Combining alpha-synuclein PET and seeded-amplification to improve diagnostic accuracy of Multiple System Atrophy

Combining alpha-synuclein PET and seeded-amplification to improve diagnostic accuracy of Multiple System Atrophy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Combining alpha-synuclein PET and seeded-amplification to improve diagnostic accuracy of Multiple System Atrophy Vikram Khurana, Diego Rodriguez, Barbara Changizi, Christine Sandiego, and 19 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4669602/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Biomarkers that facilitate early detection and track disease progression are an enormous unmet need in neurodegenerative diseases and their clinical trials. Accurate diagnosis in the early stage of Parkinsonian disorders is particularly challenging. Multiple system atrophy (MSA) and Parkinson’s disease (PD) share many clinical features and are associated with alpha-synuclein (αSyn) aggregation. However, these diseases have distinct biology and disease trajectories and are likely to respond differently to experimental therapies. Gold-standard diagnosis is only achieved at postmortem examination. Here, we combined two emerging technologies: brain imaging with αSyn [18F]ACI-12589 PET tracer with a skin αSyn seed-amplification assay (αSyn-SAA). These assays have the potential to increase diagnostic precision in vivo by delineating the spatial distribution and conformation of αSyn pathology, respectively. Of 8 clinically probable or established MSA patients, combining brain imaging with αSyn [18F]ACI-12589 PET tracer and skin αSyn-SAA helped confirm the diagnosis in 6 of the 8 patients and led to the reclassification of two cases to Parkinson’s disease and idiopathic late-onset cerebellar ataxia. Each test provided critical evidence of diagnosis even when the other was equivocal, supporting the combination of these tests. These αSyn biomarkers should now be used systematically to facilitate early and precise diagnosis across synucleinopathies. Health sciences/Neurology/Neurological disorders/Movement disorders Health sciences/Biomarkers/Diagnostic markers Health sciences/Diseases/Neurological disorders/Movement disorders Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction For decades, most clinical trials have failed to identify beneficial disease-modifying effects of experimental compounds across neurodegenerative diseases. High among many potential explanations are the heterogeneous nature of these disorders, difficulties in establishing early diagnosis, and the lack of validated biomarkers that track disease progression and treatment outcome. Indeed, definitive diagnosis has traditionally relied on neuropathological examination of postmortem brain tissue 1,2 . However, the development of disease-specific biomarkers has recently led to the inclusion of these tests as part of the clinical diagnostic criteria and disease-modifying clinical trials 3,4 . Recently, regulatory agencies have accepted data generated with imaging and biofluid biomarkers to accelerate the approval of a monoclonal antibody for Alzheimer's disease (AD) and the antisense oligonucleotide, Tofersen, for amyotrophic lateral sclerosis (ALS) 5–7 . These approvals were at least initially supported by significant changes in surrogate biomarkers, which were subsequently validated by the demonstration of clinical benefit. Specifically, beta-amyloid (Aβ) positivity measured as Aβ( 1 – 42 ) in cerebrospinal fluid (CSF) or by PET, was instrumental for the inclusion of AD subjects in the CLARITY-AD trial that ultimately led to the regulatory approval of Lecanemab 6 . The biomarkers better defined the target population of the clinical trials, translating into improved clinical outcomes. Similarly, Tofersen, an antisense oligonucleotide targeting the superoxide dismutase gene (SOD) 1, was approved after observing a decrease in CSF SOD1 and plasma neurofilament light chain (NfL) among ALS patients in the treatment arm 7 . αSyn-rich neuronal and glial aggregates are the pathologic hallmark of synucleinopathies, such as Parkinson's disease (PD) and multiple system atrophy (MSA) 8 . The hallmark pathology in PD localizes in neuronal bodies 8 . Conversely, the identification of glial cytoplasmic inclusions (GCI) in oligodendrocytes defines the neuropathology of MSA 2,9,10 . Along with autonomic dysfunction, MSA is characterized by either predominant parkinsonism (MSA-P) or cerebellar ataxia (MSA-C). In MSA-P, asymmetric parkinsonism, mild autonomic dysfunction, and a transient response to levodopa can mimic an atypical presentation of PD. Despite having distinct pathology, MSA can be easily mistaken for PD with autonomic dysfunction early in the course of the disease. Indeed, even postmortem, recent series suggest that as many as 40 percent of early-stage MSA cases can be misdiagnosed in life 11–14 . Conversely, PD with early autonomic dysfunction can also be mistaken for clinically probable MSA-P. 11 In an era of disease-modifying therapeutics for neurodegeneration, early and precise diagnosis has become imperative to triage patients to appropriate clinical trials. Because MSA is a rare orphan disease and PD a very prevalent one, misdiagnoses during clinical trial recruitment can be sufficient for MSA trials to fail in identifying modest disease-modifying effects. The field of αSyn pathology biomarkers has greatly advanced during the last few years, particularly with the development and clinical validation of the αSyn seed amplification assays (αSyn-SAA) and the first αSyn PET tracer, [18F]ACI-12589 15 . αSyn-SAA enables the capture and amplification of disease-specific αSyn conformers (or "strains") from peripheral samples such as CSF, skin, and blood 16–20 . In this assay, synthetic monomeric αSyn is co-incubated with αSyn-containing patient sample. The self-templating nature of the αSyn conformer then enables the specific amyloid to propagate and amplify in the presence of the monomer 21,22 . This biomarker can aid in diagnosing early-stage disease, and ongoing efforts promise a quantifiable metric of disease progression 18,20 . Most pertinent to differential diagnosis, αSyn-SAA can discriminate between MSA and PD αSyn strains and thus powerfully distinguish between these diseases 17,22 . MSA strains exhibit a shorter duration of the lag phase and a lower maximal fluorescence compared to PD strains 16 . These differences are associated with unique protein digestion patterns and distinct structural characteristics observed using electron microscopy 16 . Beyond CSF, ɑSyn aggregates can also be amplified from other biological samples, including olfactory mucosa, skin, and blood 17,18,20,23 . Moreover, semi-quantitative immunofluorescent assays have also been used to identify the intraepidermal localization of phosphorylated ɑSyn deposits in the skin and to differentiate MSA from other synucleinopathies 24–26 . The profound impact of SAA is manifest in new biological aSyn-based diagnostic and staging criteria proposed to facilitate precise diagnosis and clinical trial recruitment in PD 3,4 . While the αSyn seed amplification provides conformational information, the spatial distribution of αSyn deposition in the brain is missing. A PET radiotracer for imaging αSyn could fill that critical gap 15,27,28 . Moreover, PET is a noninvasive quantitative technique that has been extensively validated to measure disease progression and therapeutic responses for other diseases 15,27 . While not currently available for PD, the αSyn PET tracer [18F]ACI-12589 was recently shown to differentiate MSA from PD and other neurodegenerative disorders. Patients with MSA exhibited increased [18F]ACI-12589 retention in the cerebellar white matter (CWM) and middle cerebellar peduncles (MCPs). This was predictably more pronounced in the cerebellar (MSA-C) MSA compared to the Parkinsonian (MSA-P) subtype 15 . The MSA-P cohort also exhibited increased [18F]ACI-12589 retention in the lentiform nuclei of the striatum. However, it was not sufficiently elevated to significantly distinguish it from other synucleinopathies, such as PD and Dementia with Lewy Bodies (DLB) 15 . In the current observational cohort study, we sought to exploit exciting advances in αSyn biomarkers to improve upon current clinical consensus criteria for MSA. We recruited patients who met the consensus criteria for probable or established MSA and were eligible for the ongoing clinical trials for MSA within the Brigham and Women’s Hospital MSA Center of Excellence and Harvard Biomarkers Study MyTrial-MSA Program 29 . We combined brain imaging with the αSyn [18F]ACI-12589 PET tracer with a skin-based αSyn-SAA to simultaneously acquire conformational and spatial information about αSyn pathology. Despite the small sample size, our study suggests that the combination of both biomarkers may usefully contribute to diagnostic specificity. Indeed, we identified an MSA-specific αSyn signal in six out of eight participants, while an alternative diagnosis was considered for the remaining two cases, initially diagnosed as "clinically probable MSA-P." Additionally, all equivocal results in one assay were complemented by more definitive data from the other. We felt compelled to disseminate these early findings in order to prompt the use of αSyn-targeting biomarkers in clinical-trial recruitment for MSA, especially MSA-p. Accurate diagnosis, particularly to exclude far more common PD patients with atypical presentations, may potentially make all the difference to the outcome of disease-modifying treatments for this rare and devastating disease. Results Participants and clinical MSA diagnosis The Brigham and Women’s Hospital (BWH) P + A + MSA clinic and MSA-MyTrial Program work in close coordination to recruit MSA patients for accurate diagnosis, longitudinal biometric and biomarker assessment, with a view to pivoting from natural history to target-engagement studies for a variety of potential investigator- or industry-sponsored therapies. Fourteen participants with a clinically probable or clinically established MSA diagnosis were evaluated for participation in this study 29 . Three subjects were deemed ineligible due to insufficient diagnostic certainty or an alternative diagnosis under consideration. Two subjects were hospitalized for acute disease shortly after screening, and one participant did not tolerate the PET scan due to significant drooling and concern for respiratory compromise. Seven subjects with probable MSA and one with clinically established MSA (MSA-C = 3, MSA-P = 5) completed this study between December 2022 and December 2023. All subjects underwent dynamic scan with [18F]ACI-12589 60–90 min post-injection and PET data were analyzed as 60–90 min standardized uptake value ratio (SUVR) 15 . Twenty-three 3-mm punch skin biopsies in total from these subjects were obtained and processed for ɑSyn-SAA. Participants were evaluated for disease severity and cognitive impairment prior to enrollment. No adverse events were reported during the study. The study design and baseline demographics are presented in Fig. 1 . In addition, as a reference for [18F]ACI-12589 signal retention in MSA, we included [18F]ACI-12589 PET data from 10 previously enrolled subjects (5 healthy volunteers and five PD) 15 . The baseline demographics of these subjects (Supplementary Table 1) and a brief description of the core and supportive clinical features, as well as