Diagnostic Accuracy of a Plasma Phosphorylated Tau 217 Immunoassay for Alzheimer Disease Pathology

This cohort study examines data from 3 single-center observational cohorts to test the capabilities of a commercially available plasma phosphorylated tau 217 immunoassay to identify Alzheimer disease pathophysiology.


I
n Alzheimer disease (AD), blood biomarkers have emerged as scalable tools for clinical evaluation, trial recruitment, and disease monitoring. 1 Their anticipated implementation aims to substantially reduce the reliance on cerebrospinal fluid (CSF) or positron emission tomography (PET) scans in specialized centers. 2 Moreover, a robust and accurate bloodbased biomarker would enable a more comprehensive assessment of cognitive impairment in settings where advanced testing is limited.Therefore, use of a blood biomarker is intended to enhance an early and precise AD diagnosis, leading to improved patient management and, ultimately, timely access to disease-modifying therapies.
Phosphorylated tau (p-tau) is the leading blood biomarker candidate, demonstrating superior diagnostic accuracy and disease specificity compared with other candidates. 3,4The amyloid β 42/40 (Aβ42/40) ratio, a validated CSF biomarker, 5 has limitations in blood 6,7 and lacks the robustness required for routine clinical testing. 8,9In contrast, high-performing p-tau blood test results exhibit a substantial increase in patients with AD, 10 occurring concurrently with extracellular Aβ plaque deposition, an AD hallmark feature.6][17] Thus, p-tau is regarded as the primary blood biomarker for AD pathology throughout all stages of the disease.
Among proposed blood tau biomarkers, [18][19][20][21] phosphorylated tau at threonine 217 (p-tau217) has consistently shown high performance in differentiating AD from other neurodegenerative disorders 10,22 and in detecting AD pathology in patients with mild cognitive impairment (MCI). 22Notably, p-tau217 exhibits larger-fold changes compared with p-tau181 and p-tau231, 10 often achieving high discrimination, with areas under the curve (AUC) exceeding 90%. 19,23Additionally, p-tau217 demonstrates a unique longitudinal trajectory, showing increases associated with worsening brain atrophy and declining cognitive performance in individuals with elevated Aβ pathology. 24,25ith the imminent implementation of anti-Aβ therapies in dementia management, validated blood biomarkers are urgently needed to guide timely treatment decisions.While plasma p-tau217 has shown promise as a diagnostic tool for AD, its widespread evaluation has been hindered by limited availability of commercial assays.This study aims to address this gap by assessing the utility of ALZpath pTau217, a commercially available immunoassay, to highlight the presence of AD pathology.In addition, we aim to report reference ranges of the plasma p-tau217 that correspond to abnormal amyloid PET and CSF measures.

Methods
This study included participants from 3 observational cohorts: the Translational Biomarkers in Aging and Dementia (TRIAD), Wisconsin Registry for Alzheimer's Prevention (WRAP), and Sant Pau Initiative on Neurodegeneration (SPIN).
Participants gave written or verbal informed consent, and the studies were approved by the relevant ethics boards (eMethods in Supplement 1).A subset of patients with longitudinal follow-up consisted of 392 participants from TRIAD and WRAP defined by PET biomarkers (eTable 1 in Supplement 1).These included participants classified into 3 groups: amyloid-and taunegative (A−T−; n = 297), amyloid-positive and tau-negative (A+T−; n = 66), and amyloid-positive and tau-positive (A+T+; n = 29).In WRAP, the median number of samples collected per patient was 3 over a mean (SD) of 5.22 (1.41) years.In TRIAD, median samples per patient was 2, collected over a mean of 1.90 (0.61) years.

