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A cross-sectional study of PPMI participants using alpha-synuclein seed amplification as a biomarker

June 12, 2023
In this episode Dr. Tiago Outeiro talks with Dr. Andrew Siderowf about the exciting new results in his most recent publication on alpha-synuclein seed amplification assay. Read the article.

[00:00:00] Dr. Tiago Outeiro: Hello and welcome to the MDS Podcast, the podcast channel of the International Parkinson and Movement Disorder Society. I'm Tiago Outeiro, professor at the University Medical Center, Göttingen in Germany, and today I have the pleasure of interviewing Professor Andrew Siderowf from the Department of Neurology, University of Pennsylvania in Philadelphia.

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Andrew was the lead author of a recent manuscript in Lancet Neurology, entitled Assessment of Heterogeneity among participants in the Parkinson's Progression Markers Initiative cohort using alpha synuclein seed amplification: a cross-sectional study. The study has attracted a lot of attention and we want to dissect it here in order to understand the findings and implications.

So welcome, Andrew. It's a pleasure to host you on the podcast. I have a first question for you, and that's to ask you to please tell us briefly what the study is all about.

[00:00:56] Dr. Andrew Siderowf: Sure Tiago, thank you. The study looked at [00:01:00] samples from the Parkinson's Progression Marker Initiative, which is a longitudinal observational biomarker study involving a large number of participants. And in this study we included over 1100 participants including healthy controls, people with Parkinson's disease, including those with genetic variants related to Parkinson's disease. We also included people with so-called prodromal features, meaning those that had hyposmia and also those with REM sleep behavior disorder. And there were also a small number of these so-called scans without evidence of dopamine degeneration or sweat subjects. So a broad range of different categories, a total of 1100 subjects.

It was a cross-sectional study, and what we did is looked at synuclein seed amplification, which is a new biomarker that measures, we think, synuclein pathology in the brain of people with Parkinson's disease in a cross section of these subjects who were in PPMI. And so that's the study set up and then the main findings [00:02:00] were that overall the SAA, which is the seed amplification assay, was highly accurate. Over 95% specific and roughly 90% sensitive. So, high overall accuracy, but within, there's also a good deal of heterogeneity hence the title of the paper.

And in certain subgroups, the sensitivity, the positive rate of the SAA was quite high. And other subgroups, especially those that carried certain genetic variants or had certain clinical features, we found a much reduced rate of SAA positivity. And we think this indicates underlying pathological heterogeneity within patients with Parkinson's.

[00:02:40] Dr. Tiago Outeiro: Great. I want to get into more detail about the specifics of the study. But from the perspective of a more conservative specialist. Can you explain why we need biomarkers to identify at risk groups? And the perspective here could be, okay, we don't have a therapy at the moment. So why do we need to identify these people [00:03:00] if then we cannot really offer anything at the present moment? So how do you see this and how do you justify the need of biomarkers at this time point.

[00:03:09] Dr. Andrew Siderowf: Yeah, so to a certain extent, using biomarkers and at-risk individuals is forward-looking, looking forward to a time when there's the opportunity to identify patients at the earliest stages of degeneration and institute therapies to block progression, first off prevention, progression, and even possibly prevent the onset of Parkinson's disease.

So it's forward-looking and in a research context. In terms of therapeutics, there will be patients or individuals who are at risk, who want this information because they want to plan. They want to know if they're at risks so they can make decisions about their lives, or they just have a strong desire to know, not information for its own sake.

And that's probably mostly the current status now. They're probably some cases where this would be the case for prodromal at risk individuals for [00:04:00] patients with symptoms. I think that this will improve diagnostic accuracy and potentially could guide therapeutic decisions. But sort of along the same lines as the way we use DaT scan to guide therapeutic decisions now. I think that a lot of times you can make good therapeutic decisions for Parkinson patients now without biomarkers, but there are also certainly situations where they help.

But swinging back around to your original question, I think this is, and right now, predominantly a research tool that will allow us to understand the pathology of Parkinson patients. The information you could get potentially from postmortem, you could get to a certain extent in living patients and you can follow it serial over time.

And then there's also, obviously, you also said the information you can get in terms of risk for people who either have prodromal features or carry genetic variants and put them at a decreased risk.

[00:04:50] Dr. Tiago Outeiro: Yep. And so the readouts were done in CSF in this study. Does the assay work also in other, more easily accessible bio fluids? [00:05:00] And is this where you see the field going in the future?

[00:05:03] Dr. Andrew Siderowf: Yeah, so as you said, our study was entirely in spinal fluid and right now spinal fluid, I think, is the source that people consider to be the most standard source for doing SAA, now. As you mentioned, also, there's limitations to spinal fluid in terms of it's difficult to access. It's not really acceptable to patients, not very scalable. And so there's efforts afoot to translate SAA into more readily accessible tissue. And these tissues include skin biopsies, olfactory mucosa, and potentially even blood samples. And there have been preliminary studies looking at skin especially, which look promising.

Although I think that there need to be reproduced and standardized to a certain extent cause mostly they're in smaller and single center studies. But I think there's a good foundation for skin right now. I think it's definitely a place for you to see more work trying to refine the techniques to get [00:06:00] better sensitivity, especially.