additional clinical information for each MSA participant, can be found in “MSA Core Clinical Features and Case Descriptions” in the Supplementary Material. In vivo [18F]ACI-12589 PET signal retention in MSA [18F]ACI-12589 PET images were reconstructed and co-registered to T1-weighed MR. We first examined representative axial sections at the level of the cerebellar white matter (CWM) and middle cerebellar peduncles (MCP), since these regions significantly discriminated between MSA and PD or healthy volunteers in the initial characterization of [18F]ACI-12589 15 (Fig. 2 a). All MSA-C cases exhibited a significantly increased retention of the [18F]ACI-12589 radiotracer in the MCP compared to healthy volunteers (60–90 min SUVRs, MSA: 1.64 ± 0.10, HV: 1.08 ± 0.09; Dunn test p = 0.019, Supplementary Table 2). CWM and MCP retention were also increased in MSA-C compared to PD and MSA-P subjects, although no statistical significance was observed after adjusting for multiple comparisons (Fig. 2 b, c). While, at the individual level, all MSA-C cases demonstrated significant [18F]ACI-12589 retention in the MCP, this finding was not consistently observed across MSA-P cases. MSA-P cases #2 and #5 demonstrated borderline increased signal in this region, not strong enough to distinguish them from alternative diagnoses. Additional images for all HV and PD participants and the SUVRs for key regions across groups are presented in Supplementary Fig. 1. We next considered the lentiform nucleus of the basal ganglia, a key site of ɑSyn accumulation in MSA, especially MSA-P (Fig. 3 a) 30 . MSA-P participants trended toward increased retention of [18F]ACI-12589 in the lentiform nuclei compared to healthy volunteers, MSA-C, and PD (Fig. 3 b). However, the group comparison was not statistically significant and was primarily driven by MSA-P case #5, who exhibited a high SUVR in the globus pallidum and putamen (Fig. 3 c). Notably, despite exhibiting strikingly different [18F]ACI-12589 signals, MSA-P cases #4 and #5 demonstrated a similar severity of Parkinsonism and clinical impairment, as indicated by similar MDS-UPDRS, MDS-UPDRS part 3, and UMSARS scores (Supplementary Table 1). This finding suggests that, on an individual level, MSA-P patients with equivalent levels of Parkinsonism might exhibit highly disparate ɑSyn levels in the basal ganglia, at least as measured by [18F]ACI-12589, a point we return to below. Altogether, these data confirm the capacity of [18F]ACI-12589 PET to fully discriminate MSA-C cases from healthy controls or PD cases, attributable to its strong spatial ɑSyn distribution in the MCP and CWM in MSA-C. However, they also indicate that signal retention in MSA-P is more variable, potentially because of a higher variability of the ɑSyn levels in the basal ganglia that has been described in postmortem studies 30 . Skin ɑSyn-SAA in MSA SAA utilizes the prion-like properties of ɑSyn, whereby pathological seeds in a lysate (in this case, generated from skin) induce the aggregation of normal monomeric ɑSyn into templated fibrils, detectable via Thioflavin T (ThT) fluorescence. ɑSyn-SAA has demonstrated high diagnostic accuracy, sensitivity, and specificity for identifying ɑSyn in PD and MSA patients 16–18 . Moreover, MSA and PD strains distinctly interact with fluorescent markers and have different patterns in ɑSyn-SAA that can be used in the differential diagnosis. 16 A lower fluorescence and earlier lag time are characteristic of MSA (Fig. 4 a). While this has been well demonstrated in brain tissue and CSF, ɑSyn-SAA from peripheral sites like the skin has also shown considerable promise and similar amplification properties to central sites among synucleinopathies 18,31 The advantage of skin is that it is far more deployable for clinical trial recruitment than CSF. Twenty-three skin biopsies were processed from our MyTrial subjects into skin lysates for ɑSyn-SAA (one subject consented to only two sites). Sixteen biopsies were positive for ɑSyn amplification, meaning that at least 75% (3/4) of the experimental replicates at that site demonstrated increased ThT fluorescence during the assay (see Methods for more details). Five of the eight MSA participants had at least one positive biopsy on ɑSyn-SAA that exhibited a characteristic MSA pattern in at least 3/4 technical replicates (Fig. 4 b; Supplementary Table 3). Figure 4 b illustrates ɑSyn-SAA results for a representative skin biopsy from each subject and a corresponding [18F]ACI-12589 inset, both at the level of the cerebellum and basal ganglia. Supplementary Figs. 2 and 3 display the ɑSyn-SAA results for all skin biopsies in the MSA cohort. There were three notable outliers. For MSA-P case #1 all biopsies exhibited ɑSyn amplification but none was consistent with MSA. Instead, these three biopsies exhibited a pattern consistent with a PD ɑSyn strain (high fluorescence and late lag time). Second, for MSA-P case #3, all three biopsies were consistently negative, with less than 25% (1/4) of positive replicates despite retesting. Lastly, MSA-C case #1 had 50% (2/4) positive replicates on ɑSyn-SAA with an MSA pattern, consistent with an inconclusive SAA result. Thus, SAA was capable of confirming a synucleinopathy diagnosis in 6/8 of our cases. Advantage of combining a-Syn biomarkers in the diagnosis of MSA In isolation, [18F]ACI-12589 identified a spatial ɑSyn signal in key MSA areas in five out of eight participants and SAA confirmed synucleinopathy in 6/8 of our cases. It became clear that both tests together were superior to each test alone. [18F]ACI-12589 performed better for MSA-C, even when SAA was equivocal (MSA-C case #1), Conversely, SAA was particularly useful for MSA-P, where only two out of five MSA-P cases had visually increased [18F]ACI-12589 retention in the basal ganglia (Fig. 4 c). Two notable scenarios arose in our patients that demonstrate the utility of both tests. First, there were patients without evidence of MSA-pattern ɑSyn pathology in either skin SAA or [18F]ACI-12589 PET (Fig. 5 a), suggesting an alternative diagnosis. For instance, MSA-P #1 is a patient with prominent neurogenic orthostatic hypotension and bilateral asymmetric Parkinsonism, initially diagnosed with clinically probable MSA-P. However, [18F]ACI-12589 PET failed to identify a spatial ɑSyn signal in key MSA regions, and ɑSyn-SAA demonstrated a pattern consistent with PD. In subsequent clinical evaluations, the patient’s relatively benign course thereafter was also inconsistent with an MSA diagnosis, and this patient was re-classified as having an atypical presentation of idiopathic PD with prominent early-onset autonomic dysfunction (Fig. 5 a, top panel). Similarly, for MSA-P #3 both biomarkers failed to identify any conformational or spatial ɑSyn signature in MSA-P #3. This patient had a longstanding history of autonomic dysfunction that included orthostatic hypotension and urinary incontinence, as well as mild bilateral ataxia with Parkinsonism. He also progressed minimally over a year after the study, inconsistent with the natural history of MSA (Fig. 5 a, bottom panel). This case was re-evaluated after the study was completed and we performed a commercial test for ɑSyn visualization in skin with immunofluorescence, which was also negative in all three sites. This case has been re-classified as a non-synucleinopathy case (“idiopathic late-onset cerebellar ataxia” [ILOCA] with parkinsonism), and additional diagnostic tests are ongoing 32,33 . The second scenario comprises probable MSA-P cases in which fairly equivalent motor dysfunction is associated with a highly divergent signal on [18F]ACI-12589 PET, but a very consistent signal on SAA. For example, MSA-P #4 and MSA-P #5 are two probable MSA-P with severe, symmetric bilateral parkinsonism that is poorly responsive to dopaminergic therapy. Both cases exhibit similar severity scores across clinical rating scales (MDS-UPDRS and UMSARS). However, while there was no increased [18F]ACI-12589 retention in the basal ganglia for MSA-P #4, MSA-P #5 exhibited a robust increase of tracer retention in the putamen and globus pallidus. Thus, using [18F]ACI-12589 PET in isolation, only MSA-P #5 would have an ɑSyn signature compatible with an MSA diagnosis. However, in skin ɑSyn-SAA, both cases demonstrated an MSA pattern of ɑSyn amplification across all three skin biopsies and patients were both diagnosed with probable MSA-P (Fig. 5 b). Finally, although ɑSyn-SAA has shown to be a very sensitive test, it might not always reliably amplify the disease-specific ɑSyn strain. This was the case for MSA-C #1, a patient with clinically established disease but an inconclusive ɑSyn-SAA result. Despite an ɑSyn amplification pattern suggestive of MSA, only 50% of replicates (2/4) were positive in one out of three skin biopsies, even after re-testing. However, [18F]ACI-12589 imaging demonstrated significantly increased retention in the CWM and MCP (Fig. 5 c). Moreover, there was also increased radiotracer retention in the basal ganglia compared to HV and PD cases. Taken together, these results demonstrate the utility of both tests as mutually reinforcing biomarkers for the differential diagnosis of MSA. Discussion Clinical trials for neurodegenerative movement disorders rely on precise clinical diagnosis and rating scales as primary endpoints. Extraordinary developments in ɑSyn peripheral tests, notably SAA, have led to recent calls for new ɑSyn-based diagnosis and staging criteria for Parkinson’s disease. 3,4 . But for MSA there is already the possibility of using PET, just as in the AD field, to achieve spatial resolution diagnosis. Therefore, in this study, surmising that a surrogate for the gold-standard postmortem diagnosis of synucleinopathies could be achievable by combining spatial and conformational ɑSyn markers, we combined the brain PET ɑSyn ligand [18F]ACI-12589 with skin ɑSyn-SAA to a real-life observational cohort at our institution that seeks to deeply characterizes patients with a clinical diagnosis of MSA before pivoting them to an appropriate clinical trial 29 . Despite a small sample size, our study confirmed the well-known error rate in making an accurate MSA diagnosis, which can be up to 40% 11,14 . Importantly, the combination of these emerging ɑSyn markers appeared to be more accurate in this small study than either investigation alone. In particular, our early data suggest that the combination of these markers was able to move the needle on one of the most troublesome differential diagnoses in the movement disorders clinic, that of MSA-P versus PD. Increased [18F]ACI-12589 retention presumably correlates with the burden of ɑSyn aggregates in specific brain regions 15 . In the recent initial [18F]ACI-12589 study, increased ligand retention in the CWM and MCP differentiated patients with MSA-P and MSA-C from HV, PD, and other neurodegenerative disorders 15 . The higher retention of the [18F]ACI-12589 signal in the cerebellum of MSA-C cases was confirmed in this study (Fig. 2 a). Moreover, MSA-P cases exhibited increased [18F]ACI-12589 retention in the basal ganglia (Fig. 3 a) 15 . However, the tracer retention in the basal ganglia was more variable and present, to a lesser extent, in some HV and PD subjects (Fig. S1 ). Consistent with this, our study found variable [18F]ACI-12589 retention in probable MSA-P subjects. Indeed, only two out of five MSA-P cases exhibited a visually distinguishing signal in the basal ganglia that supported