The potential clinical value of plasma biomarkers in Alzheimer's disease

Many people with cognitive complaints or impairment never receive an accurate diagnosis of the underlying condition, potentially impacting their access to appropriate treatment. To address this unmet need, plasma biomarker tests are being developed for use in Alzheimer's disease (AD). Plasma biomarker tests span various stages of development, including in vitro diagnostic devices (or tests) (IVDs), laboratory‐developed tests (LDTs) and research use only devices (or tests) (RUOs). Understanding the differences between each test type is important for appropriate implementation into the AD diagnostic pathway and care continuum.


METHODS:
Authors reviewed scientific literature (PubMed, meeting abstracts and presentations, company press releases and websites) on AD plasma biomarkers.

RESULTS:
This article defines IVDs, LDTs, and RUOs, discusses potential clinical applications and highlights the steps necessary for their clinical implementation.DISCUSSION: Plasma biomarkers could revolutionize many areas of the AD diagnostic pathway and care continuum, but further research is needed.

K E Y W O R D S
Alzheimer's disease, diagnosis, diagnosis pathway, diagnostic test, plasma biomarkers, triage tool

HIGHLIGHTS
• There is a need for a minimally invasive Alzheimer's disease (AD) diagnostic tool.
• AD plasma biomarker tests exist at various stages of commercial development.
• Understanding the development stage of a test is important for its appropriate use.
• Plasma biomarker tests could function as a triage tool to streamline AD diagnosis.
• Further steps remain before AD plasma biomarkers can be used routinely.

INTRODUCTION
Alzheimer's disease (AD) is a neurodegenerative disorder that accounts for ≈ 60% to 80% of dementia cases. 1 It is characterized by abnormal amyloid beta (Aβ) deposition in the form of amyloid plaques (Aβ pathology), as well as neurofibrillary tangles, dystrophic neurites and neuropil threads composed of hyperphosphorylated tau proteins (tau pathology) in the brain. 2Alzheimer's Disease International estimate that, on average, > 75% of people living with cognitive complaints or impairment are undiagnosed worldwide 3 and post mortem studies report misdiagnosis rates of up to 30%. 4 Furthermore, a meta-analysis reported that the clinical judgment of general practitioners had a sensitivity of 58% and a specificity of 89% in the diagnosis of dementia, meaning that false negative diagnoses are likely. 5obally, there is no standardized AD diagnostic pathway. 6At present, symptomatic individuals with clinical suspicion of AD undergo a combination of clinical history taking, cognitive assessments, routine laboratory tests (e.g., blood and/or urine tests), and structural brain imaging (e.g., computed tomography or magnetic resonance imaging [MRI]) to rule out other causes of cognitive complaints or impairment. 7agnosis of AD can be supported by positron emission tomography (PET) and/or cerebrospinal fluid (CSF) biomarker analysis, 7 both of which are approved by the U.S. Food & Drug Administration (FDA) for the diagnosis of AD. [8][9][10][11] However, use of these confirmatory tests is limited due to perceived invasiveness, high costs, and lack of specialist equipment and/or clinical expertise. 12rtifying the presence of Aβ pathology in the brain in vivo is important for clinical decision making, particularly with the advancement of anti-Aβ disease-modifying therapies (DMTs), such as aducanumab 13 and lecanemab, 14 which are FDA approved, and donanemab, 15 which has shown promising results in a phase III clinical trial.These DMTs target disease pathology in symptomatic patients early in the AD continuum, so accurate and timely diagnosis is crucial; [13][14][15] the prescribing information and appropriate use recommendations of aducanumab and lecanemab indicate that the presence of amyloid pathology in the brain should be confirmed before DMT initiation. 16,17As DMTs become accessible, health services are unlikely to be prepared for the influx of patients requiring diagnostic testing for AD.This highlights an urgent clinical unmet need for a cost-and time-effective, minimally invasive, and easy-to-interpret diagnostic tool that can be widely implemented. Plasma