Imaging, CSF, and Plasma Biomarkers
Detailed imaging methods for TRIAD, WRAP, and SPIN are found in the eMethods in Supplement 1.In TRIAD, Aβ and tau PET were determined by [ 18 F]-AZD4694 28 and [ 18 F]-MK6240, 29 respectively.In WRAP, PET measures were determined by [ 11 C]-PiB 30 and [ 18 F]-MK6240. 31,32In SPIN, Aβ PET was determined by [ 18 F]-florbetapir or [ 18 F]-flutemetamol in a smaller subset of participants, with CSF Aβ42/40 used to define A status for most participants as described below.Tau PET was not available for the SPIN cohort, and T was defined by CSF p-tau181.Aβ-PET positivity was standardized across cohorts as a centiloid value greater than 24 (standardized uptake value ratio [SUVR] >1.55 for [ 18 F]-AZD4694 33 or distribution volume ratio >1.2 for [ 11 C]-PiB).Tau positivity with [ 18 F]-MK6240 was defined as

Key Points
Question What are the capabilities of a commercially available plasma phosphorylated tau 217 (p-tau217) immunoassay to identify Alzheimer disease pathophysiology?Findings This cohort study found that the p-tau217 immunoassay showed similar accuracies to cerebrospinal fluid biomarkers in identifying abnormal amyloid β (Aβ) and tau pathologies.A 3-range reference for detecting abnormal Aβ pathology was consistent across 3 cohorts; over 8 years, the largest change of p-tau217 was in individuals positive for both Aβ and tau.

Meaning
The wider availability of high-performing assays may expedite the use of blood biomarkers in clinical settings and benefit the research community.
meta-temporal region of interest SUVR greater than 1.24 for TRIAD 34 and SUVR greater than 1.3 in WRAP.
CSF sample collection procedures were similar across cohorts and are described in the eMethods in Supplement 1.In TRIAD and SPIN, Lumipulse G1200 or G600II was used to quantify CSF Aβ42, Aβ40, and p-tau181. 35,36Additionally, CSF p-tau217 was quantified by an in-house single-molecule array (Simoa) developed at the University of Gothenburg. 33A novel Simoa for CSF p-tau205 was measured in TRIAD only.For WRAP, CSF Aβ42, Aβ40, and p-tau181 were measured using the Roche NeuroToolKit. 37lasma samples from TRIAD, WRAP, and SPIN were analyzed at the Department of Psychiatry and Neurochemistry, University of Gothenburg.Plasma Aβ42/40, glial fibrillary acidic protein (GFAP), and neurofilament light chain (NfL) were quantified using the commercial Neurology 4-plex E kit (103670; Quanterix).Plasma p-tau231 and p-tau181 were analyzed using in-house Simoa assays developed at the University of Gothenburg, 18,38 except in WRAP where plasma p-tau181 was quantified by the commercial Advantage kit version 2.1 (104111; Quanterix). 24

Novel p-Tau217 Assay
The commercial ALZpath pTau217 assay for p-tau217 uses a proprietary monoclonal p-tau217 specific capture antibody, an N-terminal detector antibody, and a peptide calibrator.It has been validated as a fit-for-purpose assay 38 with a limit of detection of 0.0052 to 0.0074 pg/mL, a functional lower limit of quantification of 0.06 pg/mL, and a dynamic range of 0.007 to 30 pg/mL.The spike recovery for the endogenous analyte was 80%, and intrarun and interrun precision was 0.5% to 13% and 9.2% to 15.7%, respectively.Here, the assay demonstrated good repeatability (4%-8.7%)and intermediate precision (3.5%-10.7%)as shown in eTable 2 in Supplement 1.

