Plasma p-tau217, NfL, GFAP diagnostic performance and biomarker profiles in Alzheimer's disease, frontotemporal dementia, and psychiatric disorders, in a prospective unselected neuropsychiatry memory clinic

Received: 10 July 2025 Revised: 18 August 2025 Accepted: 25 August 2025 DOI: 10.1002/alz.70717 RESEARCH ARTICLE Plasma p-tau217, NfL, GFAP diagnostic performance and biomarker profiles in Alzheimer’s disease, frontotemporal dementia, and psychiatric disorders, in a prospective unselected neuropsychiatry memory clinic Dhamidhu Eratne1,2Matthew Kang1,2Charles B Malpas3,4Christa Dang5,6 Courtney Lewis7Oneil G Bhalala8,9Qiao-Xin Li10 Steven Collins10 Colin L Masters10 Samantha M Loi1,2Alexander F Santillo11 Kaj Blennow12,13,14 Henrik Zetterberg13,15,16,17 Dennis Velakoulis1,2And The MiND Study Group 1Neuropsychiatry Centre, Royal Melbourne Hospital, Melbourne, Victoria, Australia 2Department of Psychiatry, University of Melbourne, Melbourne, Victoria, Australia 3Department of Medicine (Royal Melbourne Hospital), University of Melbourne, Melbourne, Victoria, Australia 4Melbourne School of Psychological Sciences, University of Melbourne, Melbourne, Victoria, Australia 5National Ageing Research Institute, Melbourne, Victoria, Australia 6Department of General Practice, University of Melbourne Victoria, Melbourne, Victoria, Australia 7Institute of Health and Wellbeing, Federation University, Melbourne, Victoria, Australia 8Department of Medicine, Royal Melbourne Hospital, Melbourne, Victoria, Australia 9Genetics and Gene Regulation Division, The Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria, Australia 10National Dementia Diagnostics Laboratory, The Florey, Melbourne, Victoria, Australia 11Clinical Memory Research Unit, Department of Clinical Sciences, Faculty of Medicine, Lund University, Lund, Sweden 12Institute. of Neuroscience and Physiology, University of Gothenburg, Mölndal, Sweden 13Clinical Neurochemistry Laboratory, Sahlgrenska University Hospital, Mölndal, Sweden 14Neurodegenerative Disorder Research Center, Division of Life Sciences and Medicine, and Department of Neurology, Institute on Aging and Brain Disorders, University of Science and Technology of China and First Affiliated Hospital of USTC, Hefei, Anhui, P.R. China 15Department of Psychiatry and Neurochemistry, Institute of Neuroscience and Physiology, the Sahlgrenska Academy at the University of Gothenburg, Mölndal, Sweden 16Department of Pathology and Laboratory Medicine, University of Wisconsin School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin, USA 17Wisconsin Alzheimer’s Disease Research Center, University of Wisconsin School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin, USA Correspondence Dhamidhu Eratne, Neuropsychiatry Centre, The Royal Melbourne Hospital, 300 Grattan St, Parkville VIC, Melbourne, Australia. Abstract INTRODUCTION: Plasma biomarkers offer promise for improving the diagnosis of Alzheimer’s disease (AD) and differentiating AD and other neurodegenerative disorThis is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2025 The Author(s). Alzheimer’s & Dementia published by Wiley Periodicals LLC on behalf of Alzheimer’s Association. Alzheimer’s Dement. 2025;21:e70717. wileyonlinelibrary.com/journal/alz 1of15 https://doi.org/10.1002/alz.70717 2of15 ERATNE ET AL. Email: [email protected] Funding information Swedish Research, Grant/Award Numbers: 2017-00915, 2022-00732; Swedish Alzheimer Foundation, Grant/Award Numbers: AF-930351, AF-939721, AF-968270, AF-994551; Hjärnfonden, Sweden, Grant/Award Numbers: ALZ2022-0006, FO2024-0048-TK-130, FO2024-0048-HK-24; ALF agreement, Grant/Award Numbers: ALFGBG-965240, ALFGBG-1006418; European Union Joint Programme for Neurodegenerative Disorders, Grant/Award Number: JPND2019-466-236; Alzheimer’s Association 2021 Zenith Award, Grant/Award Number: ZEN-21-848495; Alzheimer’s Association 2022-2025, Grant/Award Number: SG-23-1038904 QC; the Swedish Research Council, Grant/Award Numbers: 2023-00356, 2022-01018, 2019-02397; European Union’s Horizon Europe, Grant/Award Number: 101053962; Swedish State Support for Clinical Research, Grant/Award Number: ALFGBG-71320; Alzheimer Drug Discovery Foundation (ADDF), Grant/Award Number: 201809-2016862; AD Strategic Fund and the Alzheimer’s Association, Grant/Award Numbers: ADSF-21-831376-C, ADSF-21-831381-C, ADSF-21-831377-C, ADSF-24-1284328-C; European