an MSA diagnosis (Fig. 4 b). Moreover, we observed that cases with similar clinical features and disease severity could display widely different levels of [18F]ACI-12589 retention (Fig. 5 b). One possible explanation for this finding is that clinical parkinsonism can be driven by pre-synaptic pathology in the substantia nigra, but also by post-synaptic striatal pathology with ɑSyn aggregates and neuronal loss in the caudate and pallidum 34 . While pre-synaptic pathology is the dominant mechanism in PD, both mechanisms are relevant in MSA and could partially explain different synuclein distributions in patients with similar clinical phenotypes. This variability in striatal pathology has been observed also in postmortem MSA-P of equivalent disease duration 30 . In this study, the combination of skin-based ɑSyn-SAA and brain imaging with ɑSyn [18F]ACI-12589 PET tracer was crucial for providing a biological “anchor” to the probable/established MSA diagnosis. Moreover, we identified two out of eight cases (25%) with no evidence of MSA-pattern ɑSyn pathology despite meeting the criteria for clinically probable MSA-P. We clinically re-evaluated these cases, and the diagnosis was reconsidered. Importantly, these cases would likely been included in current clinical trial designs for MSA, adding undesired noise to the collected data. Recently proposed biological classifications of PD rely on ɑSyn-based biomarkers and key prodromal features to better identify disease populations that reflect the underlying biology of the disease 3,4 . In time, ɑSyn biomarkers, including PET or peripheral seed amplification, could provide invaluable ɑSyn-specific data to MSA diagnostic criteria. While we favored the skin ɑSyn-SAA as the peripheral assay of choice here, promising recent studies have demonstrated promise in immunofluorescent analysis of peripheral nerves from the skin (ɑSyn-IF) 24–26 . This test has shown that the location of ɑSyn skin aggregates can be used to differentiate between MSA and Lewy body disease (DLB and PD) 24 . Both assays provided complementary information about ɑSyn skin pathology, and a head-to-head comparison would be valuable. The major limitation of this study is the lack of a neuropathologically confirmed diagnosis. This is a recurrent theme in the field as novel biomarkers become available but cannot be evaluated in what is considered the gold standard diagnosis. Additional limitations include the small sample size and the need for widespread validation of these novel biomarkers, including in patients with early or even prodromal disease stages. Lastly, PET and ɑSyn-SAA must be processed in specialized tertiary care centers and are not readily available across different populations. Although ɑSyn-SAA has been extensively characterized in CSF and skin, both of these tests necessitate invasive procedures for sample collection, which are resource-intensive and require significant logistical efforts to integrate into a neurology clinic 18,23,35 . The collection of CSF involves a lumbar puncture, which can cause discomfort and carries risks such as headaches and infection. Similarly, skin biopsies, while less invasive, still require careful planning and can lead to minor complications such as bleeding or infection. The olfactory mucosa has been explored as a more accessible site for sample collection; however, this procedure must be performed by an otolaryngology specialist, adding another layer of complexity and limiting its widespread application 23,36 . Ongoing efforts to develop blood-based ɑSyn-SAA present a more accessible alternative that could be applied to larger populations. However, these assays require either immunoprecipitation or the isolation of exosome-derived α-synuclein. 17,20 This approach holds promise for broader clinical use, but further optimization and validation are needed to ensure its efficacy and reliability in diverse clinical settings. Beyond aiding in accurate diagnosis, emerging ɑSyn biomarkers hold the potential for measuring disease progression and target engagement. This has been extensively validated with other brain PET tracers for targets in neurodegeneration, particularly for Aβ. Therefore, ɑSyn PET might be a key biomarker for monitoring disease progression and evaluating the engagement of ɑSyn therapies such as monoclonal antibodies and anti-sense oligonucleotides 37 . Multiple ɑSyn radiotracers are currently being tested in clinical and preclinical settings 15,28,38,39 , and our data supports the promise, among these, of [18F]ACI-12589. Recently, extensive efforts have also sought to make ɑSyn-SAA more quantitative. Certain kinetic parameters in ɑSyn-SAA may correlate with disease stage, REM sleep behavior disorder (RBD) status, and cognitive function 18,40,41 . However, further validation and standardization of ɑSyn-SAA protocols are necessary to explore these potential associations on a larger scale. Ongoing efforts to create a digital, quantitative ɑSyn-SAA may result in a more nuanced assay to help track disease progression and target engagement 37 . In conclusion, our study suggests that both ɑSyn PET imaging with [18F]ACI-12589 and ɑSyn-SAA provide complementary information in the differential diagnosis of MSA, particularly of MSA-P. Combining these spatial and conformational ɑSyn biomarkers might help achieve a precise and early MSA diagnosis anchored to a disease-specific ɑSyn signature. This approach can help biologically define the homogeneous populations suitable for clinical trial enrollment and improve the chances of detecting the efficacy of disease-modifying therapies. Methods Ethics approval This observational study complies with pertinent ethical regulations, including STROBE guidelines and the principles of the Declaration of Helsinki. The initial clinical evaluation and PET imaging were performed at Invicro, LLC, under protocol number 9105, approved by the Advarra Institutional Review Board (IRB). Skin biopsies, skin SAA, and follow-up evaluations occurred at Brigham and Women’s Hospital under protocol number 2009P000775 (Mass General Brigham IRB). Participant recruitment We included participants with a clinical diagnosis of probable or established MSA based on the most recent MDS criteria. Participants were recruited from the MSA Center of Excellence and Parkinsonism, Ataxia, and MSA (P + A + MSA) clinic at Brigham and Women’s Hospital. All MSA patients were included in our previously described MyTrial program for clinical-trial-ready cohorts in neurodegenerative disease 29 . We also recruited age-matched healthy volunteers and participants with a diagnosis of PD as determined by a movement disorders specialist following the UK Brain Bank diagnostic criteria. These subjects were recruited at Invicro and completed the [18F]ACI-12589 component of this study. The stage of the disease was evaluated using the Hoehn and Yahr (H&Y) scale. Disease severity was evaluated using the Unified Multiple System Atrophy Rating Scale (UMSARS) and the MDS-revised Unified Parkinson’s Disease Rating Scale (MDS-UPDRS). Patients were screened for mild cognitive impairment using the MoCA and for RBD using a single-question screening. [18F]ACI-12589 PET Imaging and Reconstruction Each subject underwent a 90-minute [18F]ACI-12589 PET scan on a Siemens ECAT EXACT HR + camera, centering the brain in the field of view. [18F]ACI-12589 was injected over 3 minutes through a venous catheter, followed by a 10 mL saline flush. Preceding radiotracer injection, a transmission scan was performed, which was needed for attenuation correction of the PET emission data. [18F]ACI-12589 PET images were reconstructed with all corrections applied (random, scatter, dead time, and attenuation). The dynamic PET data were reconstructed with the following frame timing: 6x30 sec, 4x1 min, 4x2 min, and 15x5 min. Image Processing and Quantification At screening, a T1-weighed MR image was collected from each subject, which was required for PET analysis. MR and PET image processing were performed in PMOD v. 3.802 (PMOD Technologies, Zurich, Switzerland). PET frames were motion corrected, rigidly aligned with the subject’s MRI, and normalized into the standard Montreal Neurological Institute (MNI) space where volumes of interest (VOIs) were defined. Brain atlas VOIs examined included amygdala, caudate, cerebellar white matter, midbrain, pallidum, pons, and putamen. A PET image-derived VOI, cerebellar white matter tracts, was delineated using an average of three subjects with high uptake within the cerebellar white matter (see Supplementary Fig. 4). Time-activity curves (TACs) were generated for each VOI, with concentration (kBq/cc) on the y-axis and time (min) on the x-axis. For visual comparison, TACs and PET images were converted from concentration to standard uptake value (SUV) units (g/mL) by normalizing with the weight of the subject and the injected dose. The SUV ratio (SUVR) was computed between 60–90 min to assess regional differences between MSA and healthy control subjects, using the cerebellar cortex as the reference 15 . The distribution volume ratio (DVR) was estimated with non-invasive Logan graphical analysis (t*=10 min), using the cerebellum as the reference region. DVR and SUVR (60–90 min) were highly correlated (R2 = 0.94), with a slight positive bias for SUVR, where SUVR = 1.13*DVR − 0.15. Skin biopsy procedure Four skin punch biopsies, each with a diameter of 3 mm, were obtained from the posterior cervical region (C7), lateral lower thigh, and two from the lower leg (ankle). One of the ankle biopsies was used to generate human fibroblast cultures for differentiation into induced pluripotent stem cell (iPSC) models matched to biospecimens as described in our MyTrial program for clinical-trial-ready cohorts in neurodegenerative disease 29 . Local anesthesia with 1% lidocaine was administered prior to the procedure. Post-biopsy wounds were dressed in non-adhesive bandages, and participants were monitored for adverse events via a follow-up call within a week. Subsequently, the biopsy specimens were flash-frozen in liquid nitrogen, coded blindly, and stored at − 80°C. Skin SAA Each biopsy was processed into a skin lysate using a similar assay procedure to the ones previously described. Briefly, two 7-mm steel beads were used to homogenize skin tissue using the TissueLyser LT device (Qiagen, Germany) to prepare 10% skin lysates. The lysates were further diluted 20-fold, and 2 µl were added to a reaction buffer containing 1 mg/mL C-terminus 6xHis-tagged recombinant wild-type α-syn, 500 µM NaCl, 100 mM PIPES, 10 µM thioflavin T (ThT), and 6+-2 silica glass beads (OPS Diagnostics) 16,18,23 . Recombinant wild-type α-syn with 6xhistidine-tag at C-terminal was produced in the Rudolf–Virchow Center, University of Würzburg, Germany, recombinant protein expression facility 18 . Each skin biopsy was tested in quadruplicate by adding 100 µl of the reaction buffer to a 96-well plate for incubation at 37°C with cycles of 1 min double orbital shaking at 400 rpm and 5 min rest. The assay spanned 48 hours, during which fluorescence readings were recorded at 448 nm/482 nm wavelengths every 45 minutes using a FLUOstar Omega microplate reader (BMG Labtech, Germany). Maximal fluorescence is expressed as a percent of the maximum relative fluorescent units (RFU) in our assay. Samples were deemed positive if at least 75% of the replicates demonstrated increased fluorescence from baseline. Skin αSyn-SAA