DEFINING IVDS, LDTS, AND RUOS
All three categories of biomarker test, IVDs, LDTs, and RUOs, serve their own purposes in the diagnostic field; the main differences among the three are summarized in Table 1.A summary of selected plasma biomarker tests for use in the AD diagnostic pathway is provided in Table 2.
The majority of plasma biomarker tests currently available, and in development, for use in the AD field are RUOs, so can only be used for research purposes.RUOs are devices that are in the laboratory research-based phase of development. 19They are valuable tools for gathering real-world evidence on a particular biomarker or disease, supporting product development, and conducting non-clinical laboratory research, but they must not be used for clinical decision making or diagnosis and must be labeled as "for research use only." 18,19Ts are designed, manufactured, and used in a single laboratory or laboratory network 18,40 and are important for accelerating research progress as they can often be developed more quickly and cheaply than IVDs because they only undergo limited analytical and clinical validation and regulatory review. 40,41In contrast to RUOs and LDTs, IVDs are commercially available tests intended to support clinical decision making.They have undergone extensive regulatory review to obtain approval, meaning that they produce reliable test results and are widely validated with respect to their analytical and clinical performance in the intended use population. 18,22,20In December 2022, Sysmex Corporation fulfilled regulatory requirements and was granted IVD status in Japan for the HISCL β-Amyloid 1-42 and β-Amyloid 1-40 Assay Kits.The kits measure Aβ42 and Aβ40 in human plasma and use the Aβ42/Aβ40 ratio to detect Aβ pathology in the brain of symptomatic people. 25e Elecsys ® Amyloid Plasma Panel (Roche Diagnostics International Ltd) and the PrecivityAD test (C 2 N Diagnostics) have been granted FDA Breakthrough Device Designation, 45,46 which is a program that can accelerate the assay obtaining IVD status by facilitating interactions between assay developers and FDA experts and prioritizing FDA review. 47The Elecsys Amyloid Plasma Panel combines measurement of phosphorylated tau181 (p-tau181) and apolipoprotein E4 to identify people that would benefit from further confirmatory testing for AD.Both LDTs and RUOs can be further developed and reclassified as IVDs once extensive analytical and clinical validation have been conducted and regulatory requirements are satisfied. 19,40,48Analytical validation assesses whether the test is technically robust and will produce reliable and consistent results when conducted in different laboratories under different test conditions.Typical experiments performed during analytical validation include, among others: (1) testing for the degree of agreement between replicate measurements of the same sample under the same conditions (precision), ( 2) testing for substances affecting the measurable concentration of the analyte sample (interferences), 49 and (3) testing for the ability to obtain results that are proportional to the concentration of analyte being measured (linearity). 50High robustness indicates that diagnostic performance is unaffected by small differences in test conditions such as pre-analytical sample handling techniques, analytical variability, lab-to-lab, batch-to-batch, and operator differences, all of which are unavoidable in practice. 51Clinical validation assesses whether the test serves its purpose in the intended use population, that is, whether the test is able to reliably diagnose people with the condition and, in doing so, inform clinical decision making.In real-world terms, a widely validated test with high robustness means that patients are less likely to be misclassified and, hence, are more likely to receive the correct support and treatment.As such, IVDs tend to be one of the most widely used clinical tests 52

POTENTIAL CLINICAL VALUE OF PLASMA BIOMARKER TESTING IN THE AD DIAGNOSTIC PATHWAY AND CARE CONTINUUM
Many of the plasma biomarker tests currently available, and in development, for use in the AD field have diagnostic capabilities that would allow them to act as a triage tool to aid in the diagnosis of symptomatic people; however, with ongoing development, plasma biomarker tests have the potential to be used in many different contexts along the AD diagnostic pathway and care continuum (Table 3).