Statistical Analysis
Between-group comparisons were conducted using linear models, adjusting for age and sex.Determining Aβ-PET and tau-PET positivity and other outcomes was done using receiver operating characteristics AUC and compared with those of other established biomarkers with the DeLong test.Correlations were always evaluated using Spearman ρ.A binary reference point for Aβ-PET positivity was derived based on the Youden index.
Alternatively, a 3-range strategy comprised a lower reference point to rule out AD (95% sensitivity) and a higher reference point to rule in AD (95% specificity).In both strategies, we evaluated the concordance of a negative p-tau217 result with Aβ-PET negativity (negative percent agreement), and the concordance of a positive plasma p-tau217 with Aβ-PET positivity (positive percent agreement), as well as the overall percent agreement.In the latter strategy, individuals with p-tau217 levels between the reference point were classified as intermediate risk and would constitute the population referred to confirmatory testing. 39e evaluated the longitudinal trajectories of plasma p-tau217 in participants with no cognitive impairment and those with MCI according to their amyloid (A) and tau (T) status.We used linear mixed-effects models with plasma p-tau217 as the response variable, including as predictors time (since first plasma collection), AT status, age at first plasma collection, years of education, sex, and cognitive status at first visit, as well as an interaction between AT status and time.The model contained random intercepts and random slopes for each participant, and time was modeled as a continuous variable.Post hoc pairwise contrasts were conducted to compare the slopes for group × time interactions.
All analyses were performed using R version 4.2.2 (R Project for Statistical Computing), with a 2-sided α of .05.No adjustments for multiple comparisons were performed. 40eported results include 95% confidence intervals when applicable.

Participant Characteristics
A total of 786 participants (mean [SD] age, 66.3 [9.7]  ) also had information on tau status (TRIAD and WRAP: tau PET; SPIN: CSF p-tau181), as described in Table 1 alongside demographic and clinical information for all crosssectional analyses.eTable 2 in Supplement 1 describes the TRIAD and WRAP longitudinal subsets.

p-Tau217 Levels by Amyloid and Tau Status
When stratified by AT status, regardless of clinical diagnosis, plasma p-tau217 significantly increased in a stepwise manner in all cohorts (Figure 1

Comparing p-Tau217 With Other Plasma Biomarkers
Plasma p-tau217 alone or p-tau217 plus demographic variables (age, sex, and APOE status) outperformed all other plasma biomarkers (p-tau181, p-tau231, Aβ42/40, GFAP, and NfL), and their optimal combinations, for predicting both amyloid and tau status in all cohorts (eTables 4 and 5 and eFigure 7 in Supplement 1).A minimal improvement in model metrics of goodness-of-fit (Akaike information criterion) was observed in p-tau217 plus demographic data but not in discriminatory performance.The correlations of plasma p-tau217 with Aβ PET, tau PET, and CSF p-tau217 are shown in eFigures 9 and 10 in Supplement 1.