analysis After determining whether each replicate was positive for α-syn aggregation, the following parameters were calculated: maximal ThT fluorescence, time (h) for a reaction to reach 50% of the maximum fluorescence (T50), duration of lag phase (the reaction time [h] required to cross the fluorescence threshold), and area under the ThT fluorescence curve (AUC). Mean and standard deviation across positive replicates were computed for each variable per biopsy. Positive samples with a mean maxRFU under 20% of the maximal fluorescence were considered to have an MSA αSyn aggregation pattern. Those over 60% of the maximal fluorescence were deemed as a PD aggregation pattern. The αSyn-SAA test was considered positive if at least one of the biopsy samples yielded a positive result with an MSA or PD pattern. Statistical Analysis Statistical analyses were conducted using the R software (R version 4.3.2, RStudio 2023.12.0.369; Boston, MA, USA). For intergroup comparisons of non-normally distributed [18F]ACI-12589 data we used the Kruskal-Wallis test with a significance level of 0.05. When significant, we adjusted for multiple comparisons using the Dunn test with the Bonferroni method. Data in supplementary tables is represented by means and standard deviations. Figures were created in ggplot2 version 3.4.4 and Affinity Designer 2 (Serif, 2022). Declarations Competing interests AC Immune SA provided the radiotracer used in this study [18F]ACI-12589. AC Immune SA was not involved in the study design, data collection, analysis, interpretation of data, the writing of this article, or the decision to submit it for publication. The authors had full access to all the data in this study and take complete responsibility for the integrity of the data and the accuracy of the data analysis. E.V., J.M., I.K.D., V.H., M.K.-V., N.M., and F.C. are employees of AC Immune SA. C.S., A.G., J.A., A.K., and D.S.R. are employees of Invicro, LLC. V.K. is a co-founder of and senior advisor to DaCapo Brainscience, a company focused on CNS diseases. D.R., B.K.C., A.K., O.L., K.J., D.C., J.M., S.P., and S.R. report no disclosures. References Postuma RB et al (2015) MDS clinical diagnostic criteria for Parkinson’s disease. Mov Disord Off J Mov Disord Soc 30:1591–1601 Wenning GK et al (2022) The Movement Disorder Society Criteria for the Diagnosis of Multiple System Atrophy. Mov Disord 37:1131–1148 Simuni T et al (2024) A biological definition of neuronal α-synuclein disease: towards an integrated staging system for research. 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Clifton NJ 35–44 (2019) Shahnawaz M et al (2017) Development of a Biochemical Diagnosis of Parkinson Disease by Detection of α-Synuclein Misfolded Aggregates in Cerebrospinal Fluid. JAMA Neurol 74:163–172 Kuzkina A et al (2023) Combining skin and olfactory α-synuclein seed amplification assays (SAA)-towards biomarker-driven phenotyping in synucleinopathies. NPJ Park Dis 9:79 Gibbons C et al (2023) Cutaneous α-Synuclein Signatures in Patients With Multiple System Atrophy and Parkinson Disease. Neurology 100:e1529–e1539 Gibbons CH et al (2024) Skin Biopsy Detection of Phosphorylated α-Synuclein in Patients With Synucleinopathies. JAMA 331:1298–1306 Gibbons CH, Garcia J, Wang N, Shih LC, Freeman R (2016) The diagnostic discrimination of cutaneous α-synuclein deposition in Parkinson disease. Neurology 87:505–512 Korat Š et al (2021) Alpha-Synuclein PET Tracer Development—An Overview about Current Efforts. Pharmaceuticals 14:847 Xiang J et al (2023) Development of an α-synuclein positron emission tomography tracer for imaging synucleinopathies. Cell 186:3350–3367e19 Ndayisaba A et al (2024) Clinical Trial-Ready Patient Cohorts for Multiple System Atrophy: Coupling Biospecimen and iPSC Banking to Longitudinal Deep-Phenotyping. Cerebellum Lond Engl 23:31–51 Brettschneider J et al (2018) Converging Patterns of α-Synuclein Pathology in Multiple System Atrophy. J Neuropathol Exp Neurol 77:1005–1016 Liguori R et al (2023) A comparative blind study between skin biopsy and seed amplification assay to disclose pathological α-synuclein in RBD. Npj Park Dis 9:1–6 Khurana V, De Gusmao CM, Glover M, Helgager J (2021) Case 20-2021: A 69-Year-Old Man with Ataxia. N Engl J Med 385:165–175 Lin DJ, Hermann KL, Schmahmann JD (2016) The Diagnosis and Natural History of Multiple System Atrophy, Cerebellar Type. Cerebellum Lond Engl 15:663–679 Ghaemi M, Hilker R, Rudolf J, Sobesky J, Heiss W (2002) Differentiating multiple system atrophy from Parkinson’s disease: contribution of striatal and midbrain MRI volumetry and multi-tracer PET imaging. J Neurol Neurosurg Psychiatry 73:517–523 Coughlin DG, Irwin DJ (2023) Fluid and Biopsy Based Biomarkers in Parkinson’s Disease. Neurotherapeutics 20:932–954 Bongianni M et al (2022) Olfactory swab sampling optimization for α-synuclein aggregate detection in patients with Parkinson’s disease. Transl Neurodegener 11:37 Gilboa T et al (2024) Toward the quantification of α-synuclein aggregates with digital seed amplification assays. Proc. Natl. Acad. Sci. U. S. A. 121, e2312031121 Wang C (2024) PET Imaging Evaluation of [11C]SY08 . https://clinicaltrials.gov/study/NCT06098612 Kim HY et al (2023) A Novel PET Radiotracer for Imaging Alpha Synuclein Fibrils in Multiple System Atrophy (MSA). J Nucl Med 64:P1402–P1402 Russo MJ et al (2021) High diagnostic performance of independent alpha-synuclein seed amplification assays for detection of early Parkinson’s disease. Acta Neuropathol Commun 9:179 Bräuer S et al (2023) Kinetic parameters of alpha-synuclein seed amplification assay correlate with cognitive impairment in patients with Lewy body disorders. Acta Neuropathol Commun 11:162 Additional Declarations Yes there is potential Competing Interest. AC Immune SA provided the radiotracer used in this study [18F]ACI-12589. AC Immune SA was not involved in the study design, data collection, analysis, interpretation of data, the writing of this article, or the decision to submit it for publication. The authors had full access to all the data in this study and take complete responsibility for the integrity of the data and the accuracy of the data analysis. E.V., J.M., I.K.D., V.H., M.K.-V., N.M., and F.C. are employees of AC Immune SA. C.S., A.G., J.A., A.K., and D.S.R. are employees of Invicro, LLC. D.R., B.K.C., A.K., O.L., K.J., D.C., J.M., S.P., S.R., and V.K. report no disclosures. Supplementary Files Supplementarymaterial.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4669602","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":327257579,"identity":"44d8280c-994c-472d-8fa9-00b7fa8ba608","order_by":0,"name":"Vikram 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SA","correspondingAuthor":false,"prefix":"","firstName":"Francesca","middleName":"","lastName":"Capotosti","suffix":""}],"badges":[],"createdAt":"2024-07-01 17:20:09","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":true,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-4669602/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4669602/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":60530926,"identity":"d974cef9-3f15-4965-80a0-766e197cb531","added_by":"auto","created_at":"2024-07-17 20:14:37","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":593398,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e. Study design and recruitment strategy. \u003cstrong\u003eb. \u003c/strong\u003eBaseline demographics of the MSA-P and MSA-C cohorts. See also “MSA Core Clinical Features and Case Descriptions” in the Supplemental Material. \u0026nbsp;All cases were evaluated with the MDS-UPDRS and MoCA prior to [\u003csup\u003e18\u003c/sup\u003eF]ACI-12589 PET. Additional clinical scales (UMSARS and BARS) are shown when available.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4669602/v1/b5f872c7c7dacb6698003aa4.jpeg"},{"id":60530655,"identity":"81073322-7cf7-4f44-a3ec-4d12863ca4d9","added_by":"auto","created_at":"2024-07-17 20:06:37","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":975144,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea.\u003c/strong\u003e Axial images of [18F]ACI-12589 at the level of the CWM and MCP across the MSA participants. \u003cstrong\u003eb.\u003c/strong\u003e Average axial image at the level of the CWM and MCP for each patient and control group. As noted in the initial study\u003csup\u003e15\u003c/sup\u003e, [18F]ACI-12589 \u0026nbsp;exhibits a strong variable background signal in the pons. \u003cstrong\u003ec.\u003c/strong\u003e SUVR values across all groups (HV = 5, MSA-C = 3, MSA-P = 5, PD = 5). A Kruskal-Wallis test was conducted to compare group-level effects (HV, PD, MSA-P, MSA-C) on SUVRs of the CWM (chi-squared = 10.33, p = 0.016) and MCP (chi-squared = 9.96, p = 0.019). The Dunn test was used for evaluating pairwise comparisons with p-values adjusted using the Bonferroni method across diagnoses (* p \u0026lt; 0.05). MSA-C: Multiple System Atrophy Cerebellar type, MSA-P: Multiple System Atrophy Parkinsonian type, CWM: Cerebellar white matter, MCP: middle cerebellar peduncles, HV: Healthy volunteers, PD: Parkinson’s disease, SUVR: standardized uptake value ratio.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4669602/v1/2ed3fb28c21d6aaf0a279cea.jpeg"},{"id":60530658,"identity":"4b9fbb21-4c27-46b0-931a-ee3fef2924c8","added_by":"auto","created_at":"2024-07-17 20:06:37","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":965962,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea.\u003c/strong\u003e Axial images of [18F]ACI-12589 at the level of the basal ganglia across the MSA participants. \u003cstrong\u003eb.\u003c/strong\u003e Average axial image at the level of the basal ganglia for each disease group \u003cstrong\u003ec.\u003c/strong\u003e SUVR values across all groups (HV = 5, MSA-C = 3, MSA-P = 5, PD = 5). A Kruskal-Wallis test was conducted to compare group-level effects on SUVRs of the pallidum (chi-squared = 1.52, p = 0.68) and putamen (chi-squared = 1.31, p = 0.73). There were no significant differences at the group level, error bars display the interquartile range. MSA-C: Multiple System Atrophy Cerebellar type, MSA-P: Multiple System Atrophy Parkinsonian type, CWM: Cerebellar white matter, MCP: middle cerebellar peduncles, HV: Healthy volunteers, PD: Parkinson’s disease, SUVR: standardized uptake value ratio.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4669602/v1/7a1e36a2d7fbd67ba36b92ab.jpeg"},{"id":60530656,"identity":"2876d64e-a3d1-4c3c-8e6f-58d290045038","added_by":"auto","created_at":"2024-07-17 20:06:37","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":981239,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea.\u003c/strong\u003e Schematic representation of the ɑSyn-SAA assay and the kinetic patterns of PD and MSA strains. A high maximal thioflavin T (ThT) fluorescence is suggestive of a PD strain. ɑSyn strains from subjects with MSA pathology demonstrate lower ThT fluorescence and a shorter lagging phase (lag time) \u003csup\u003e18,22,23\u003c/sup\u003e. \u003cstrong\u003eb.\u003c/strong\u003e Representative skin aSyn-SAA and [18F]ACI-12589 PET inset for all subjects, the brackets indicate the number of positive replicates on each sample (four replicates per sample). Patients had three samples each, a select site with the clearest SAA pattern is shown. Complete SAA results are displayed in figures S2 and S3. \u003cstrong\u003ec.