Acting as a triage tool by ruling out symptomatic individuals with a low likelihood of Aβ pathology or ruling in symptomatic individuals with a high likelihood of Aβ pathology
In both primary and secondary care settings, plasma biomarker testing could function as a triage tool alongside clinical and neurological investigations to rule out symptomatic individuals with a low likelihood of Aβ pathology or rule in symptomatic individuals with a high likelihood of Aβ pathology.Several of the plasma biomarker tests currently or imminently available are intended to be rule-out triage tools. 35,36 is suggested that a rule-out test would need to have a negative predictive value (NPV) of > 90% in the intended use population, whereas a rule-in test would need to have a positive predictive value (PPV) of > 90% in the intended use population.A triage tool would assist clinical decision making around whether to: refer the individual for confirmatory PET/CSF biomarker analysis, investigate a different cause of the symptoms, or develop an appropriate plan for treatment and and up to 30% of people are misdiagnosed. 4e a greater need for a triage tool to help identify patients who would benefit from such therapies.
Plasma Aβ42 concentration and the Aβ42/Aβ40 ratio have been widely researched as triage tools, with several tests reporting good diagnostic performances. 39,55Both the HISCL β-Amyloid 1-42 and β-Amyloid 1-40 Assay Kits (Sysmex Corporation) and the PrecivityAD test (C 2 N Diagnostics) use the Aβ42/Aβ40 ratio to identify people with amyloid pathology.In a direct comparison study, the HISCL assays showed high correlation with immunoprecipitation mass spectrometry (Pearson r: 0.82 and 0.91 for Aβ42 and Aβ40, respectively). 56The PrecivityAD test has also been shown to have good diagnostic performance in retrospective studies using clinical samples; 46,57,58 however, the diagnostic performance of plasma biomarkers may be lower in prospective settings and the test requires mass spectrometry equipment, which may not be easily scalable.Furthermore, the Aβ42/Aβ40 ratio has been shown to have an inherently small dynamic range and hence low clinical robustness, 59 meaning that small technical differences when using the tests may result in patients being misclassified. 59 addition, a 2023 publication found that including age, sex, and/or apolipoprotein E4 carrier status into a risk model did not improve the diagnostic performance compared to the plasma Aβ42/Aβ40 ratio alone, 60 a finding that should be considered when assessing the performance of tests that rely on the Aβ42/Aβ40 ratio in combination with age, sex, and/or apolipoprotein E4 carrier status.
Plasma p-tau and total tau (t-tau) assays are also under investigation as potential triage tools.Several head-to-head studies have been conducted between a range of plasma p-tau and t-tau assays in a variety of cohorts and settings and report that p-tau181, p-tau217, and p-tau231 are all promising plasma biomarkers for identifying people with Aβ pathology. 28,31,61A 2021 study reported that plasma p-tau231 levels increased earlier than plasma p-tau181 levels, indicating that plasma p-tau231 may be particularly useful for identifying symptomatic individuals who are early in the AD continuum. 62Two further studies reported that p-tau231 increases and reaches abnormal levels at lower thresholds of brain amyloidosis than p-tau217 or p-tau181. 63,64e Elecsys Amyloid Plasma Panel (Roche Diagnostics International Ltd) is a triage tool that comprises a combination of biomarkers. 35e Elecsys Amyloid Plasma Panel is based on a retrospective study that assessed a panel of Elecsys prototype immunoassays for clinical performance and robustness in three cohorts.It was found that the p-tau181 and apolipoprotein E4 composite plasma biomarker score was the best-performing combination in terms of meeting the clinical performance and robustness requirements to rule out symptomatic individuals with a low likelihood of Aβ pathology. 51

Enriching clinical trial populations by screening out people without Aβ pathology
Several clinical trials are already benefiting from population enrichment using plasma biomarker tests to identify symptomatic individuals with a high likelihood of Aβ pathology, and thereby reducing the number of unnecessary PET and/or CSF biomarker analyses that often take place during pre-trial screening. 12,55For example, using plasma p-tau181 and Aβ42/Aβ40 measurements to identify suitable trial participants has been found to improve trial screening burden and reduce timelines for participant recruitment. 65

Acting as a diagnostic test, replacing confirmatory PET and/or CSF biomarker analysis
Plasma biomarker tests would ideally replace confirmatory PET and/or CSF biomarker analysis in the future for symptomatic individuals, particularly as they are less invasive and easily scalable so are likely to be more accessible. 12,20Plasma biomarkers could reduce health disparities by improving access, lowering the cost, and eliminating hesitancy around the invasiveness of testing, leading to earlier diagnosis of symptomatic AD, particularly in underserved populations, compared to PET and/or CSF biomarker analysis. 12,66To replace PET and/or CSF biomarker analysis, a plasma biomarker test would need to be evaluated in prospective multicenter studies and have high diagnostic performance similar to that of PET and/or CSF biomarker analysis.
Importantly, the plasma biomarker test would need to provide diagnostic certainty such that a positive result could lead to initiation of DMT treatment.The assays included in Table 2 are not yet able to replace PET and/or CSF biomarker analysis; however, as technology advances and assays become more sensitive and validated in a wide range of populations, it is hoped this will become a reality.