Union’s Horizon Europe Research and Innovation Programme; NEuroBioStand, Grant/Award Number: 22HLT07; Erling–Persson Family Foundation, Grant/Award Number: FO2022-0270; European Union’s Horizon 2020, Grant/Award Number: 860197; European Union Joint Programme–Neurodegenerative Disease Research, Grant/Award Number: JPND2021-00694; UK Dementia Research Institute, Grant/Award Number: UKDRI-1003; AFS, Grant/Award Number: ALF 2022 YF 0017 ders (NDs) like frontotemporal dementia (FTD) from primary psychiatric disorders (PPDs), particularly in younger patients. METHODS: In this prospective study, we investigated plasma phosphorylated tau 217 (p-tau217), neurofilament light chain (NfL), and glial fibrillary acidic protein (GFAP) in 341 unselected participants from a neuropsychiatry memory clinic, including AD (n=40),behavioralvariantFTD(bvFTD)(n=15),PPD(n=69),otherNDs,andcontrols. RESULTS: Plasma p-tau217 showed strong diagnostic performance for distinguishing AD from bvFTD (96% accuracy) and PPD (93% accuracy). NfL best distinguished all NDs from PPD, while GFAP did not bring additional value. Biomarker profiles using predefined cut-offs and age-adjusted z-scores further clarified group differences. DISCUSSION: Plasma p-tau217 and NfL have strong diagnostic utility in real-world, diagnostically complex cohorts. These findings support implementation of scalable blood-based biomarkers to improve early and accurate diagnosis in memory clinical settings. KEYWORDS age-adjusted reference ranges, Alzheimer’s disease, blood-based biomarkers, diagnostic accuracy, frontotemporal dementia, GFAP, memory clinic, neurodegeneration, neurofilament light chain, plasma biomarkers, primary psychiatric disorders, prospective cohort, psychiatric misdiagnosis, p-tau217 Highlights ∙Plasma p-tau217 was significantly elevated in AD compared to other disorders. ∙P-tau217 distinguished AD from bvFTD with high accuracy. ∙P-tau217 distinguished AD from PPDs with high accuracy. ∙NfL/p-tau217 ratio and GFAP added limited diagnostic value compared to p-tau217 and NfL. ∙Findings support blood biomarkers in younger, real-world clinical cohorts. 1BACKGROUND Blood-based biomarkers show great potential to transform diagnosis and clinical care of patients presenting with cognitive, psychiatric, and neurological symptoms. Their potential includes improving early and accurate diagnosis of Alzheimer’s disease (AD) and resolving realworld diagnostic challenges, such as distinguishing AD from other neurodegenerative disorders (NDs) like behavioral variant frontotemporal dementia (bvFTD) and from common mimics such as primary psychiatric disorders (PPDs).1–6 Early, accurate diagnosis is increasingly important with the emergence of disease-specific treatments, such as monoclonal antibodies targeting amyloid beta. Two of the most promising biomarkers are plasma phosphorylated tau 217 (p-tau217) and neurofilament light chain (NfL) protein. P-tau217 shows strong diagnostic performance and specificity to distinguish AD from non-AD disorders (including non-AD dementias and PPD).7–10 Within AD and control cohorts, plasma levels of p-tau217 correlate with tau positron emission tomography (PET) pathology11 andtangleloadatautopsy 12 and are elevated in disorders with tangles but without amyloid plaques,13 supporting its reflection of tau phosphorylation and pathology. NfL, a non-specific marker of neurodegeneration and acute neuronal injury, particularly in long myelinated axons, distinguishes neurodegenerativedisorders(NDs) from ND mimics, including PPDs and non-NDs,3,4,14,15 and has utility in clinical settings.14,16–19 Glial fibrillary acidic protein (GFAP), a marker of astrocytic activation and neuroinflammation, is an additional biomarker of interest in a range of disorders.14,20–26 However, the utility of GFAP for the clinical differentiation between AD, non-AD NDs, and PPDs, especially when compared to p-tau217 and NfL is not yet clear. We previously published diagnostic performance, cut-offs, and reference ranges for plasma p-tau217 and NfL,10,14,18,27–29 but we have not investigated their combined use. Furthermore, while we and oth- ERATNE ET AL.3of15 ers have published data showing the superiority of NfL compared to GFAP to distinguish bvFTD from PPDs,30,31 we have not yet investigated all three, p-tau217, NfL, and GFAP, in a younger real-world clinical neuropsychiatric cohort with