 epitope. (iii) For the N‐p‐tau181 assay, the capture antibody specifically recognizes phosphorylation at threonine‐181 and the detection antibody targets the N‐terminal 6‐QEFEVMEDHAGT‐18 epitope. (iv) The Mid‐p‐tau181 assay (commercially available from INNOTEST) uses a capture antibody that is specific to tau phosphorylated at threonine‐181 and a detection antibody directed at the 159‐PPGQK‐163 epitope. The Lumipulse Mid‐p‐tau181 assay uses a similar antibody combination as the INNOTEST assay but the identity and exact epitopes of these antibodies are not published. ⁴⁰ B, A schematic illustration of full‐length tau‐441 with the different regions and epitopes of the N‐p‐tau217 antibody pair marked (iii). (i) In human CSF immunoprecipitated with the p‐tau217 antibody, an endogenous peptide (amino acid 212‐224) was identified (blue line); this peptide was phosphorylated at threonine‐217 as indicated by the purple circle. In (ii), the range of tryptic peptides identified from glycogen synthase kinase (GSK)‐3β‐phosphorylated full‐length tau‐441 (the assay calibrator; SignalChem #TO8‐50FN), immunoprecipitated using the p‐tau217 antibody. Cleavage positions for trypsin are indicated with vertical lines and the identified peptides are indicated in black while sequence portions not detected are shown in gray. (iv) The range of tryptic peptides identified from human cerebrospinal fluid (CSF) immunoprecipitated using the detection antibody, Tau12. The detected peptide sequence was from the acetylated N‐terminus up to amino acid 254, including peptides phosphorylated at threonine‐217. C, The illustration shows a schematic of full‐length tau‐441 with the antibodies used in the N‐p‐tau181 assay shown in (iv). In human CSF immunoprecipitated with AT270, only endogenous peptides phosphorylated at threonine‐181 were identified (i). These peptides were in the amino acid range 155 to 196 (blue lines with phosphorylation site indicated by purple circles). (ii) The range of tryptic peptides identified from GSK‐3β‐phosphorylated full‐length tau‐441 (the assay calibrator; SignalChem # TO8‐50FN) immunoprecipitated using the AT270 antibody. (iii) The range of tryptic peptides identified from human CSF, both immunoprecipitated using the AT270 antibody. Cleavage positions for trypsin are indicated with vertical lines, the identified peptides are indicated in black while sequence portions not detected are gray. In (v) is shown the range of tryptic peptides identified from human CSF immunoprecipitated using Tau12. Here, the detected range was from the acetylated N‐terminus up to amino acid 254, including peptides phosphorylated at threonine‐181. D, Aliquots from two different CSF samples were analyzed untreated (neat) or immunodepleted with the capture and detection antibodies (IP'ed) used in the N‐p‐tau217 assay. More than 95% of the measurable N‐p‐tau217 levels were lost after immunodepletion. E, Immunodepletion of two different CSF samples with the N‐p‐tau181 assay antibodies led to the removal of more than 94% of the available N‐p‐tau181 signal in each sample

FIGURE 2

Concentrations of p‐tau biomarkers in the three cohorts. (A), (D), and (G) show N‐p‐tau217 concentrations in the discovery, Ljubljana, and Paris cohorts, respectively. The levels of N‐p‐tau181 in the discovery, Ljubljana, and Paris cohorts are given in (B), (E), and (H), respectively. The plots in (C), (F), and (I) show Mid‐p‐tau181 concentrations in the discovery, Ljubljana, and Paris cohorts, respectively. Participants in each cohort were classified according to clinical diagnosis and amyloid pathology. Group differences were compared using a two‐tailed Mann‐Whitney test (the discovery cohort) or Kruskal‐Wallis test followed by the Dunn's multiple comparison test (Ljubljana and Paris cohorts). Note that p‐tau concentrations were estimated from known concentrations of the assay calibrators. Assays that measure different p‐tau epitopes, those quantified on different analytical platforms as well as assays targeting different tau fragments (N‐terminal versus mid‐region p‐tau species) are therefore likely to give non‐identical values. For these reasons, the p‐tau concentrations measured by the different assays should not be compared by absolute pg/ml levels but rather according to their diagnostic performances and associations with Alzheimer's disease–type pathophysiologies

FIGURE 3

Accuracies of p‐tau biomarkers in identifying increased amyloid pathology, separating mild cognitive impairment‐Alzheimer's disease (MCI‐AD) from non‐AD MCI, and distinguishing AD dementia from amyloid beta (Aβ)– non‐AD. A, Area under the curves (AUC) comparing the predictive capacities of N‐p‐tau217, N‐p‐tau181, and Mid‐p‐tau181 to correctly identify individuals with increased Aβ pathology (assessed by cerebrospinal fluid [CSF] Aβ42/Aβ40 ratio). (B) and (C) depict AUC showing the abilities of the different p‐tau biomarkers to distinguish between individuals with MCI‐AD and Aβ– cognitively unimpaired (CU) groups and the MCI‐AD and non‐AD MCI groups, respectively. D, Diagnostic accuracies of p‐tau variants in separating between AD dementia and non‐AD dementia (including dementia with Lewy bodies, frontotemporal dementia, and vascular dementia patients). AUC values representing diagnostic accuracies for the different p‐tau biomarkers were statistically compared head‐to‐head using the DeLong test package in the MedCalc software. P values < .05 were considered statistically significant

FIGURE 4

Concordance among the three p‐tau assays. (A) N‐p‐tau217 versus N‐p‐tau181, (B) N‐p‐tau217 versus Mid‐p‐tau181, and (C) N‐p‐tau181 versus Mid‐p‐tau181 in the Ljubljana cohort. On each plot, the percentage of concordant cases are given in the upper right and lower left quadrants while the percent of discordant cases are shown in the upper left and lower right quadrants. Assay cut‐offs were set as the concentrations of the 95th percentage individual in the amyloid beta–negative cognitively unimpaired group.