Predicting disease progression
Plasma biomarker testing may one day be able to predict disease progression in symptomatic individuals.Several studies have examined the relationship between baseline plasma biomarkers and the development of AD.In a study of people without cognitive impairment (CI), people with mild cognitive impairment (MCI) and people with confirmed AD, baseline plasma p-tau181 levels from people with MCI predicted the development of AD 12 months later and were strongly associated with cognitive decline and gray matter loss. 67A study examining glial fibrillary acidic protein (GFAP) in cognitively normal individuals found that high baseline GFAP levels were associated with an increased risk of developing dementia, thus progressing from asymptomatic to symptomatic disease state. 68Furthermore, a systematic review concluded that plasma p-tau217 is a sensitive and specific marker for the clinical manifestation of AD and its progression, making it a promising biomarker for predicting disease progression. 69

Monitoring disease progression and/or treatment response
Disease progression may be monitored by serial plasma biomarker testing. 12In a study of people without CI, people with MCI and people with confirmed AD, serial measurements of p-tau181 correlated well with disease progression in all three groups. 70A further study examining neurofilament light chain (NfL) levels in cognitively normal people found that NfL rose more steeply in individuals who developed dementia (i.e., progressed from an asymptomatic state to a symptomatic disease state) compared to those who did not. 68Monitoring plasma biomarker levels could also help physicians identify whether a decline in a patient's condition is due to disease progression, or due to another cause, such as an infection, or an adverse reaction to a new medication.
Plasma biomarker levels are also altered in patients treated with DMTs so could be used to monitor treatment response.A significant decrease in plasma p-tau217 compared to baseline and placebo was observed in patients treated with donanemab. 71Numerical improvements were also observed for all of the plasma biomarkers examined (Aβ42/Aβ40 ratio, p-tau181, GFAP, and NfL) in patients treated with lecanemab versus placebo in a phase III clinical trial. 14Similarly, in patients treated with aducanumab, serial reductions in plasma p-tau181 were observed compared to placebo. 13Plasma biomarker testing is particularly suited to serial monitoring because it is minimally invasive and scalable.It would be useful to guide clinicians about whether a course of treatment is effective and should be continued, or whether it is not slowing disease progression and an alternative treatment should be tried.Furthermore, it would also be of interest to investigate whether there is an association between plasma biomarkers and adverse events due to DMTs to help identify which patients are more likely to experience them.

Acting as a screening test for cognitively normal individuals or individuals at risk of developing AD
To act as a screening test that can be widely used in primary care, the plasma biomarker test would need to be extremely reliable, with an NPV of >99%, as well as being cost effective. 20A population screening test of AD is not yet in development, but may be in the future as assays become more accurate and the availability of DMTs increases. 20th further development, plasma biomarkers may be used as routine screening tools to identify high-risk asymptomatic individuals.They