diverse NDs and PPDs. Some studies have investigated combinations of these markers. Benussi et al. investigated NfL, p-tau217, NfL/p-tau217 ratios, and other biomarkers in 374 participants (97 AD, 278 FTD).32 They found strong diagnostic performance of p-tau217 and slightly superior performance of NfL/p-tau217 to distinguish between AD and FTD. However, they lacked a PPD group, which is critical to appreciate the use of biomarkers for bvFTD diagnosis. Rousset et al. assessed p-tau217 and NfL in an unselected memory clinic cohort, showing strong utility of p-tau217 for AD diagnosis and describing biomarker profiles (high/low p-tau217 and NfL) across clinical groups.17 To our knowledge, no study to date has jointly investigated p-tau217, NfL, GFAP, and biomarker profiles, with a specific focus on AD, bvFTD, PPD, in younger people, where differential diagnoses, diagnostic uncertainty, misdiagnosis, and delay are greater.1,33–35 Further research is needed to properly establish the roles of single and combination biomarkers in real-world clinicalsettings with diverseneuropsychiatric cohorts. The primary aim of this study was to compare levels and diagnostic performances of plasma p-tau217, NfL, NfL/p-tau217 ratio, and GFAP for distinguishing between AD, bvFTD, and PPDs. We hypothesized that combining p-tau217 and NfL would improve diagnostic accuracy compared to either alone. We also aimed to describe ptau217/NfL biomarker profiles of AD pathology and neuronal injury (e.g., low AD/low neuronal injury, high AD/high neuronal injury) in an unselected population of patients from a neuropsychiatry memory clinic. We focused on p-tau217 and NfL using our previously described cut-off for plasma p-tau21710 and age-adjusted z-score reference range models for plasma NfL.27,30 We wanted to explore whether GFAP could improve any distinctions, for example, between bvFTD and PPDs. Although some studies reported age-binned reference ranges for GFAP in serum36 and plasma,37 we are unaware of any studies that have described GFAP reference ranges using continuous modeling to derive precise age-adjusted percentiles and z-scores, as we did for NfL. As an exploratory aim, we applied a novel z-score model for plasma GFAP to define biomarker profiles reflecting AD pathology, neuronal injury, and neuroinflammation (p-tau217/NfL/GFAP). 2METHODS The current study included participants prospectively recruited between June 2019 and April 2023 who had provided a blood sample for biomarker analysis. This is a follow-up to our previous study cohort in which we investigated plasma and cerebrospinal fluid (CSF) NfL.14 In the current study, we included participants who had plasma p-tau217, NfL, and GFAP biomarker results available. In the current study the focus was on AD, bvFTD, and PPD diagnostic groups, with comparator groups being control participants and other NDs (both described in the previous study). Data were available in the current study for addiRESEARCH IN CONTEXT 1. Systematic review: We reviewed the PubMed literature on plasma p-tau217, NfL, and GFAP in AD, FTD, and PPDs. Although several studies support the individual diagnostic value of p-tau217 and NfL, few examined all three biomarkers together in real-world, diagnostically heterogeneous clinical cohorts, including PPDs. 2. Interpretation: This prospective study demonstrates the strong diagnostic performance of plasma p-tau217 in distinguishing AD from FTD and PPDs and other non-AD disordersandconfirms NfLas auseful markerofneurodegeneration. GFAP added limited value. These findings add to growing evidence on the clinical application of bloodbased biomarkers in younger and diagnostically complex populations. 3. Future directions: Future research should validate these findings in larger, more diverse cohorts, develop multimodal diagnostic algorithms, and evaluate the clinical utility and cost-effectiveness of implementing blood biomarker testing in psychiatric, primary care, and memory clinic settings. tional comparator groups (not previously described): people with mild cognitive impairment (MCI) and people with presymptomatic genetic NDs. The bvFTD group included patients meeting diagnostic criteria for possible bvFTD. The participants in this study did not overlap with our previous study on NfL and GFAP in bvFTD, mood, and psychotic disorders.30 Participants were recruited from the Neuropsychiatry