The accuracy and robustness of plasma biomarker models for amyloid PET positivity

Therapies targeting amyloid beta (Aβ), a defining feature in the pathophysiology of Alzheimer’s disease (AD) [1], have recently been developed and proven to reduce Aβ plaque load in the brain [2‐5]. However, the cognitive benefit to symptomatic patients is either very mild or, in most cases, inconclusive. The reasons for these findings are unclear, but it is hypothesized that anti-Aβ trials target a population too advanced in the disease course or that the trial duration does not have the length to observe a conclusive cognitive benefit. Nonetheless, therapeutic trials that target any phase of the AD continuum require confirmatory evidence of Aβ burden—which is of principal importance in trials that will target preclinical AD. Cerebrospinal fluid (CSF) Aβ42/40 and Aβ positron emission tomography (PET) imaging are highly representative of Aβ burden, and the latter is likely a fundamental obligation to prove target engagement throughout an intervention trial. Still, neither CSF nor PET biomarkers have the capacity to serve as a population screening tool for eligibility to anti-Aβ trials.

A blood biomarker would act as a widely accessible and simplified triage of large and diverse populations to indicate appropriate individuals for therapeutic trial recruitment—irrespective of disease stage. Furthermore, in a clinical setting, an indication that mild cognitive symptoms are accompanied by Aβ pathology is of importance for the specialist delivering a diagnosis and symptomatic treatment and, soon, determining which disease-modifying treatment would be more suitable. The development of plasma biomarkers has been driven by targeting candidates proven to be successful in CSF. Novel mass spectrometry and ultra-sensitive immunoassay methods have recently allowed for the measurement of the Aβ42/Aβ40 ratio and concentrations of phosphorylated tau (p-tau), glial fibrillary acidic protein (GFAP), and neurofilament light (NfL) in blood.

In this context, plasma Aβ42/40 has been shown to be associated with CSF and PET measures of Aβ and to be capable of identifying Aβ-positive individuals with high accuracy [6, 7]. However, this is suggested to be assay-dependent given the emerging data highlighting the superior accuracy of immunoprecipitation mass spectrometry (IP-MS) compared with ultrasensitive immunoassays for the detection of cerebral Aβ [8]. In contrast, immunoassays for the detection of p-tau181 (as well as other epitopes; p-tau217 [9] and p-tau231 [10]) in plasma have been shown to be most valuable in identifying AD in heterogeneous dementia population [11‐14] and in predicting cognitive decline [11, 15, 16], besides also being highly correlated with cerebral Aβ burden. GFAP, a biomarker of astrocyte reactivity, increases in preclinical AD and is a promising plasma biomarker for this stage of the disease [17‐19]. While CSF GFAP is seemingly associated with Aβ pathology only in symptomatic individuals, plasma GFAP continues to rise during disease evolution in parallel with clinical syndrome severity and Aβ accumulation [17, 19]. These recent findings suggest that plasma GFAP is more closely related to abnormal Aβ accumulation due to AD, whereas CSF GFAP may also incorporate changes independent of Aβ pathology. Increases in plasma NfL are a widely reported finding in AD [20, 21] and are also observed in pre-symptomatic familial AD [22]. Contrasting to Aβ and p-tau, NfL is not specific to AD pathology and is increased in many other neurodegenerative disorders [23] and acute neurological conditions [24]. Hence, plasma biomarkers for AD are either directly (Aβ42/40) or indirectly (e.g., tau phosphorylation, astrocyte reactivity and neurodegeneration) associated with presence of Aβ pathology and could be used to indicate elevated Aβ burden for therapeutic trials. They could be used as standalone tests or in a combinational biomarker panel, but different configurations and accuracies will likely depend on disease stage; Aβ42/40 and GFAP are likely to be more associated with preclinical Aβ, whereas p-tau181 and NfL may be later markers with increases more apparent in the transition between preclinical and prodromal AD.

In this brief report, we studied the available plasma biomarker results from the Alzheimer Disease Neuroimaging Initiative (ADNI), Aβ42/40, p-tau181, GFAP, and NfL, to suggest which biomarker(s) models would be best suited as a population prescreen for Aβ burden in a clinically heterogeneous population (i.e., all participants independent of disease stage), composed by cognitively unimpaired (CU) participants and cognitively impaired (CI) patients. Further, we sought to determine the robustness of single or multi-biomarker models to identify Aβ burden by assessing whether simulated changes in biomarker concentration (0–20%) values would significantly impact on the predictive power or model selection.