\u003c/strong\u003e Diagnostic reclassification of clinically defined MSA cases in our study. MSA-C: Multiple System Atrophy Cerebellar type, MSA-P: Multiple System Atrophy Parkinsonian type, MCP: middle cerebellar peduncles, ILOCA: Idiopathic late-onset cerebellar ataxia\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4669602/v1/157f163449d43a34010fbfc2.jpeg"},{"id":60530657,"identity":"66f53447-02cf-4fa2-8ea2-da5be7850aba","added_by":"auto","created_at":"2024-07-17 20:06:37","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1011035,"visible":true,"origin":"","legend":"\u003cp\u003eDiagnostic reevaluation based on spatial and conformational synuclein biomarkers in patients fulfilling a clinical MSA diagnosis. \u003cstrong\u003ea. \u003c/strong\u003eCases where the combination of [18F]ACI-12589 and aSyn-SAA led to the consideration of an alternative diagnosis. \u003cstrong\u003eb. \u003c/strong\u003eCases with equivalent clinical severity, diagnostic certainty, and disease stage exhibiting a disparate [18F]ACI-12589 retention in the basal ganglia but a consistent MSA signal on aSyn-SAA. \u003cstrong\u003ec.\u003c/strong\u003e Indeterminate or weak aSyn-SAA (2/4 positive replicates) but the clearly increased MCP and striatal [18F]ACI-12589 retention reinforces the initial MSA diagnosis. MSA: Multiple System Atrophy, MSA-P: Multiple System Atrophy Parkinsonian type, nOH: neurogenic orthostatic hypotension\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4669602/v1/aa08308bd5b8c5ae3164179d.png"},{"id":62395680,"identity":"a37eca48-405d-4d94-8b6e-b2bbefdc38cc","added_by":"auto","created_at":"2024-08-13 17:03:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5273304,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4669602/v1/5fa0752c-d579-4141-b004-af2237fdee23.pdf"},{"id":60530659,"identity":"efdc6cde-9c25-4c72-8c6b-c34806b2ee44","added_by":"auto","created_at":"2024-07-17 20:06:37","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4542990,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-4669602/v1/13c640ff2f9f37eae0e72ce0.docx"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nAC Immune SA provided the radiotracer used in this study [18F]ACI-12589. AC Immune SA was not involved in the study design, data collection, analysis, interpretation of data, the writing of this article, or the decision to submit it for publication. The authors had full access to all the data in this study and take complete responsibility for the integrity of the data and the accuracy of the data analysis. E.V., J.M., I.K.D., V.H., M.K.-V., N.M., and F.C. are employees of AC Immune SA. C.S., A.G., J.A., A.K., and D.S.R. are employees of Invicro, LLC. D.R., B.K.C., A.K., O.L., K.J., D.C., J.M., S.P., S.R., and V.K. report no disclosures.","formattedTitle":"Combining alpha-synuclein PET and seeded-amplification to improve diagnostic accuracy of Multiple System Atrophy","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFor decades, most clinical trials have failed to identify beneficial disease-modifying effects of experimental compounds across neurodegenerative diseases. High among many potential explanations are the heterogeneous nature of these disorders, difficulties in establishing early diagnosis, and the lack of validated biomarkers that track disease progression and treatment outcome. Indeed, definitive diagnosis has traditionally relied on neuropathological examination of postmortem brain tissue\u003csup\u003e1,2\u003c/sup\u003e. However, the development of disease-specific biomarkers has recently led to the inclusion of these tests as part of the clinical diagnostic criteria and disease-modifying clinical trials\u003csup\u003e3,4\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRecently, regulatory agencies have accepted data generated with imaging and biofluid biomarkers to accelerate the approval of a monoclonal antibody for Alzheimer's disease (AD) and the antisense oligonucleotide, Tofersen, for amyotrophic lateral sclerosis (ALS)\u003csup\u003e5\u0026ndash;7\u003c/sup\u003e. These approvals were at least initially supported by significant changes in surrogate biomarkers, which were subsequently validated by the demonstration of clinical benefit. Specifically, beta-amyloid (Aβ) positivity measured as Aβ(\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5 CR6 CR7 CR8 CR9 CR10 CR11 CR12 CR13 CR14 CR15 CR16 CR17 CR18 CR19 CR20 CR21 CR22 CR23 CR24 CR25 CR26 CR27 CR28 CR29 CR30 CR31 CR32 CR33 CR34 CR35 CR36 CR37 CR38 CR39 CR40 CR41\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e) in cerebrospinal fluid (CSF) or by PET, was instrumental for the inclusion of AD subjects in the CLARITY-AD trial that ultimately led to the regulatory approval of Lecanemab\u003csup\u003e6\u003c/sup\u003e. The biomarkers better defined the target population of the clinical trials, translating into improved clinical outcomes. Similarly, Tofersen, an antisense oligonucleotide targeting the superoxide dismutase gene (SOD) 1, was approved after observing a decrease in CSF SOD1 and plasma neurofilament light chain (NfL) among ALS patients in the treatment arm\u003csup\u003e7\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eαSyn-rich neuronal and glial aggregates are the pathologic hallmark of synucleinopathies, such as Parkinson's disease (PD) and multiple system atrophy (MSA)\u003csup\u003e8\u003c/sup\u003e. The hallmark pathology in PD localizes in neuronal bodies\u003csup\u003e8\u003c/sup\u003e. Conversely, the identification of glial cytoplasmic inclusions (GCI) in oligodendrocytes defines the neuropathology of MSA\u003csup\u003e2,9,10\u003c/sup\u003e. Along with autonomic dysfunction, MSA is characterized by either predominant parkinsonism (MSA-P) or cerebellar ataxia (MSA-C). In MSA-P, asymmetric parkinsonism, mild autonomic dysfunction, and a transient response to levodopa can mimic an atypical presentation of PD. Despite having distinct pathology, MSA can be easily mistaken for PD with autonomic dysfunction early in the course of the disease. Indeed, even postmortem, recent series suggest that as many as 40 percent of early-stage MSA cases can be misdiagnosed in life\u003csup\u003e11\u0026ndash;14\u003c/sup\u003e. Conversely, PD with early autonomic dysfunction can also be mistaken for clinically probable MSA-P.\u003csup\u003e11\u003c/sup\u003e In an era of disease-modifying therapeutics for neurodegeneration, early and precise diagnosis has become imperative to triage patients to appropriate clinical trials. Because MSA is a rare orphan disease and PD a very prevalent one, misdiagnoses during clinical trial recruitment can be sufficient for MSA trials to fail in identifying modest disease-modifying effects.\u003c/p\u003e \u003cp\u003eThe field of αSyn pathology biomarkers has greatly advanced during the last few years, particularly with the development and clinical validation of the αSyn seed amplification assays (αSyn-SAA) and the first αSyn PET tracer, [18F]ACI-12589\u003csup\u003e15\u003c/sup\u003e. αSyn-SAA enables the capture and amplification of disease-specific αSyn conformers (or \"strains\") from peripheral samples such as CSF, skin, and blood\u003csup\u003e16\u0026ndash;20\u003c/sup\u003e. In this assay, synthetic monomeric αSyn is co-incubated with αSyn-containing patient sample. The self-templating nature of the αSyn conformer then enables the specific amyloid to propagate and amplify in the presence of the monomer\u003csup\u003e21,22\u003c/sup\u003e. This biomarker can aid in diagnosing early-stage disease, and ongoing efforts promise a quantifiable metric of disease progression\u003csup\u003e18,20\u003c/sup\u003e. Most pertinent to differential diagnosis, αSyn-SAA can discriminate between MSA and PD αSyn strains and thus powerfully distinguish between these diseases\u003csup\u003e17,22\u003c/sup\u003e. MSA strains exhibit a shorter duration of the lag phase and a lower maximal fluorescence compared to PD strains\u003csup\u003e16\u003c/sup\u003e. These differences are associated with unique protein digestion patterns and distinct structural characteristics observed using electron microscopy\u003csup\u003e16\u003c/sup\u003e. Beyond CSF, ɑSyn aggregates can also be amplified from other biological samples, including olfactory mucosa, skin, and blood\u003csup\u003e17,18,20,23\u003c/sup\u003e. Moreover, semi-quantitative immunofluorescent assays have also been used to identify the intraepidermal localization of phosphorylated ɑSyn deposits in the skin and to differentiate MSA from other synucleinopathies\u003csup\u003e24\u0026ndash;26\u003c/sup\u003e. The profound impact of SAA is manifest in new biological aSyn-based diagnostic and staging criteria proposed to facilitate precise diagnosis and clinical trial recruitment in PD\u003csup\u003e3,4\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWhile the αSyn seed amplification provides conformational information, the spatial distribution of αSyn deposition in the brain is missing. A PET radiotracer for imaging αSyn could fill that critical gap\u003csup\u003e15,27,28\u003c/sup\u003e. Moreover, PET is a noninvasive quantitative technique that has been extensively validated to measure disease progression and therapeutic responses for other diseases\u003csup\u003e15,27\u003c/sup\u003e. While not currently available for PD, the αSyn PET tracer [18F]ACI-12589 was recently shown to differentiate MSA from PD and other neurodegenerative disorders. Patients with MSA exhibited increased [18F]ACI-12589 retention in the cerebellar white matter (CWM) and middle cerebellar peduncles (MCPs). This was predictably more pronounced in the cerebellar (MSA-C) MSA compared to the Parkinsonian (MSA-P) subtype\u003csup\u003e15\u003c/sup\u003e. The MSA-P cohort also exhibited increased [18F]ACI-12589 retention in the lentiform nuclei of the striatum. However, it was not sufficiently elevated to significantly distinguish it from other synucleinopathies, such as PD and Dementia with Lewy Bodies (DLB)\u003csup\u003e15\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn the current observational cohort study, we sought to exploit exciting advances in αSyn biomarkers to improve upon current clinical consensus criteria for MSA. We recruited patients who met the consensus criteria for probable or established MSA and were eligible for the ongoing clinical trials for MSA within the Brigham and Women\u0026rsquo;s Hospital MSA Center of Excellence and Harvard Biomarkers Study MyTrial-MSA Program\u003csup\u003e29\u003c/sup\u003e. We combined brain imaging with the αSyn [18F]ACI-12589 PET tracer with a skin-based αSyn-SAA to simultaneously acquire conformational and spatial information about αSyn pathology. Despite the small sample size, our study suggests that the combination of both biomarkers may usefully contribute to diagnostic specificity. Indeed, we identified an MSA-specific αSyn signal in six out of eight participants, while an alternative diagnosis was considered for the remaining two cases, initially diagnosed as \"clinically probable MSA-P.\" Additionally, all equivocal results in one assay were complemented by more definitive data from the other. We felt compelled to disseminate these early findings in order to prompt the use of αSyn-targeting biomarkers in clinical-trial recruitment for MSA, especially MSA-p. Accurate diagnosis, particularly to exclude far more common PD patients with atypical presentations, may potentially make all the difference to the outcome of disease-modifying treatments for this rare and devastating disease.