Role of alpha-synuclein seed amplification assay in Parkinson’s disease clinical trials: A case of misdiagnosis

Highlights

• •Early differentiation between PD and atypical parkinsonism is challenging.
• •Variability in diagnosis can weaken statistical power in trials.
• •Precise diagnostic tools are needed for trial population homogeneity.
• •This patient initially diagnosed with PD, was diagnosed with MSA via CSF αSyn-SAA.
• •Adding αSyn-SAA to inclusion criteria could enhance subject selection in trials.

Despite robust preclinical evidence, numerous clinical trials have consistently failed to discover a treatment capable of slowing Parkinson's disease (PD) progression. In recent years, only 14 % of PD clinical trials have progressed to Phase 3. This ongoing challenge highlights fundamental concerns related to study design for disease-modifying therapies, including target populations, trial duration, and outcome measures [1].

The clinical heterogeneity of PD makes diagnosis challenging during recruitment for clinical trials, especially in early-stage cases where dopaminergic treatment response and atypical features are unclear [2]. Diagnostic accuracy for PD varies widely, achieving 73.8 % accuracy in non-experts and up to 79.6 % in movement disorder specialists [3]. Although standardized clinical criteria, such as the Movement Disorder Society (MDS) Parkinson's Disease Diagnostic Criteria and the UK Parkinson's Disease Society Brain Bank criteria, have enhanced diagnostic accuracy in PD research, distinguishing between typical and atypical parkinsonism—such as Dementia with Lewy Bodies, Multiple System Atrophy (MSA), and Progressive Supranuclear Palsy—remains difficult. This challenge is primarily due to the need to observe symptom progression over time to confirm a definitive PD diagnosis [4]. Recently, the cerebrospinal fluid (CSF) alpha-synuclein seed amplification assay (αSyn-SAA), using the Protein Misfolding Cyclic Amplification (PMCA) method, has shown notable potential in differentiating PD from other alpha-synucleinopathies, such as MSA [5]. Thus, implementing this assay could help reduce misdiagnosis, thereby improving trial participant consistency and statistical power.

We present the case of a 63-year-old Hispanic male initially diagnosed with PD, who participated in a Phase 2b clinical trial evaluating human allogenic bone marrow-derived mesenchymal stem cell therapy as a potential disease-modifying therapy for PD (NCT04506073). The patient's medical history was notable for coronary artery disease and partial erectile dysfunction. The patient was exposed to paraquat while working in the Coast Guard and reported no family history of neurodegenerative diseases. Symptom onset occurred at age 56 with Rapid Eye Movement Sleep Behavior Disorder (RBD), subsequently progressing to the development of rigidity and bradykinesia, primarily affecting his right side. These symptoms led to a PD diagnosis at age 59. Two years later, he underwent evaluation for inclusion in the aforementioned PD trial. During the screening visit, the patient fulfilled the MDS Parkinson's Disease Diagnostic Criteria and the UK Parkinson's Disease Society Brain Bank criteria assessed by a movement disorder specialist, did not exhibit atypical parkinsonism red flags (Fig. 1A), had unremarkable neuroimaging (Fig. 1B-D), and demonstrated a 52 % improvement with levodopa treatment in the MDS-Unified Parkinson's Disease Rating Scale (MDS-UPDRS) motor subscale. The screening assessment revealed orthostatic hypotension without compensatory tachycardia (Fig. 1A) and absence of a “cold hands sign.” Additionally, the patient showed normal cognitive function with a Montreal Cognitive Assessment (MoCA) of 28, normal olfaction with The University of Pennsylvania Smell Identification Test (UPSIT) of 36, hypomimia, mild dysarthria, sialorrhea, mild bradykinesia, bilateral rigidity (more pronounced on the right), postural instability, reduced arm swing on the right, normal tandem gait, and normal ocular movements. Several clinical scales assessing motor and non-motor symptoms were performed and are summarized in Fig. 1A. It is noteworthy that the MDS-UPDRS was assessed in the levodopa OFF state, defined by withholding dopaminergic medications 12 h prior to the evaluation.

Fig. 1.

Clinical and neuroimaging characteristics in a subject initially misdiagnosed with Parkinson’s Disease whose diagnosis was subsequently corrected as Multiple System Atrophy during a Phase 2b clinical trial using alpha-synuclein seed amplification (αSyn-SAA). A. Longitudinal changes in motor and non-motor assessments. B-D. Baseline magnetic resonance imaging (MRI) without contrast sequences in T2 with axial sections of the cerebellum, cerebellar nuclei, middle cerebellar peduncles (B), midbrain (C), and basal ganglia (D), showing age-expected mild atrophy with no other relevant changes. E. α-Syn-SAA from the patient’s CSF sample using the protein misfolding cyclic amplification (PMCA) method showing a typical MSA aggregation pattern5: faster aggregation but lower maximum fluorescence compared to a standard PD control. A.U.: Arbitrary Units. DBP: Diastolic Blood Pressure. HR: Heart Rate. MDS-UPDRS: The Movement Disorder Society Unified Parkinson's Disease Rating Scale. MSA: Multiple System Atrophy. NA: Not Available. NMSQ: Non-Motor Symptoms Questionnaire. T₅₀: Time (hours) to reach 50% of maximum aggregation, and F_max: The maximum ThT fluorescence at the plateau. SBP: Systolic Blood Pressure. PD: Parkinson’s Disease. Error bars indicate the standard error of the mean (SEM).