In this study, our results denote that plasma Aβ42/40 as determined by IP-MS was the best predictor of Aβ-positivity, followed by p-tau181 and GFAP. In a novel approach, preparing for such tests in clinical chemistry routine, we were interested in how variations in the biomarker measurements would impact the robustness of these biomarker performances. Random variations on the CV indicated that, around a simulated CV of 5%, the accuracy of IP-MS Aβ42/40 drops below to that of GFAP and p-tau181. In contrast, GFAP and p-tau181 performances remain stable even at a 20% CV. When biomarkers were evaluated in several combinations of models, IP-MS Aβ42/40 was the most significant contributor in predicting Aβ-positivity at the preclinical stages of AD, and adding p-tau181, GFAP, or NfL did not significantly improve this finding. At the CI stages of the AD continuum, however, a model combing IP-MS Aβ42/40, GFAP, and p-tau181 was found to be the best indicator of Aβ-positivity and results in very high accuracy. In general, models that included IP-MS Aβ42/40 significantly outperformed model selections that included Simoa Aβ42/40 as an alternative. We then investigated how the variations in biomarker CV would impact on the optimal model selection. With small variations in biomarker measurements, all selected models were preserved and shown to be robust. However, for CU participants, IP-MS measurements were not able to withstand a larger variation (CV > 10%), being subsequently replaced by GFAP in the majority of model iterations. Originally selected Aβ-positivity models which included all participants and CI were robust, i.e., were most frequently selected, up to 15%.

The use of plasma biomarkers to highlight underlying cerebral Aβ pathology is greatly anticipated in clinical routine and disease-modifying trials, for both symptomatic and preclinical stages of AD. An increasing number of plasma biomarkers, shown to be related to Aβ pathology, have now been reported [7, 15, 30, 31], but it is yet to be determined which combinations are best suited in a heterogeneous population (e.g., diagnosis independent), preclinical or symptomatic stages. In this study, we show that IP-MS Aβ42/40 have high accuracy in the detection of Aβ pathology at all stages of the AD continuum and, in combination with GFAP and p-tau181, had a very high accuracy to determine Aβ-positivity in CI (> 93%). There is a mixture of reports about the use plasma Aβ42/40 in the literature [32]. While immunoassay results of p-tau from differing platforms are seemingly concordant with reproducible results and measures of plasma NfL and GFAP tend to utilize the same Simoa technology [33], methods to determine plasma Aβ42/40 varies. This study shows the importance of method choice for the detection of brain amyloidosis by plasma Aβ since, when IP-MS measures of Aβ42/40 were not included, Aβ-positivity was best represented by GFAP and p-tau181 and not by immunoassay determinations of Aβ42/40. It is also important to signify that models that included IP-MS significantly outperformed models without it.

It is unlikely that Aβ PET will be replaced from the recruitment process in anti-Aβ trials, as target engagement and possible termination of Aβ removal agents are necessary to determine participant’s baseline and subsequent changes in Aβ burden relative to the intervention process [4]. However, the plasma biomarker models demonstrated in this study may act, with good accuracy, as important initial screening tools to enrich a population for a larger success rate of Aβ PET scan or tau PET scans [4] in the recruitment process. Our aim was to report the best plasma models for this process while acknowledging that IP-MS technology currently has constraints on availability and costs in comparison to semi-automated immunoassay methods. Thus, we included a commercially available immunoassay which did not significantly add to any biomarker model and was inferior to IP-MS Aβ42/40, GFAP, and p-tau181 at the single biomarker level. Therefore, at this time, it is important to disseminate that IP-MS Aβ42/40 measurements cannot simply be replaced by immunoassay Aβ42/40 and, if IP-MS is not a viable option, Aβ-positivity is best represented by surrogate measures of Aβ pathology, e.g., GFAP and p-tau181, as shown in this study. This difference between Aβ methods could be explained by IP-MS being less prone to matrix effects that are particularly noticeable in complex biological fluids such as blood.