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eParticipants and clinical MSA diagnosis\u003c/h2\u003e \u003cp\u003eThe Brigham and Women\u0026rsquo;s Hospital (BWH) P\u0026thinsp;+\u0026thinsp;A\u0026thinsp;+\u0026thinsp;MSA clinic and MSA-MyTrial Program work in close coordination to recruit MSA patients for accurate diagnosis, longitudinal biometric and biomarker assessment, with a view to pivoting from natural history to target-engagement studies for a variety of potential investigator- or industry-sponsored therapies. Fourteen participants with a clinically probable or clinically established MSA diagnosis were evaluated for participation in this study\u003csup\u003e29\u003c/sup\u003e. Three subjects were deemed ineligible due to insufficient diagnostic certainty or an alternative diagnosis under consideration. Two subjects were hospitalized for acute disease shortly after screening, and one participant did not tolerate the PET scan due to significant drooling and concern for respiratory compromise. Seven subjects with probable MSA and one with clinically established MSA (MSA-C\u0026thinsp;=\u0026thinsp;3, MSA-P\u0026thinsp;=\u0026thinsp;5) completed this study between December 2022 and December 2023. All subjects underwent dynamic scan with [18F]ACI-12589 60\u0026ndash;90 min post-injection and PET data were analyzed as 60\u0026ndash;90 min standardized uptake value ratio (SUVR)\u003csup\u003e15\u003c/sup\u003e. Twenty-three 3-mm punch skin biopsies in total from these subjects were obtained and processed for ɑSyn-SAA. Participants were evaluated for disease severity and cognitive impairment prior to enrollment. No adverse events were reported during the study.\u003c/p\u003e \u003cp\u003eThe study design and baseline demographics are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. In addition, as a reference for [18F]ACI-12589 signal retention in MSA, we included [18F]ACI-12589 PET data from 10 previously enrolled subjects (5 healthy volunteers and five PD)\u003csup\u003e15\u003c/sup\u003e. The baseline demographics of these subjects (Supplementary Table\u0026nbsp;1) and a brief description of the core and supportive clinical features, as well as additional clinical information for each MSA participant, can be found in \u0026ldquo;MSA Core Clinical Features and Case Descriptions\u0026rdquo; in the Supplementary Material.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eIn vivo [18F]ACI-12589 PET signal retention in MSA\u003c/h2\u003e \u003cp\u003e[18F]ACI-12589 PET images were reconstructed and co-registered to T1-weighed MR. We first examined representative axial sections at the level of the cerebellar white matter (CWM) and middle cerebellar peduncles (MCP), since these regions significantly discriminated between MSA and PD or healthy volunteers in the initial characterization of [18F]ACI-12589\u003csup\u003e15\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). All MSA-C cases exhibited a significantly increased retention of the [18F]ACI-12589 radiotracer in the MCP compared to healthy volunteers (60\u0026ndash;90 min SUVRs, MSA: 1.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10, HV: 1.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09; Dunn test p\u0026thinsp;=\u0026thinsp;0.019, Supplementary Table\u0026nbsp;2). CWM and MCP retention were also increased in MSA-C compared to PD and MSA-P subjects, although no statistical significance was observed after adjusting for multiple comparisons (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, c). While, at the individual level, all MSA-C cases demonstrated significant [18F]ACI-12589 retention in the MCP, this finding was not consistently observed across MSA-P cases. MSA-P cases #2 and #5 demonstrated borderline increased signal in this region, not strong enough to distinguish them from alternative diagnoses. Additional images for all HV and PD participants and the SUVRs for key regions across groups are presented in Supplementary Fig.\u0026nbsp;1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe next considered the lentiform nucleus of the basal ganglia, a key site of ɑSyn accumulation in MSA, especially MSA-P (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea)\u003csup\u003e30\u003c/sup\u003e. MSA-P participants trended toward increased retention of [18F]ACI-12589 in the lentiform nuclei compared to healthy volunteers, MSA-C, and PD (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). However, the group comparison was not statistically significant and was primarily driven by MSA-P case #5, who exhibited a high SUVR in the globus pallidum and putamen (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Notably, despite exhibiting strikingly different [18F]ACI-12589 signals, MSA-P cases #4 and #5 demonstrated a similar severity of Parkinsonism and clinical impairment, as indicated by similar MDS-UPDRS, MDS-UPDRS part 3, and UMSARS scores (Supplementary Table\u0026nbsp;1). This finding suggests that, on an individual level, MSA-P patients with equivalent levels of Parkinsonism might exhibit highly disparate ɑSyn levels in the basal ganglia, at least as measured by [18F]ACI-12589, a point we return to below.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAltogether, these data confirm the capacity of [18F]ACI-12589 PET to fully discriminate MSA-C cases from healthy controls or PD cases, attributable to its strong spatial ɑSyn distribution in the MCP and CWM in MSA-C. However, they also indicate that signal retention in MSA-P is more variable, potentially because of a higher variability of the ɑSyn levels in the basal ganglia that has been described in postmortem studies\u003csup\u003e30\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eSkin ɑSyn-SAA in MSA\u003c/h2\u003e \u003cp\u003eSAA utilizes the prion-like properties of ɑSyn, whereby pathological seeds in a lysate (in this case, generated from skin) induce the aggregation of normal monomeric ɑSyn into templated fibrils, detectable via Thioflavin T (ThT) fluorescence. ɑSyn-SAA has demonstrated high diagnostic accuracy, sensitivity, and specificity for identifying ɑSyn in PD and MSA patients\u003csup\u003e16\u0026ndash;18\u003c/sup\u003e. Moreover, MSA and PD strains distinctly interact with fluorescent markers and have different patterns in ɑSyn-SAA that can be used in the differential diagnosis. \u003csup\u003e16\u003c/sup\u003e A lower fluorescence and earlier lag time are characteristic of MSA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). While this has been well demonstrated in brain tissue and CSF, ɑSyn-SAA from peripheral sites like the skin has also shown considerable promise and similar amplification properties to central sites among synucleinopathies\u003csup\u003e18,31\u003c/sup\u003e The advantage of skin is that it is far more deployable for clinical trial recruitment than CSF. Twenty-three skin biopsies were processed from our MyTrial subjects into skin lysates for ɑSyn-SAA (one subject consented to only two sites). Sixteen biopsies were positive for ɑSyn amplification, meaning that at least 75% (3/4) of the experimental replicates at that site demonstrated increased ThT fluorescence during the assay (see Methods for more details).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFive of the eight MSA participants had at least one positive biopsy on ɑSyn-SAA that exhibited a characteristic MSA pattern in at least 3/4 technical replicates (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb; Supplementary Table\u0026nbsp;3). Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb illustrates ɑSyn-SAA results for a representative skin biopsy from each subject and a corresponding [18F]ACI-12589 inset, both at the level of the cerebellum and basal ganglia. Supplementary Figs.\u0026nbsp;2 and 3 display the ɑSyn-SAA results for all skin biopsies in the MSA cohort. There were three notable outliers. For MSA-P case #1 all biopsies exhibited ɑSyn amplification but none was consistent with MSA. Instead, these three biopsies exhibited a pattern consistent with a PD ɑSyn strain (high fluorescence and late lag time). Second, for MSA-P case #3, all three biopsies were consistently negative, with less than 25% (1/4) of positive replicates despite retesting. Lastly, MSA-C case #1 had 50% (2/4) positive replicates on ɑSyn-SAA with an MSA pattern, consistent with an inconclusive SAA result. Thus, SAA was capable of confirming a synucleinopathy diagnosis in 6/8 of our cases.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eAdvantage of combining a-Syn biomarkers in the diagnosis of MSA\u003c/h2\u003e \u003cp\u003eIn isolation, [18F]ACI-12589 identified a spatial ɑSyn signal in key MSA areas in five out of eight participants and SAA confirmed synucleinopathy in 6/8 of our cases. It became clear that both tests together were superior to each test alone. [18F]ACI-12589 performed better for MSA-C, even when SAA was equivocal (MSA-C case #1), Conversely, SAA was particularly useful for MSA-P, where only two out of five MSA-P cases had visually increased [18F]ACI-12589 retention in the basal ganglia (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003eTwo notable scenarios arose in our patients that demonstrate the utility of both tests. First, there were patients without evidence of MSA-pattern ɑSyn pathology in either skin SAA or [18F]ACI-12589 PET (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea), suggesting an alternative diagnosis. For instance, MSA-P #1 is a patient with prominent neurogenic orthostatic hypotension and bilateral asymmetric Parkinsonism, initially diagnosed with clinically probable MSA-P. However, [18F]ACI-12589 PET failed to identify a spatial ɑSyn signal in key MSA regions, and ɑSyn-SAA demonstrated a pattern consistent with PD. In subsequent clinical evaluations, the patient\u0026rsquo;s relatively benign course thereafter was also inconsistent with an MSA diagnosis, and this patient was re-classified as having an atypical presentation of idiopathic PD with prominent early-onset autonomic dysfunction (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, top panel). Similarly, for MSA-P #3 both biomarkers failed to identify any conformational or spatial ɑSyn signature in MSA-P #3. This patient had a longstanding history of autonomic dysfunction that included orthostatic hypotension and urinary incontinence, as well as mild bilateral ataxia with Parkinsonism. He also progressed minimally over a year after the study, inconsistent with the natural history of MSA (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, bottom panel). This case was re-evaluated after the study was completed and we performed a commercial test for ɑSyn visualization in skin with immunofluorescence, which was also negative in all three sites. This case has been re-classified as a non-synucleinopathy case (\u0026ldquo;idiopathic late-onset cerebellar ataxia\u0026rdquo; [ILOCA] with parkinsonism), and additional diagnostic tests are ongoing\u003csup\u003e32,33\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe second scenario comprises probable MSA-P cases in which fairly equivalent motor dysfunction is associated with a highly divergent signal on [18F]ACI-12589 PET, but a very consistent signal on SAA. For example, MSA-P #4 and MSA-P #5 are two probable MSA-P with severe, symmetric bilateral parkinsonism that is poorly responsive to dopaminergic therapy. Both cases exhibit similar severity scores across clinical rating scales (MDS-UPDRS and UMSARS). However, while there was no increased [18F]ACI-12589 retention in the basal ganglia for MSA-P #4, MSA-P #5 exhibited a robust increase of tracer retention in the putamen and globus pallidus. Thus, using [18F]ACI-12589 PET in isolation, only MSA-P #5 would have an ɑSyn signature compatible with an MSA diagnosis. However, in skin ɑSyn-SAA, both cases demonstrated an MSA pattern of ɑSyn amplification across all three skin biopsies and patients were both diagnosed with probable MSA-P (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eFinally, although ɑSyn-SAA has shown to be a very sensitive test, it might not always reliably amplify the disease-specific ɑSyn strain. This was the case for MSA-C #1, a patient with clinically established disease but an inconclusive ɑSyn-SAA result. Despite an ɑSyn amplification pattern suggestive of MSA, only 50% of replicates (2/4) were positive in one out of three skin biopsies, even after re-testing. However, [18F]ACI-12589 imaging demonstrated significantly increased retention in the CWM and MCP (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). Moreover, there was also increased radiotracer retention in the basal ganglia compared to HV and PD cases. Taken together, these results demonstrate the utility of both tests as mutually reinforcing biomarkers for the differential diagnosis of MSA.