Centre at The Royal Melbourne Hospital, a quaternary service receiving referrals for diagnostically complex cases from primary care and other specialist services within Australia. Patients, as part of routine clinical care through the Neuropsychiatry Centre, received comprehensive multidisciplinary assessments and multimodal investigations, including CSF AD biomarker analysis, with gold standard consensus diagnosis based on established diagnostic criteria, as previously described in detail.14,18,28 Diagnostic group categorization was determined based on the most recent diagnosis, at longitudinal follow-up, based on established diagnostic criteria, blinded to plasma biomarker levels, as previously described.14,18,28 Control participants were people recruited from the community. Cognitive screening data are still being collected and not yet available for this study; however, no control participants had symptoms or diagnoses of neurological or NDs and no active psychiatric symptoms or conditions. EDTA plasma samples were collected during patients’ diagnostic work-up and at first visit for community controls. Samples were stored at −80◦C. Plasma NfL and GFAP were measured using N2PB kits on a Quanterix Single molecule array (Simoa) HD-X analyzer, according to the manufacturer’s instructions (Quanterix Corp., Billerica, MA, 4of15 ERATNE ET AL. USA). Plasma p-tau217 was measured using an in-house University of Gothenburg (UGOT) p-tau217 assay, as previously described in detail.38 The measurements were performed in one round of experiments using one batch of reagents by analysts blinded to clinical data and diagnoses, thereby reducing potential batch effects. This study, part of the Markers in Neuropsychiatric Disorders Study (The MiND Study, https://themindstudy.org), was approved by the human research ethics committee at Melbourne Health (2016.038, 2017.090, 2018.371, 2020.142). 2.1 Statistical analyses Statistical analyses were performed using R version 4.5.0 (202504-11). Biomarker levels in different groups were compared using standardized bootstrapped general linear models (GLMs), with age at blood sample and sex as additional covariates. Receiver operating characteristic (ROC) curve analyses were then performed to investigate diagnostic utility between different combinations of groups. Bootstrapped differences in area under the curve (AUC) were used to compare ROC curves. Optimal cut-offs were selected based on Youden’s Jstatistic. Additional diagnostic test parameters were computed: positive and negative likelihood ratios, positive and negative predictive values, overall accuracy, and diagnostic odds ratio. Additional sensitivity analyses were performed: excluding extreme outliers, excluding patients with Creutzfeldt–Jakob disease (CJD), and performing all GLMs with weight included as a covariate. As results were similar, the results excluding weight and including outliers and patients with CJD were presented to maximize the sample sizes for analyses and presented results (since not all participants had weight data). We focused on describing p-tau217 and NfL biomarker profiles. These were low p-tau217/low NfL (a low AD pathology and low neuronal injury profile – a “normal” profile) and abnormal profiles: low p-tau217/high NfL (low AD pathology but elevated neuronal injury), high p-tau217/low NfL (AD pathology but low neuronal injury), and high p-tau217/high NfL (high AD pathology and high neuronal injury). Plasma p-tau217 and NfL biomarker levels were dichotomized in to “high” and “low” based on our previously published data and cut-offs that used the same assays.10,27 For p-tau217, we used a cut-off of 2.35, which was optimal at distinguishing AD from non-AD, as previously described and published.10 For plasma NfL, given the strong non-linear association with age, we used age-based percentiles and z-scores derived from the generalized additive models for location, scale, and shape (GAMLSS) model of a large reference control cohort that we developed, previously described and published,14 defining the 95th percentile as the cut-off between high and low. For this study, we created a novel age-adjusted GFAP reference range model using (GAMLSS) using this study’s control group. This allowed us to derive precise age-based percentiles and z-scores and thus more precise categorization,again giventhe significant strong and non-linearassociation with age compared to coarse age-binned cut-offs. The 95th percentile was defined as the cut-off for high and low GFAP levels. 