During the first year of the clinical trial, the participant's reported motor and non-motor symptoms remained largely stable, except for worsening orthostatic hypotension (Fig. 1A). By the second year of the trial (follow-up period), the patient’s autonomic dysfunction had significantly worsened and now included complete impotence, severe urinary frequency, and neurogenic orthostatic hypotension that resulted in multiple hospitalizations for syncopal episodes. This worsening prompted a clinical work-up for MSA, including a tilt-table test consistent with clinically probable MSA. Despite these findings and because of his clinical response to levodopa and his initial presentation, his providers remained unsure of the diagnosis. As part of the study's secondary outcomes, CSF αSyn-SAA was conducted at baseline and 49 weeks later, revealing a typical MSA aggregation pattern (Fig. 1E): a faster aggregation, evidenced by a time to reach 50 % of maximum aggregation (T₅₀) of 115.3 ± 5.367 h at baseline and 156.3 ± 2.371 h at 49 weeks, and a lower maximum fluorescence, confirmed by a maximum ThT fluorescence at the plateau (F_max) of 554.5 ± 76.57 Arbitrary Units (A.U.) at baseline and 1171.669 ± 15.34 A.U. at 49 weeks. The findings are in contrast with a standard PD control where T₅₀ was at 226.8 ± 1.846 h at baseline and 224.4 ± 1.901 h at 49 weeks, as well as a higher F_max of 4097.124 A.U. ± 27.70 at baseline and 5006.091 ± 270.5 A.U. at 49 weeks.

Consequently, the revised diagnosis led to the exclusion from the final statistical analysis, which required the incorporation of strategies for addressing missing data, thereby constraining the comprehensive analysis within what was already a limited sample size characteristic of PD Phase 2 trials.

The failure to discover effective therapies that slow the progression of PD in past research may be linked to disease heterogeneity and overlapping symptoms within alpha-synucleinopathies [2]. This case underscores the essential role of precise diagnosis in achieving homogeneous participant enrollment in clinical trials, especially with early-stage populations and small sample sizes. Although the patient initially met all inclusion criteria for a PD trial, subsequent assessments were consistent with a diagnosis of probable MSA. The limited accuracy of clinical PD diagnosis [3], the low sensitivity of diagnosing MSA in its early stages, and the fact that about half of patients with MSA-Parkinsonian type have a beneficial response to levodopa [4] can result in frequent misdiagnoses among alpha-synucleinopathies [5]. Because of the ambiguity surrounding diagnosis in patients with early disease, recent research has focused on distinguishing these pathologies based on objective biomarkers. CSF αSyn-SAA using PMCA has emerged as a reliable way to distinguish between MSA and PD, with our lab’s technique showing a sensitivity of 95.4 %. Although CSF αSyn-SAA has proven to be a vital tool for distinguishing between PD and MSA when the diagnosis is unclear, it has not been adopted as an inclusion criterion to increase diagnostic certainty in PD trials.

The incorporation of αSyn-SAA into eligibility criteria could significantly enhance the accurate identification of the intended target population, thereby increasing the probability of detecting therapeutic effects. A notable limitation of using this assay in PD diagnosis is the need for a lumbar puncture, which may discourage potential participants or disqualify those with conditions like lumbar arthrosis. Nevertheless, based on these findings, we advocate for integrating αSyn-SAA as a standard inclusion criterion in PD clinical trials.

Ethical compliance statement

This study was approved by The University of Texas Health Science Center Institutional Review Board (IRB) with identification number HSC-MS-20-0150. The participant provided written informed consent before undergoing any trial-specific screening tests or evaluations.

Funding

This work was supported by the 2019 Clinical Therapeutic Pipeline Program Award by the Michael J. Fox Foundation for Parkinson’s Research (Grant ID: MJFF-009634 and MJFF-023299), the Adriana Blood Distinguished Chair in Neurology Endowment, and philanthropic contributions from individuals affected by Parkinson's Disease and their families.

CRediT authorship contribution statement

Emily Tharp: Writing – original draft, Methodology, Investigation, Conceptualization. Juan D. Martinez-Lemus: Writing – original draft, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Mya C. Schiess: Writing – review & editing, Visualization, Supervision, Resources, Project administration, Conceptualization. Timothy M. Ellmore: Writing – review & editing, Visualization, Supervision, Methodology. Jessika Suescun: Writing – original draft, Methodology, Investigation, Formal analysis, Data curation. Mohammad Shahnawaz: Writing – review & editing, Resources, Investigation, Formal analysis, Conceptualization.

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Mya C. Schiess and Mohammad Shahnawaz have received financial support from grants provided by the Michael J. Fox Foundation (MJFF) for Parkinson’s Research. Mohammad Shahnawaz is the inventor of the patent for PMCA under #US10989718B2, licensed to the University of Texas Health Science Center at Houston and Amprion Inc. The other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.