Discussion
In 3 independent cohorts, this study presents the performance of a commercially available plasma assay targeting p-tau217.Our findings demonstrate high accuracy in identifying abnormal Aβ and tau pathologies, comparable with CSF measures and superior to brain atrophy assessments.A 3-range approach demonstrated high negative and positive concordance with Aβ status, with approximately 20% of individuals in an intermediate zone that would require confirmatory CSF or PET, as previously proposed. 41Longitudinally, this assay exhibited increases solely in individuals with Aβ pathology at baseline, and those with both elevated Aβ and tau pathologies demonstrated a greater rate of annual increase.Plasma biomarkers have emerged as important tools for AD evaluation.Their specificity to underlying pathology offers great potential for rapid screening, reducing the dependence on advanced confirmatory tests.A clinical AD diagnosis often lacks sensitivity and specificity, resulting in many individuals with MCI (40%-60%) or dementia (20%-30%) who exhibit typical AD symptoms lacking Aβ pathology. 1In primary care, it is estimated that more than 50% of patients with cognitive impairment remain undiagnosed or incorrectly diagnosed because of the lack of accessible and cost-effective tools. 1 Thus, blood biomarkers are set to revolutionize clinical care by providing  objective biomarker-based information.As anti-Aβ trials move toward targeting a preclinical population with lower prevalence of Aβ abnormalities, 42 a cost-effective screening strategy becomes paramount.In previous studies, targeting p-tau217 in blood has yielded the best results as a diagnostic and prognostic tool that tracks longitudinal change.
There has been limited access to immunoassays targeting p-tau217 for broader evaluation.This study evaluates a commercially available assay for p-tau217 that exhibits similar advantageous features to those previously reported.Consistent with Palmqvist et al, 19 this assay outperformed magnetic resonance imaging and showed comparable performance with CSF biomarkers in detecting Aβ PET positivity and tau PET positivity. 43Further, significant superiority to other plasma p-tau epitopes, Aβ42/40, NfL, and GFAP and their optimal combinations was shown.When combined with APOE status and age, only modest improvements in diagnostic accuracy were observed, whereas other plasma biomarkers relied more heavily on these variables for their performance.Notably, the assay demonstrated high accuracy in identifying tau pathology within Aβ-positive individuals.This is particularly important as antiamyloid therapies may be less effective in patients with advanced tau pathology. 44,45Our findings suggest that p-tau217 has the potential to identify elevated tau-PET uptake and promising utility in early AD trials.Our study did not define elevated tau in the same manner as the TRAILBLAZER trials but warrants further studies applying p-tau217 to intermediatetau trial inclusion designs. 45ntegrating blood biomarkers into diagnostic workflows remains challenging despite their promise.Therefore, this study also aimed to establish reference points based on abnormal Aβ pathology.The study evaluated a 3-range approach as recommended by Alzheimer's Association guidelines 39 and recently proposed by Brum et al, 41 which suggests confirmatory testing for patients with uncertain plasma p-tau217 results.Evaluating this approach using a commercial immunoassay showed high negative and positive predictive accuracy at screening, indicating only 12% to 23% of individuals warranted advanced testing, depending on the clinical stage.However, we acknowledge that the cohorts used in this study may not fully represent real-world clinical settings.Importantly, the reported negative and positive predictive accuracy of these reference ranges can vary based on the prevalence of the outcome in the target population.Lower positive percent agreements are expected in settings with lower prevalence, 46 as observed in the preclinical WRAP cohort compared with the higher prevalence seen in TRIAD and SPIN cohorts.Therefore, future studies should prospectively evaluate plasma p-tau217 reference points in memory clinic populations with wider diversity to ensure optimized implementation, accounting for higher rates of important comorbidities. 47

Limitations
This study is not without limitations.First, one-third of our participants were classified as cognitively impaired, and this may limit our generalizability to the symptomatic stages of the disease but highlights promise for future preclinical recruitment.In addition, our results cannot be generalized to all individuals without detailed examination in cohorts with a larger representation of diverse ethnic populations.We acknowledge that CSF p-tau181, utilized as a T marker in SPIN, is not interchangeable with other methods that more accurately reflect neurofibrillary tangle pathology. 48

Conclusions
This study highlights the effectiveness of a commercially available plasma p-tau217 assay in identifying AD pathology.Our findings demonstrate the substantial reduction of confirmatory testing, by approximately 80%, by implementing a 3-range approach for Aβ positivity based on plasma p-tau217.These results emphasize the important role of plasma p-tau217 as an initial screening tool in the management of cognitive impairment by underlining those who may benefit from antiamyloid immunotherapies.

Diagnostic
Accuracy of a Plasma Phosphorylated Tau 217 Immunoassay for Alzheimer Disease Pathology Original Investigation Research jamaneurology.com (Reprinted) JAMA Neurology March 2024 Volume 81, Number 3

Table 2 .
Binary Reference and Three-Range Reference for Aβ Positivity a The table shows key metrics for the evaluation of a binary and 3-range reference point for Aβ positivity.The binary reference point was based in the Youden index derived in the WRAP cohort and cross-validated in the TRIAD and SPIN cohorts.Three-range reference points for Aβ positivity were derived in WRAP based on 95% sensitivity (lower reference point) and 95% specificity (upper reference point) and cross-validated in TRIAD and SPIN.The OPA for p-tau217 negative and positive indicates the combined NPA of those below the lower reference point and the PPA for those above the upper reference point, not accounting for the intermediate zone.In WRAP and TRIAD, Aβ positivity was determined with Aβ positron emission tomography, whereas in SPIN, Aβ positivity was determined with cerebrospinal fluid Aβ42/Aβ40. a