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eClinical trials for neurodegenerative movement disorders rely on precise clinical diagnosis and rating scales as primary endpoints. Extraordinary developments in ɑSyn peripheral tests, notably SAA, have led to recent calls for new ɑSyn-based diagnosis and staging criteria for Parkinson\u0026rsquo;s disease.\u003csup\u003e3,4\u003c/sup\u003e. But for MSA there is already the possibility of using PET, just as in the AD field, to achieve spatial resolution diagnosis. Therefore, in this study, surmising that a surrogate for the gold-standard postmortem diagnosis of synucleinopathies could be achievable by combining spatial and conformational ɑSyn markers, we combined the brain PET ɑSyn ligand [18F]ACI-12589 with skin ɑSyn-SAA to a real-life observational cohort at our institution that seeks to deeply characterizes patients with a clinical diagnosis of MSA before pivoting them to an appropriate clinical trial\u003csup\u003e29\u003c/sup\u003e. Despite a small sample size, our study confirmed the well-known error rate in making an accurate MSA diagnosis, which can be up to 40%\u003csup\u003e11,14\u003c/sup\u003e. Importantly, the combination of these emerging ɑSyn markers appeared to be more accurate in this small study than either investigation alone. In particular, our early data suggest that the combination of these markers was able to move the needle on one of the most troublesome differential diagnoses in the movement disorders clinic, that of MSA-P versus PD.\u003c/p\u003e \u003cp\u003eIncreased [18F]ACI-12589 retention presumably correlates with the burden of ɑSyn aggregates in specific brain regions\u003csup\u003e15\u003c/sup\u003e. In the recent initial [18F]ACI-12589 study, increased ligand retention in the CWM and MCP differentiated patients with MSA-P and MSA-C from HV, PD, and other neurodegenerative disorders\u003csup\u003e15\u003c/sup\u003e. The higher retention of the [18F]ACI-12589 signal in the cerebellum of MSA-C cases was confirmed in this study (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Moreover, MSA-P cases exhibited increased [18F]ACI-12589 retention in the basal ganglia (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea)\u003csup\u003e15\u003c/sup\u003e. However, the tracer retention in the basal ganglia was more variable and present, to a lesser extent, in some HV and PD subjects (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Consistent with this, our study found variable [18F]ACI-12589 retention in probable MSA-P subjects. Indeed, only two out of five MSA-P cases exhibited a visually distinguishing signal in the basal ganglia that supported an MSA diagnosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Moreover, we observed that cases with similar clinical features and disease severity could display widely different levels of [18F]ACI-12589 retention (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). One possible explanation for this finding is that clinical parkinsonism can be driven by pre-synaptic pathology in the substantia nigra, but also by post-synaptic striatal pathology with ɑSyn aggregates and neuronal loss in the caudate and pallidum\u003csup\u003e34\u003c/sup\u003e. While pre-synaptic pathology is the dominant mechanism in PD, both mechanisms are relevant in MSA and could partially explain different synuclein distributions in patients with similar clinical phenotypes. This variability in striatal pathology has been observed also in postmortem MSA-P of equivalent disease duration\u003csup\u003e30\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, the combination of skin-based ɑSyn-SAA and brain imaging with ɑSyn [18F]ACI-12589 PET tracer was crucial for providing a biological \u0026ldquo;anchor\u0026rdquo; to the probable/established MSA diagnosis. Moreover, we identified two out of eight cases (25%) with no evidence of MSA-pattern ɑSyn pathology despite meeting the criteria for clinically probable MSA-P. We clinically re-evaluated these cases, and the diagnosis was reconsidered. Importantly, these cases would likely been included in current clinical trial designs for MSA, adding undesired noise to the collected data. Recently proposed biological classifications of PD rely on ɑSyn-based biomarkers and key prodromal features to better identify disease populations that reflect the underlying biology of the disease\u003csup\u003e3,4\u003c/sup\u003e. In time, ɑSyn biomarkers, including PET or peripheral seed amplification, could provide invaluable ɑSyn-specific data to MSA diagnostic criteria. While we favored the skin ɑSyn-SAA as the peripheral assay of choice here, promising recent studies have demonstrated promise in immunofluorescent analysis of peripheral nerves from the skin (ɑSyn-IF)\u003csup\u003e24\u0026ndash;26\u003c/sup\u003e. This test has shown that the location of ɑSyn skin aggregates can be used to differentiate between MSA and Lewy body disease (DLB and PD)\u003csup\u003e24\u003c/sup\u003e. Both assays provided complementary information about ɑSyn skin pathology, and a head-to-head comparison would be valuable.\u003c/p\u003e \u003cp\u003eThe major limitation of this study is the lack of a neuropathologically confirmed diagnosis. This is a recurrent theme in the field as novel biomarkers become available but cannot be evaluated in what is considered the gold standard diagnosis. Additional limitations include the small sample size and the need for widespread validation of these novel biomarkers, including in patients with early or even prodromal disease stages. Lastly, PET and ɑSyn-SAA must be processed in specialized tertiary care centers and are not readily available across different populations. Although ɑSyn-SAA has been extensively characterized in CSF and skin, both of these tests necessitate invasive procedures for sample collection, which are resource-intensive and require significant logistical efforts to integrate into a neurology clinic\u003csup\u003e18,23,35\u003c/sup\u003e. The collection of CSF involves a lumbar puncture, which can cause discomfort and carries risks such as headaches and infection. Similarly, skin biopsies, while less invasive, still require careful planning and can lead to minor complications such as bleeding or infection. The olfactory mucosa has been explored as a more accessible site for sample collection; however, this procedure must be performed by an otolaryngology specialist, adding another layer of complexity and limiting its widespread application\u003csup\u003e23,36\u003c/sup\u003e. Ongoing efforts to develop blood-based ɑSyn-SAA present a more accessible alternative that could be applied to larger populations. However, these assays require either immunoprecipitation or the isolation of exosome-derived α-synuclein.\u003csup\u003e17,20\u003c/sup\u003e This approach holds promise for broader clinical use, but further optimization and validation are needed to ensure its efficacy and reliability in diverse clinical settings.\u003c/p\u003e \u003cp\u003eBeyond aiding in accurate diagnosis, emerging ɑSyn biomarkers hold the potential for measuring disease progression and target engagement. This has been extensively validated with other brain PET tracers for targets in neurodegeneration, particularly for Aβ. Therefore, ɑSyn PET might be a key biomarker for monitoring disease progression and evaluating the engagement of ɑSyn therapies such as monoclonal antibodies and anti-sense oligonucleotides\u003csup\u003e37\u003c/sup\u003e. Multiple ɑSyn radiotracers are currently being tested in clinical and preclinical settings\u003csup\u003e15,28,38,39\u003c/sup\u003e, and our data supports the promise, among these, of [18F]ACI-12589. Recently, extensive efforts have also sought to make ɑSyn-SAA more quantitative. Certain kinetic parameters in ɑSyn-SAA may correlate with disease stage, REM sleep behavior disorder (RBD) status, and cognitive function\u003csup\u003e18,40,41\u003c/sup\u003e. However, further validation and standardization of ɑSyn-SAA protocols are necessary to explore these potential associations on a larger scale. Ongoing efforts to create a digital, quantitative ɑSyn-SAA may result in a more nuanced assay to help track disease progression and target engagement\u003csup\u003e37\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn conclusion, our study suggests that both ɑSyn PET imaging with [18F]ACI-12589 and ɑSyn-SAA provide complementary information in the differential diagnosis of MSA, particularly of MSA-P. Combining these spatial and conformational ɑSyn biomarkers might help achieve a precise and early MSA diagnosis anchored to a disease-specific ɑSyn signature. This approach can help biologically define the homogeneous populations suitable for clinical trial enrollment and improve the chances of detecting the efficacy of disease-modifying therapies.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eEthics approval\u003c/h2\u003e \u003cp\u003e This observational study complies with pertinent ethical regulations, including STROBE guidelines and the principles of the Declaration of Helsinki. The initial clinical evaluation and PET imaging were performed at Invicro, LLC, under protocol number 9105, approved by the Advarra Institutional Review Board (IRB). Skin biopsies, skin SAA, and follow-up evaluations occurred at Brigham and Women\u0026rsquo;s Hospital under protocol number 2009P000775 (Mass General Brigham IRB).