CLINICAL IMPLEMENTATION OF PLASMA BIOMARKER TESTING IN THE AD DIAGNOSTIC PATHWAY AND CARE CONTINUUM
Most plasma biomarker studies have been performed in retrospective studies in well-controlled research-based settings in populations with low heterogeneity and limited comorbidities. 12The diagnostic performances of the plasma biomarker tests may therefore be skewed as the intended use population in clinical practice is likely to have lower rates of AD than the participants of a retrospective study.Furthermore, retrospective batch analyses of plasma samples are not affected by between-assay and between-batch variability, which are present in clinical routine use.As such, for a plasma biomarker test to gain IVD status and be deployed globally for routine clinical use, the test must be extensively validated in large, prospective studies in diverse, heterogenous populations and in settings that accurately reflect realworld clinical practice.Clinical utility will also need to be established; the plasma biomarker test will need to have a positive impact on patient management to secure future reimbursement and wide-scale adoption.
In a field-test study conducted in a memory clinic, plasma biomarkers demonstrated excellent diagnostic accuracy compared to current confirmatory diagnostic tests (MRI, PET, and CSF biomarker analysis); it was estimated that plasma biomarker testing reduced the number of confirmatory diagnostic tests needed by up to 49%, highlighting their potential clinical utility as diagnostic tools. 72plementation studies should be undertaken to gather evidence on how plasma biomarker testing can be incorporated in various different clinics and contexts and to examine the most effective ways to use plasma biomarker testing in clinical settings.The expectation is that this will vary depending on the type of clinic and the standard of care within the clinic or geographical region in question but more research is needed.It is likely that a plasma biomarker test will be implemented in secondary care first because test results will need to be interpreted in the context of thorough clinical and neurological examination.At present, the Alzheimer's Association cautiously recommends the use of plasma biomarker tests in specialist memory clinics as part of the diagnostic workup for symptomatic individuals; 73 however, such tests will need to be extensively validated before they can be routinely implemented.For implementation into primary care, training and education of primary care providers would be needed to ensure that tests are conducted in suitable patients and the results are properly interpreted and communicated.Evidence-based appropriate use criteria and clinical implementation guidelines could support this.
From a practical perspective, standardized pre-analytical handling protocols 74,75 and reference standards will be needed to ensure reliable and consistent results are obtained.In addition, further plasma biomarker development that enables researchers to understand and take into consideration co-pathologies, such as Lewy body disease, limbic-predominant age-related TAR DNA-binding protein 43 encephalopathy, or cerebrovascular co-pathology are required.In addition, it has also been shown that chronic kidney disease, hypertension, stroke, and myocardial infarction, which are likely to be prevalent in the intended use population for AD plasma biomarker testing, have an impact on p-tau181 and p-tau217 concentrations, so will need to be considered. 76Understanding co-pathologies will allow for further patient stratification beyond amyloid and tau proteins to include other proteins that may contribute to cognitive impairment and progression to dementia-like symptoms.The frequency of amyloid deposition and the accumulation of multiple brain pathologies rise with age.
Therefore, in a 50-year-old person, a positive plasma biomarker test would indicate the presence of amyloid pathology that is highly likely to be contributing to their cognitive symptoms, whereas, in a 90-yearold person, a positive plasma biomarker test would still indicate brain amyloidosis, but there may be other pathologies contributing to or causing cognitive symptoms, as cognitive decline and dementia are often due to mixed pathology at that age.More research is needed on how the diagnostic pathway and clinical management may change with age.