3RESULTS The final cohort consisted of 341 participants: 40 with AD (median age 62 years, 53% female), 15 with bvFTD (median age 57 years, 27% female), and 69 with PPDs (median age 55 years, 48% female). There were 119 controls (median age 63, 74% female), and in the additional comparator groups: 67 with other NDs, 13 with MCI, and 18 with presymptomatic genetic NDs (see Table 1for full details). PPDs consisted of major depressive disorder (MDD, n=21), bipolar disorder (n=6), functional neurological/cognitive disorder (n=8), schizophrenia spectrum disorder (n=16), bvFTD “phenocopy” syndrome (n=2), and other PPDs (n=16, which included anxiety, personality, obsessive-compulsive, post-traumatic stress, and undifferentiated psychiatric disorders). Other NDs consisted of CJD (n=2), dementia with Lewy bodies (n=5), dementia not otherwise specified (n=6), Huntington’s disease (HD, n=15), mixed AD/vascular (n=3), substancerelated cognitive impairment/dementia (n=2), vascular dementia (n=5),andarangeofotherNDs(n=29, which included autoimmune encephalitis, cerebral amyloid angiopathy, corticobasal syndrome, central nervous system vasculitis, Down syndrome, Fahr disease, metabolic disorders, Niemann–Pick Type C, Parkinson’s disease, and cerebellar degenerative disorder). The presymptomatic genetic ND group consisted of AD (PSEN1,n=2), genetic CJD (n=6), HD (n=6), CADASIL (n=1), bvFTD (C9orf72 n =1andGRN n=2). CSF biomarker analysis, including AD proteins CSF AB42 and p-tau181 (Supporting Information Table 1), was conducted on 80 patients. Briefly, 27/40 (68%) of AD patients had CSF AD biomarkers. Most (18/27, 67%) had a CSF AD biomarker profile consistent with AD, as defined by an amyloid-positive, p-tau-positive (A+T+) profile, categorizedusing established cut-offs,as described in ourpreviousstudy.10 Other profiles in the AD group were 8/27 with A+T−and 1/27 with A−T+. Two patients with AD had amyloid PET (but not CSF), and both were positive for amyloid plaques. Further information, including phenotype information for AD, are in the Supporting Information Tables 1and 2. Forty-seven percent (7/15) of bvFTD patients had CSF AD biomarkers (none with an A+T+profile, 4/7 with A−T−,and3/7with A+T−). None of the patients in the other groups who had CSF AD biomarkers (20 in the PPD group, 21 in other NDs, and five MCI) had A+T+profiles (further details in Supporting Information Tables 1and 2). 3.1 Levels of plasma p-tau217, NfL, NfL/p-tau217 ratio, GFAP, in AD, bvFTD, and PPD 3.1.1 Plasma p-tau 217 As demonstrated in Table 1and Figure 1, plasma p-tau217 levels were significantly elevated in AD compared to bvFTD (standardized boot- ERATNE ET AL.5of15 TABLE 1 Study cohort details and biomarker levels. Characteristic N AD N=40a bvFTD N=15a PPD N=69a Control N=119a Other ND N=67a MCI N=13a Presymptomatic genetic ND N=18a Age 341 62 (58, 65) 57 (56, 62) 55 (45, 62) 63 (55, 70) 61 (45, 67) 65 (56, 67) 51 (43, 62) Sex 341 Female 21/40 (53%) 4/15 (27%) 33/69 (48%) 88/119 (74%) 29/67 (43%) 3/13 (23%) 13/18 (72%) Male 19/40 (48%) 11/15 (73%) 36/69 (52%) 31/119 (26%) 38/67 (57%) 10/13 (77%) 5/18 (28%) Weight 270 73 (59, 83) 84 (63, 101) 84 (73, 99) 75 (65, 85) 75 (65, 88) 84 (78, 90) 79 (70, 98) Unknown 15 2 14 16 12 4 8 p-tau217 341 3.63 (2.90, 4.41) 1.07 (0.72, 1.50) 0.92 (0.58, 1.32) 0.91 (0.65, 1.31) 1.11 (0.69, 1.68) 1.04 (0.78, 1.40) 0.83 (0.65, 1.25) Nfl 341 24 (18, 28) 21 (12, 55) 11 (8, 13) 12 (9, 17) 29 (16, 42) 15 (13, 20) 12 (10, 19) NfL/p-tau217 ratio 341 7 (5, 9) 25 (10, 48) 12 (9, 20) 13 (9, 22) 24 (11, 47) 16 (12, 27) 14 (11, 27) GFAP 341 212 (151, 305) 79 (53, 192) 86 (56, 117) 115 (89, 177) 132 (77, 216) 144 (86, 174) 97 (61, 125) log p-tau217 341 0.56 (0.46, 0.64) 0.03 (−0.14, 0.18) −0.04 (−0.24, 0.12) −0.04 (−0.19, 0.12) 0.05 (−0.16, 0.23) 0.02 (−0.11, 0.15) −0.08 (−0.19, 0.10) log NfL 341 1.38 (1.25, 1.45) 1.31 (1.08, 1.74) 1.02 (0.90, 1.11) 1.09 (0.94, 1.24) 1.46 (1.20, 1.62) 1.19 (1.11, 1.30) 1.06 (0.99, 1.27) log GFAP 341 2.33 (2.18, 2.48) 1.90 (1.72, 2.28) 1.93 (1.75, 2.07) 2.06 (1.95, 2.25) 2.12 (1.88, 2.33) 2.16 (1.93, 2.24) 1.99 (1.78, 2.10) Abbreviations:AD, Alzheimer’s disease; bvFTD, behavioral variant frontotemporal dementia; GFAP, glial fibrillary acidic protein; MCI, mild cognitive impairment; NfL, neurofilament light chain; ND, neurodegenerative disorder; PPD, primary psychiatric disorder; p-tau217, phosphorylated tau 217 aMedian (Q1, Q3); n/N(%). 