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eParticipant recruitment\u003c/h2\u003e \u003cp\u003e We included participants with a clinical diagnosis of probable or established MSA based on the most recent MDS criteria. Participants were recruited from the MSA Center of Excellence and Parkinsonism, Ataxia, and MSA (P\u0026thinsp;+\u0026thinsp;A\u0026thinsp;+\u0026thinsp;MSA) clinic at Brigham and Women\u0026rsquo;s Hospital. All MSA patients were included in our previously described MyTrial program for clinical-trial-ready cohorts in neurodegenerative disease\u003csup\u003e29\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWe also recruited age-matched healthy volunteers and participants with a diagnosis of PD as determined by a movement disorders specialist following the UK Brain Bank diagnostic criteria. These subjects were recruited at Invicro and completed the [18F]ACI-12589 component of this study. The stage of the disease was evaluated using the Hoehn and Yahr (H\u0026amp;Y) scale. Disease severity was evaluated using the Unified Multiple System Atrophy Rating Scale (UMSARS) and the MDS-revised Unified Parkinson\u0026rsquo;s Disease Rating Scale (MDS-UPDRS). Patients were screened for mild cognitive impairment using the MoCA and for RBD using a single-question screening.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e[18F]ACI-12589 PET Imaging and Reconstruction\u003c/h2\u003e \u003cp\u003eEach subject underwent a 90-minute [18F]ACI-12589 PET scan on a Siemens ECAT EXACT HR\u0026thinsp;+\u0026thinsp;camera, centering the brain in the field of view. [18F]ACI-12589 was injected over 3 minutes through a venous catheter, followed by a 10 mL saline flush. Preceding radiotracer injection, a transmission scan was performed, which was needed for attenuation correction of the PET emission data. [18F]ACI-12589 PET images were reconstructed with all corrections applied (random, scatter, dead time, and attenuation). The dynamic PET data were reconstructed with the following frame timing: 6x30 sec, 4x1 min, 4x2 min, and 15x5 min.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eImage Processing and Quantification\u003c/h2\u003e \u003cp\u003eAt screening, a T1-weighed MR image was collected from each subject, which was required for PET analysis. MR and PET image processing were performed in PMOD v. 3.802 (PMOD Technologies, Zurich, Switzerland). PET frames were motion corrected, rigidly aligned with the subject\u0026rsquo;s MRI, and normalized into the standard Montreal Neurological Institute (MNI) space where volumes of interest (VOIs) were defined. Brain atlas VOIs examined included amygdala, caudate, cerebellar white matter, midbrain, pallidum, pons, and putamen. A PET image-derived VOI, cerebellar white matter tracts, was delineated using an average of three subjects with high uptake within the cerebellar white matter (see Supplementary Fig.\u0026nbsp;4). Time-activity curves (TACs) were generated for each VOI, with concentration (kBq/cc) on the y-axis and time (min) on the x-axis. For visual comparison, TACs and PET images were converted from concentration to standard uptake value (SUV) units (g/mL) by normalizing with the weight of the subject and the injected dose.\u003c/p\u003e \u003cp\u003eThe SUV ratio (SUVR) was computed between 60\u0026ndash;90 min to assess regional differences between MSA and healthy control subjects, using the cerebellar cortex as the reference\u003csup\u003e15\u003c/sup\u003e. The distribution volume ratio (DVR) was estimated with non-invasive Logan graphical analysis (t*=10 min), using the cerebellum as the reference region. DVR and SUVR (60\u0026ndash;90 min) were highly correlated (R2\u0026thinsp;=\u0026thinsp;0.94), with a slight positive bias for SUVR, where SUVR\u0026thinsp;=\u0026thinsp;1.13*DVR \u0026minus;\u0026thinsp;0.15.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eSkin biopsy procedure\u003c/h2\u003e \u003cp\u003eFour skin punch biopsies, each with a diameter of 3 mm, were obtained from the posterior cervical region (C7), lateral lower thigh, and two from the lower leg (ankle). One of the ankle biopsies was used to generate human fibroblast cultures for differentiation into induced pluripotent stem cell (iPSC) models matched to biospecimens as described in our MyTrial program for clinical-trial-ready cohorts in neurodegenerative disease\u003csup\u003e29\u003c/sup\u003e. Local anesthesia with 1% lidocaine was administered prior to the procedure. Post-biopsy wounds were dressed in non-adhesive bandages, and participants were monitored for adverse events via a follow-up call within a week. Subsequently, the biopsy specimens were flash-frozen in liquid nitrogen, coded blindly, and stored at \u0026minus;\u0026thinsp;80\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eSkin SAA\u003c/h2\u003e \u003cp\u003eEach biopsy was processed into a skin lysate using a similar assay procedure to the ones previously described. Briefly, two 7-mm steel beads were used to homogenize skin tissue using the TissueLyser LT device (Qiagen, Germany) to prepare 10% skin lysates. The lysates were further diluted 20-fold, and 2 \u0026micro;l were added to a reaction buffer containing 1 mg/mL C-terminus 6xHis-tagged recombinant wild-type α-syn, 500 \u0026micro;M NaCl, 100 mM PIPES, 10 \u0026micro;M thioflavin T (ThT), and 6+-2 silica glass beads (OPS Diagnostics)\u003csup\u003e16,18,23\u003c/sup\u003e. Recombinant wild-type α-syn with 6xhistidine-tag at C-terminal was produced in the Rudolf\u0026ndash;Virchow Center, University of W\u0026uuml;rzburg, Germany, recombinant protein expression facility\u003csup\u003e18\u003c/sup\u003e. Each skin biopsy was tested in quadruplicate by adding 100 \u0026micro;l of the reaction buffer to a 96-well plate for incubation at 37\u0026deg;C with cycles of 1 min double orbital shaking at 400 rpm and 5 min rest. The assay spanned 48 hours, during which fluorescence readings were recorded at 448 nm/482 nm wavelengths every 45 minutes using a FLUOstar Omega microplate reader (BMG Labtech, Germany). Maximal fluorescence is expressed as a percent of the maximum relative fluorescent units (RFU) in our assay. Samples were deemed positive if at least 75% of the replicates demonstrated increased fluorescence from baseline.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eSkin αSyn-SAA analysis\u003c/h2\u003e \u003cp\u003eAfter determining whether each replicate was positive for α-syn aggregation, the following parameters were calculated: maximal ThT fluorescence, time (h) for a reaction to reach 50% of the maximum fluorescence (T50), duration of lag phase (the reaction time [h] required to cross the fluorescence threshold), and area under the ThT fluorescence curve (AUC). Mean and standard deviation across positive replicates were computed for each variable per biopsy. Positive samples with a mean maxRFU under 20% of the maximal fluorescence were considered to have an MSA αSyn aggregation pattern. Those over 60% of the maximal fluorescence were deemed as a PD aggregation pattern. The αSyn-SAA test was considered positive if at least one of the biopsy samples yielded a positive result with an MSA or PD pattern.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were conducted using the R software (R version 4.3.2, RStudio 2023.12.0.369; Boston, MA, USA). For intergroup comparisons of non-normally distributed [18F]ACI-12589 data we used the Kruskal-Wallis test with a significance level of 0.05. When significant, we adjusted for multiple comparisons using the Dunn test with the Bonferroni method. Data in supplementary tables is represented by means and standard deviations. Figures were created in ggplot2 version 3.4.4 and Affinity Designer 2 (Serif, 2022).\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAC Immune SA provided the radiotracer used in this study [18F]ACI-12589. AC Immune SA was not involved in the study design, data collection, analysis, interpretation of data, the writing of this article, or the decision to submit it for publication. The authors had full access to all the data in this study and take complete responsibility for the integrity of the data and the accuracy of the data analysis. E.V., J.M., I.K.D., V.H., M.K.-V., N.M., and F.C. are employees of AC Immune SA. C.S., A.G., J.A., A.K., and D.S.R. are employees of Invicro, LLC. V.K. is a co-founder of and senior advisor to DaCapo Brainscience, a company focused on CNS diseases. D.R., B.K.C., A.K., O.L., K.J., D.C., J.M., S.P., and S.R. report no disclosures.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePostuma RB et al (2015) MDS clinical diagnostic criteria for Parkinson\u0026rsquo;s disease. Mov Disord Off J Mov Disord Soc 30:1591\u0026ndash;1601\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWenning GK et al (2022) The Movement Disorder Society Criteria for the Diagnosis of Multiple System Atrophy. Mov Disord 37:1131\u0026ndash;1148\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSimuni T et al (2024) A biological definition of neuronal α-synuclein disease: towards an integrated staging system for research. Lancet Neurol 23:178\u0026ndash;190\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eH\u0026ouml;glinger GU et al (2024) A biological classification of Parkinson\u0026rsquo;s disease: the SynNeurGe research diagnostic criteria. 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Acta Neuropathol Commun 11:162\u003c/span\u003e\u003c/li\u003e \u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4669602/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4669602/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBiomarkers that facilitate early detection and track disease progression are an enormous unmet need in neurodegenerative diseases and their clinical trials. Accurate diagnosis in the early stage of Parkinsonian disorders is particularly challenging. Multiple system atrophy (MSA) and Parkinson\u0026rsquo;s disease (PD) share many clinical features and are associated with alpha-synuclein (αSyn) aggregation. However, these diseases have distinct biology and disease trajectories and are likely to respond differently to experimental therapies. Gold-standard diagnosis is only achieved at postmortem examination. Here, we combined two emerging technologies: brain imaging with αSyn [18F]ACI-12589 PET tracer with a skin αSyn seed-amplification assay (αSyn-SAA). These assays have the potential to increase diagnostic precision \u003cem\u003ein vivo\u003c/em\u003e by delineating the spatial distribution and conformation of αSyn pathology, respectively. Of 8 clinically probable or established MSA patients, combining brain imaging with αSyn [18F]ACI-12589 PET tracer and skin αSyn-SAA helped confirm the diagnosis in 6 of the 8 patients and led to the reclassification of two cases to Parkinson\u0026rsquo;s disease and idiopathic late-onset cerebellar ataxia. Each test provided critical evidence of diagnosis even when the other was equivocal, supporting the combination of these tests. These αSyn biomarkers should now be used systematically to facilitate early and precise diagnosis across synucleinopathies.\u003c/p\u003e","manuscriptTitle":"Combining alpha-synuclein PET and seeded-amplification to improve diagnostic accuracy of Multiple System Atrophy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-17 20:06:32","doi":"10.21203/rs.3.rs-4669602/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3293f891-f3e4-4356-a140-dbc00afb0b37","owner":[],"postedDate":"July 17th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":34635495,"name":"Health sciences/Neurology/Neurological disorders/Movement disorders"},{"id":34635496,"name":"Health sciences/Biomarkers/Diagnostic markers"},{"id":34635497,"name":"Health sciences/Diseases/Neurological disorders/Movement disorders"}],"tags":[],"updatedAt":"2024-08-13T16:55:28+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-17 20:06:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4669602","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4669602","identity":"rs-4669602","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}