CONCLUSIONS
Plasma

BOX 1
biomarker tests could have different applications along the AD diagnostic pathway and care continuum (Box 1).Such tests could add clinical value by potentially streamlining diagnosis, improving the patient journey, and alleviating pressure on health-care systems.There are a range of plasma biomarker tests at different stages of development for use in AD research and as part of the AD diagnostic pathway and care continuum, including in vitro diagnostic devices (or tests; IVDs), laboratory-developed tests (LDTs; also known as in-house devices), and research use only devices (or tests; RUOs). 18As IVDs, LDTs, and RUOs have different intended uses and are subject to different regulatory requirements, understanding the differences among them is vital so that they can be appropriately implemented into the AD diagnostic pathway and care continuum.The scope of this review is to define IVDs, LDTs, and RUOs by their relative advantages and disadvantages, discuss potential clinical applications of plasma biomarkers, and highlight the steps necessary for implementation of plasma biomarker testing into the intended use population.Current and imminent applications for plasma biomarkers in AD: • Acting as a triage tool by ruling out symptomatic individuals with a low likelihood of Aβ pathology or ruling in symptomatic individuals with a high likelihood of Aβ pathology • Enriching clinical trial populations by screening out people without Aβ pathology Potential future applications for plasma biomarkers in AD: • Acting as a diagnostic test, replacing confirmatory PET and/or CSF biomarker analysis • Predicting disease progression • Monitoring disease progression and/or treatment response, including monitoring for adverse events • Acting as a screening test for cognitively normal individuals or individuals at risk of developing AD are particularly well suited to screening as they are minimally invasive and scalable.Unlike their applications in symptomatic individuals, such as discriminating AD from other neurological pathologies, the use of plasma biomarker tests in asymptomatic individuals may help to identify high-risk individuals and enable timely implementation of prevention strategies, such as lifestyle interventions, with the potential of delaying and/or preventing cognitive decline.Notably, current FDAapproved DMTs are not indicated for the treatment of asymptomatic individuals.
biomarker tests have the potential to revolutionize the AD diagnostic pathway and care continuum and could provide more patients with accurate and timely diagnosis and/or access to treatment, while reducing the burden on health-care facilities.The volume of the scientific literature on plasma biomarkers in AD has increased steeply over the last few years, with almost double the number of publications indexed on PubMed in 2022 as there was in 2019.The purpose of this review was to provide a clinical context for this increasing scientific interest in plasma biomarkers for AD, such that there are important stages of assay development (RUO, LDT, IVD), which are completed to varying degrees and with various regulatory requirements before these plasma biomarker assays are ready for use in clinical routine within the contexts discussed here.Several clinical trials are already using plasma biomarker testing for population enrichment; however, more research and development are needed before patients and health-care facilities will tangibly benefit from plasma biomarker testing in routine use.AUTHOR CONTRIBUTIONS All authors have made a substantial contribution to the conception or design of this manuscript, have reviewed it critically for important intellectual content, have provided final approval of the version to be published, and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.Siemens Healthineers, Triplet Therapeutics, and Wave.He has given lectures in symposia sponsored by Alzecure, Biogen, Cellectricon, Fujirebio, and Roche and is a co-founder of Brain Biomarker Solutions in Gothenburg AB (BBS), which is part of the GU Ventures Incubator Program.Prof. Zetterberg is also chair of the Alzheimer's Association Global Biomarker Standardization Consortium and is a Wallenberg Scholar supported by grants from the AD Strategic Fund and the Alzheimer's Association (#ADSF-21-831376-C, #ADSF-21-831381-C, and #ADSF-21-831377-C), the Alzheimer Drug Discovery Foundation USA (#201809-2016862), the Bluefield Project, the European Union Horizon Europe Research and Innovation programme (grant agreement #101053962), the European Union's Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant (grant agreement #860197; MIRIADE), the European Union Joint Program -Neurodegenerative Disease Research (JPND2021-00694), the Erling-Persson Family Foundation, Hjärnfonden (Sweden; #FO2022-0270), the Olav Thon Foundation, the Swedish Research Council (#2022-01018), the Swedish state (the ALF agreement; #ALFGBG-71320), and the UK Dementia Research Institute at UCL (UKDRI-1003).Author disclosures are available in the supporting information. 45 22,21rences between selected categories of biomarker tests.Table created using information from references Cummings and Kinney, 18 U.S. Food & Drug Administration,19and Teunissen et al.20Abbreviations: IVD, in vitro diagnostic; LDT, laboratory-developed test; RUO, research use only.aIn the U.S., IVDs require review by the Food & Drug Administration;18,21the European Union has its own Conformitè Europëenne mark of approval for IVD devices.22 TA B L E 1 TA B L E 2 Selected AD plasma biomarker tests that are commercially available or in development.Potential clinical value of plasma biomarkers in the AD diagnostic pathway and care continuum.
54Test category and status as of April 2023.support.This could streamline referrals for PET/CSF biomarker analysis and reduce people undergoing unnecessary procedures.In turn, this could improve patient waiting times and help alleviate the burden on specialist physicians, something that would be particularly useful in countries in which patient waiting times are a concern.53Asystematicreviewconducted in the U.S. reported that combining plasma biomarker test results with Mini-Mental State Examination could improve efficiency of identifying patients who are eligible for DMT treatment, while reducing waiting lists and average annual US health-care costs.54AsDMTs become increasingly available, there will TA B L E 3 a Proposed minimum acceptance criteria are the opinions of the authors.While it is appreciated that NPVs and PPVs are dependent on the prevalence of amyloid positivity in the population in question, any estimate of prevalence is limited by the fact that > 75% of people are undiagnosed globally 3