6of15 ERATNE ET AL. (A) (B) (C) (D) FIGURE 1 Plasma p-tau217, NfL, NfL/p-tau217 ratio, and GFAP levels, in AD, bvFTD, PPDs, controls, and other comparator groups. Plasma p-tau217 levels were significantly elevated in AD compared to all other disorders and controls. To improve readability, seven outliers were not displayed in plot B for NfL (two for AD [141 and 294 pg/mL], two bvFTD [101 and 214 pg/mL], three other NDs [106, 352, and 1154 pg/mL]), and six in plot C for NfL/p-tau217 ratio (one bvFTD [143], two controls [699, 772], three other NDs [190, 198, and 604]). Dashed red lines =predefined optimal cut-offs from our previous studies, for p-tau217 (2.35pg/mL). AD, Alzheimer’s disease; bvFTD, behavioral variant frontotemporal dementia; GFAP, glial fibrillary acidic protein; ND, neurodegenerative disorder; NfL, neurofilament light chain; PPD, primary psychiatric disorder; p-tau217, phosphorylated tau 217. strapped GLM with age and sex as additional covariates; β=1.30, 95% CI: [0.71, 1.73], p<0.001), and AD compared to PPDs (β=1.59 [1.34, 1.80], p<0.001). Levels were also elevated in AD compared to the other groups (controls, Other NDs, MCI, and Presymptomatic ND, and all p<0.001). 3.1.2 Plasma NfL Plasma NfL was elevated in AD compared to PPD (β=0.87 [0.59, 1.13], p<0.001), but not for AD compared to bvFTD (β=−0.10 [−0.82, 0.58], p=0.788). There were no significant differences in plasma NfL levels across the ND groups (AD, bvFTD, Other NDs, all p>0.05). Levels werealsosimilarbetweenPPDs,control,MCI,andpresymptomaticND groups. 3.1.3 Plasma NfL/p-tau217 ratio The plasma NfL/p-tau217 ratio was reduced in AD compared to bvFTD (β=−1.57 [−2.16, −0.94], p<0.001) and PPD (β=−0.99 [−0.58, −1.34], p<0.001). The ratio was also lower in AD compared to all the other groups (controls, Other NDs, MCI, Presymptomatic NDs, and all p<0.002). 3.1.4 Plasma GFAP GFAP levels were elevated in AD compared to bvFTD (β=0.80 [0.23, 1.39], p=0.010), and PPD (β=0.82 [0.36, 1.21], p<0.001). GFAP levels were also elevated in AD compared to some other groups (controls, MCI,Presymptomatic ND,allp<0.038), butnot between ADand other NDs (p=0.108). ERATNE ET AL.7of15 FIGURE 2 ROC analyses for diagnostic performance of plasma p-tau217, NfL, NfL/p-tau217 ratio, and GFAP. Plasma p-tau217 had the very strong diagnostic performance for AD versus PPD, AD versus bvFTD, AD versus non-AD, AD versus controls, outperforming other biomarkers and the NfL/p-tau217 ratio. NfL’s diagnostic performance was for bvFTD versus PPD and all NDs versus PPDs. AD, Alzheimer’s disease; bvFTD, behavioral variant frontotemporal dementia; GFAP, glial fibrillary acidic protein; NfL, neurofilament light chain; PPD, primary psychiatric disorder; p-tau217, phosphorylated tau 217; ROC, receiver operating characteristic. 3.2 Biomarker diagnostic performance to distinguish AD from PPDs, AD from bvFTD, and bvFTD from PPD 3.2.1 Distinguishing AD from PPDs P-tau217 demonstrated the strongest diagnostic performance to distinguish AD from PPDs, as demonstrated in Figure 2and Supporting Information Table 3. P-tau217 had an AUC of 0.97, outperforming NfL, NfL/p-tau217 ratio, and GFAP (AUCs 0.89, 0.77, 0.86 respectively; AUC differences all p<0.016). P-tau217 had the highest accuracy (93%), specificity (91%), and sensitivity (95%), at an optimal cut-off of 1.84pg/mL. Further details of all diagnostic performance metrics are available in Supporting Information Table 3. 3.2.2 Distinguishing AD from bvFTD To distinguish AD from bvFTD, p-tau217 once again demonstrated the highest AUC (0.93) and the strongest diagnostic performance (93% specificity, 98% sensitivity, 96% accuracy, and cut-off 1.64) compared to the other biomarkers. NfL did not have significant diagnostic performance to distinguish AD from bvFTD (AUC 0.51 [0.30, 0.74]). The AUC difference was not statistically different between p-tau217 and the NfL/p-tau217 ratio (AUC 0.88, AUC difference p=0.509) or GFAP (AUC 0.77, AUC difference p=0.10). However, compared to p-tau217, both the ratio and GFAP demonstrated poorer specificity (87% and 67%, respectively), sensitivity (80% and 85%), and accuracy (82% and 80%) and much lower diagnostic odds ratios (26 and 11.33, vs p-tau217’s 546). 