However, there are constraints to Aβ42/40 as a plasma biomarker which could be significant limiting factor in clinical chemistry routine. As Aβ42/40 is suggested to change by only 10% in Aβ-positivity individuals, compared with 50% in CSF [7], a moderate change in assay variability could greatly influence the result. Our first robustness analysis, which focused on random variation (not bias) on the single biomarker level, denoted a diminishing performance of IP-MS Aβ42/40 as the CV increased. While IP-MS Aβ42/40 was the best performing biomarker, random variations ~ 5% lowered the accuracy below GFAP in CU participants and p-tau181 in CI participants. As the CV increased to 15%, an accepted level of intra-assay variation in clinical chemistry, IP-MS Aβ42/40 produced AUC’s only around 60% to predict Aβ-positivity. In contrast, GFAP and p-tau181 maintained the same level of accuracy regardless of intra-assay variation. This demonstrates that plasma measures Aβ42/40 need to have a very low-level variability in order to maintain maximum accuracy. Given the more complex nature of IP-MS protocol and heterogeneous sample collections, we feel that an analytical variability of 10% or higher is likely across laboratories, particularly in ad hoc sampling in routine testing. While simulated variations showed clear shifts of best performance for single biomarkers, models incorporating biomarker combinations were more robust, remaining relatively stable with greater variations—IP-MS Aβ42/40 in combination with either GFAP (all participants) and p-tau and GFAP (CI) were relatively robust up to 20%. Again, however, in CU, where IP-MS Aβ42/40 alone was the best biomarker, higher variability affects this model selection, opting for GFAP at > 10% CV.

Despite both being antibody-based assays, the Simoa and IP-MS Aβ assays have somewhat different biochemical properties. However, it is unknown if these technical differences contribute to the observed performances. The Simoa assay utilizes the same principle as a sandwich immunoassay, where the target analyte is first bound by a capture antibody and this immunocomplex further refined by binding of a detection antibody following washing steps to remove unspecific binding. In the Simoa Aβ40 and Aβ42 assays, the same capture antibody common to both analytes is used while antibodies specific to either peptide are used for detection [34]. The Aβ40 and Aβ42 assays in the Simoa Neuro 4-plex E kit and Advantage kit are based on the same biochemical principle except that (1) different Aβ antibodies are used in either kit, and (2) the latter kit provides multiplexing advantages that allow Aβ40 and Aβ42 to be measured alongside NfL and GFAP concurrently in the same sample. The IP-MS assay enriches for Aβ in plasma by precipitating the analyte signal by binding to an Aβ-specific antibody or a cocktail of Aβ antibodies coated onto paramagnetic beads. Following elution of the bound analytes, the signal is read with a mass spectrometer, using labeled synthetic peptides as quantification [32] standards. The biochemistry of different plasma Aβ assays have been summarized in a recent review [32].

The foremost constraint in this study is that the sample size of the ADNI participants with all plasma biomarkers was limited (total, n = 118; CU, n = 50; CI, n = 68), which could have led to slightly reduced overall biomarker performance. Furthermore, it is known that preanalytical procedures and protocol variations may affect biomarker analysis and results, and therefore, we strongly advise the replication of these findings in larger independent cohorts with these available biomarker methods. However, we are encouraged that these results are in line with developing evidence from the recent literature [30]—namely, IP-MS Aβ being a strong predictor of amyloidosis [6, 7], particularly at CU [6] and p-tau181 being more important at CI [15]. In studies where IP-MS Aβ has not been included, GFAP has emerged as the principal candidate for amyloidosis [18, 19, 31, 35]. It must be noted that plasma p-tau217 and p-tau231 are variables not included in the ADNI cohort at this time. These additional p-tau biomarkers have both been shown to have high accuracy, together with a high fold change in AD, in determining Aβ pathology at both the preclinical and symptomatic phases of the disease and therefore may significantly contribute to the model selections, if available [10, 36].

In this report, utilizing participants in the ADNI database, we demonstrate that plasma Aβ, as indexed by IP-MS, is the simplest model that best determines Aβ burden at the preclinical stage. At the symptomatic phase, IP-MS Aβ in combination with GFAP and p-tau181 was found to be the simplest model with the highest accuracy. However, the accuracy of plasma IP-MS Aβ42/40 to indicate Aβ burden deteriorates with only a modest increase in analytical variation, which will pose as an issue in ad hoc testing in clinical routine or multicenter laboratory testing in trials. In contrast, despite lower overall accuracies, GFAP and p-tau181 are highly robust. In the absence of IP-MS Aβ measures, GFAP is the best predictor of amyloidosis at the preclinical stage of AD and, in combination with p-tau181, best predicts amyloidosis at the symptomatic phase of the disease.