3.2.3 Distinguishing AD from non-AD disorders P-tau217demonstratedhighdiagnosticperformancetodistinguishAD from other non-AD disorders (consisting of bvFTD, PPDs, Other NDs), withanAUCof0.94.Itwas superiortoNfL, NfL/p-tau217(all p≤0.001) and had 88% specificity, 93% sensitivity, 90% accuracy, and a cut-off of 2.19. 8of15 ERATNE ET AL. P-tau217 demonstrated very strong diagnostic performance to distinguish AD from controls (AUC 0.98, 96% specificity, and 93% sensitivity). 3.2.4 Distinguishing bvFTD from PPD To distinguish bvFTD from PPD, NfL had the highest AUC (0.78) and strongest diagnostic performance (81% specificity, 67% sensitivity, 79% accuracy, and cut-off 15.15pg/mL), although the AUC difference between NfL and NfL/p-tau217 ratio was not significant (p=0.14). Ptau217 and GFAP had no diagnostic utility (AUC confidence intervals both crossed 0.50). 3.3 Biomarker diagnostic performance to distinguish all NDs from PPDs As a follow-on from our previous studies that focused on NfL in distinguishing NDs from PPDs, we investigated the ability of p-tau217 and GFAP to distinguish NDs as a group from PPDs. We created an “all NDs” group by combining AD, bvFTD, and Other NDs. NfL demonstrated the highest AUC (0.87) and strongest diagnostic performance (81% specificity, 85% sensitivity, 84% accuracy, and cut-off 14.35) to distinguishAll NDs fromPPDs, significantly outperformingGFAP(AUC 0.73, AUC difference p<0.0001) and p-tau217 (AUC 0.72, AUC difference p<0.001). The NfL/p-tau217 ratio did not have diagnostic utility for All NDs versus PPDs (AUC confidence interval crossed 0.50).xxx 3.4 Biomarker profiles in diagnostic groups 3.4.1 AD pathology/neuronal injury (P-tau217/NfL) biomarker profiles We described p-tau217 and NfL biomarker profiles in the diagnostic groups, using our previously described cutoff for plasma p-tau217, and age-adjusted percentiles model for NfL.10,27 As demonstrated in Figure 3, the high p-tau217 profiles were most commonly seen in AD (total 88% of AD, 60% high p-tau217/high NfL, and 28% high p-tau217/low NfL biomarker profiles). By comparison less than 10% of each of the other groups exhibited high p-tau217 profile. bvFTD only had 7% with high p-tau217 profiles (all high ptau217/high NfL), PPD only 6% (3% high p-tau217/high NfL, 3% high p-tau217/low NfL), controls only 3% (all high p-tau217/low NfL), and MCI and presymptomatic ND 8% and 6% respectively. Other NDs had 13% with high p-tau217 profiles (10% high p-tau217/high NfL, 3% high p-tau217/low NfL). 68% of AD had high NfL profiles (high p-tau217/high NfL, low ptau217/high NfL), and AD had the lowest proportion of “normal” (low p-tau217/lowNfL) profiles.ForbvFTDonthe otherhand, only53% had high NfL profiles, and the remaining 47% had low p-tau217/low NfL profiles. Most PPD had a normal profile (72%), with 25% showing high NfL profiles. Looking at individual PPD (details in Supporting Information Figures 1and 2), there was a greater proportion of patients with high NfL profiles in schizophrenia (37%) and Other PPD (33%), compared to other groups (MDD 19%, FND 12.5%, BPAD 0%), although theseexploratory findingsshouldbe interpretedwith caution giventhe small sub-group numbers. 83% of controls had a normal profile, and only 14% having high NfL profiles. Other ND had the highest proportionsofhigh NfLlevels(74%,64%lowp-tau217/high NfL,and 10%high p-tau217/highNfL).Most participantsin MCI,andPresymptomatic ND had low p-tau217/low NfL profiles (72%, 77%, and 67%, respectively), similar to PPD and controls. These findings are further demonstrated in Figure 4, a plot for different diagnostic groups of log p-tau217 versus NfL age-adjusted z-score, and distributions. The scatter plot and distributions demonstratethatalmostallADpatientshadelevatedp-tau217levelsandhigh p-tau217/high NfL profiles (most in upper right in scatter plot), as compared to non-AD disorders, where almost all had low p-tau217 levels. Most NDs had high NfL levels (most in lower right in scatter plot), compared to a small proportion of PPDs and controls (most in lower left in scatter plot). 3.4.2 AD pathology/neuronal injury/neuroinflammation (p-tau217/NfL/GFAP) biomarker profiles As an exploratory aim, we developed a novel GAMLSS age-based model for plasma GFAP to derive percentiles and z-scores, based on our control group. As demonstrated in Figure 5, this shows a similar non-linear association with age and a U-shaped pattern similar to the found in other studies.36,37 This enabled complex p-tau217/NfL/GFAP (AD pathology/neuronal injury/neuroinflammation) biomarker profiling, which is demonstrated in Figure 6. Inclusion of GFAP to create this three-biomarker profile resulted in some differences compared to ptau217/NfL only profiling. The proportion of “normal” profiles using three biomarkers (i.e., low p-tau217/low NfL/low GFAP) was slightly lower in bvFTD (40%, compared to 47% low p-tau217/low NfL profiles). The proportion of normal p-tau217/NfL/GFAP profiles in PPDs and controls were similar to proportions of normal p-tau217/NfL profiles. Looking at individual PPDs (details in Supporting Information Figures 1and 2), high GFAP profiles did not appear to be different between groups, although these findings should once again be interpreted with caution given the small subgroup numbers. We performed some additional exploratory analyses. To investigate whether there were differences in levels of severity and biomarkers between AD and bvFTD, we first compared MMSE scores between these two groups (calculated from total scores on the Neuropsychiatry Unit Cognitive Assessment Tool (NUCOG), the cognitive screening instrument used in the Neuropsychiatry Centre).39 MMSE scores were not statistically different (median [interquartile range, IQR]: 21.9 [16.5 to 24.9] for AD vs 23.4 [20.9 to 25.0] for bvFTD, p=0.093), Supporting Information Table 1. Next, we ran GLMs exploring whether diagnosis of AD or bvFTD, MMSE, and diagnosis ×MMSE interaction ERATNE ET AL.9of15 FIGURE 3 P-tau217 and NfL biomarker profiles, classified based on previously described cut-offs, in different diagnostic groups. NfL, neurofilament light chain; p-tau217, phosphorylated tau 217. influenced NfL levels, adjusting for age and sex. Diagnosis (p=0.394), MMSE (p=0.700), and the interaction term (p=0.634) were all non-significant. Taken together, these findings indicate that the higher proportion of “high NfL” profiles in AD was unlikely to be explained by more advanced cognitive impairment compared to bvFTD. Finally, we investigated for any associations between biomarkers and cognitive impairment in AD. In AD cases only, higher plasma NfL showed a trend toward association with lower MMSE, in GLMs also adjusting for age and sex, but it was not statistically significant (p=0.076). No significant associations were observed in AD between MMSE scores and plasma p-tau217 (p=0.928) and GFAP (p=0.614). These suggest that within our AD group, biomarker levels did not significantly vary with the degree of cognitive impairment. 4DISCUSSION We investigated multiple biomarkers in an unselected real-world clinical cohort of younger participants seen in a neuropsychiatry memory clinic, focusing on AD, bvFTD, and PPDs. The main findings were as follows: (1) very strong diagnostic performance of plasma p-tau217 to distinguish AD from bvFTD, and AD from PPDs; (2) superior performance of p-tau217 for AD diagnosis on its own, compared to NfL/p-tau217 ratio, NfL, and GFAP; (3) strongest performance of NfL for distinguishing bvFTD from PPDs, and all NDs from PPDs, consistent with previous studies.30,31 This adds important further evidence to the limited literature so far on roles for different biomarkers in a real-worldclinicalcohort, withp-tau217 beingaverysensitiveandspecific test for AD and NfL having the strongest diagnostic performance to distinguish neurodegeneration from PPDs/non-NDs, with little role for GFAP in any of these distinctions. The key strengths of this study included a real-world, unselected diverse, clinical, and younger cohort – a relatively underinvestigated group. Establishing the role for these biomarkers in younger people, who face greater rates of misdiagnosis and diagnostic uncertainty, is important for increasing clinical and research access, where arguably the clinical, treatment, and broader psychosocial benefits could be greater. This study builds on our previous work on p-tau217,10 providing further evidence of very strong diagnostic performance for some of the most common diagnostic distinctions, distinguishing AD from bvFTD, and AD from PPDs. We saw very high specificity and sensitivity (91% and 95%), establishing a role for such an accurate test (93%) in younger people. We found no benefit of NfL/p-tau217 ratio over ptau217 alone in terms of improving the distinction between AD and