And then olfactory mucosa has been identified as a potential source in their preliminary studies. Smaller pilot studies that show promise. But again, I think getting the sensitivity up and really finding a way to get the tissue that contains the SACs, which is probably olfactory mucosa rather than just nasal mucosa. Remains a challenge. It's rather relatively invasive to get olfactory mucosa. And then blood would be ideal. And I think that there's efforts looking at neurally derived exosomes and I think there's other techniques which are even simpler that are being explored. And I think this is something that we're gonna see a lot more of in the next six months, twelve months.

I think this is with proof of concept for spinal fluid, but scalability is gonna depend on other matrices, especially blood.

[00:06:47] Dr. Tiago Outeiro: Great. So now let's get more into the specifics. So can the SAA that you guys perform distinguish between different synucleinopathies? Meaning does it give a positive signal, for [00:07:00] example, in incidental Lewy body disease? I don't know if you had these types of samples, but what do you think would happen in those cases?

[00:07:07] Dr. Andrew Siderowf: That's a good question. We did look at this specifically. And, incidental Lewy body cases would be included among our controls. And we do know that the specificity for healthy controls was about 96%. So 4% of cases were positive. And we also know that the specificity among SWEDDs and these are people that have mild parkinsonian signs, generally, sort of borderline parkinsonism and DaT scans, which are normal or at least read as the normal, although the quantification can be borderline. Among the SWEDDs, the specificity was 90%, so about 10% positive among the SWEDDs and about 5% positive, more or less among the healthy controls.

And so what's going on with these healthy controls, it's a little bit of an open question. The thing that's interesting about them is that if you look at the healthy controls [00:08:00] carefully in our study, you can see that I think about 5% of the normosmic healthy controls were positive, but about 30% of the hyposmic healthy controls were positive, so a certain fraction of the healthy controls also had an impaired sense of smell, which was strongly related to being SAA positive among the Parkinson patients. And these hyposmics were dramatically overrepresented among the healthy controls that were positive. And so I think the take home from this is there's probably some just random lab error among you know, the accounts for the less than a hundred percent specificity, but probably it's only part of it. And probably there are some true positives in the healthy controls and they may represent incidental Lewy body cases. And one thing that's kind of interesting is that if you sort of do the math, the number of these hyposmic healthy controls who are assay positive, if you sort of like multiply it out to represent the population, it's substantially higher than the frequency of Parkinson's disease in the population.

And it may indicate that SAA positivity is substantially more common than Parkinson's [00:09:00] diseases. And there's a number of people in the general population who have, biomarker evidence of a synucleinopathy that never go on to develop Parkinson's. And this I think is, it's not really the main thrust of the data.

I think it's an area which probably merits follow up in population studies.

[00:09:13] Dr. Tiago Outeiro: Yeah. And this connection between SAA positivity and hyposmia is very interesting. But here I was thinking about MSA, where Hyposmia is considered as an exclusion criteria, but then in SAA assays, it seems to be positive. So, I mean, clearly we need to study MSA in more detail and also at the structural level of the seeds.

But how do you see the seeding amplification assay help here in more difficult situation as MSA and where there seems to be disconnect with some of the features like hyposmia.

[00:09:50] Dr. Andrew Siderowf: Yeah, so the way the assay was implemented in our study, it was sensitive to Parkinson's or, Lewy body like synucleinopathy, but not as [00:10:00] sensitive to MSA. And in our run, although it was reported in a different paper, there was one case that had a negative SAA and came to autopsy and had MSA at autopsy.

So this was a case that was read as negative by the SSA assay that we used in our paper. But at autopsy had MSA we think that's probably the case is that some of the negatives some of the SAA negative cases, especially among the normosmic, non LRRK-2 patients may turn out to have MSA.

We don't know that for sure. They haven't come to autopsy, but I think there were 16 patients that came to autopsy that were included in our group and the SAA one that was positive was always Lewy pathology. And the one case where the SAA negative was LRRK-2 positive Parkinson patient who had cell loss, but no Lewy pathology.

So no synuclein aggregates seen at autopsy, but was not MSA. But I think that in the future we'll get a sense of whether this is more common that we see a negative SAA and[00:11:00] it turns out to have the MSA pathology. I should note that Amprion, which is our partner in doing the assay for this study and other groups also are working on calibrating the seed amplification assay so that there's three levels.

One would be, truly synuclein negative, one level would be an MSA type of amplification. And the third level would be a lewy pathology type of amplification. And it depends on the conditions where the amplification assays run. And I think that this is something that's of interest and being worked on, I know by Amprion and other groups.

[00:11:31] Dr. Tiago Outeiro: So that would mean that with the same sample, then they would kind of run three assays depending on the calibration of the assay. Is that the idea?

[00:11:39] Dr. Andrew Siderowf: Yeah, so they would run it under slightly different conditions, which would allow for a differentiation between lewy pathology, Parkinson's, and DLB versus MSA. And you could differentiate it based on kinetic parameters. And the way the amplification assay works is that it takes a certain amount of time for the amplification to occur.

So that time is one [00:12:00] parameter, and then it also reaches a maximum fluorescence, like how much amplification actually takes place at the end of the day. And so you can measure the peak fluorescence and maximal fluorescence, and you can measure some time parameters. And the key take home is that MSA produces a maximal fluorescence, which is sort of intermediate between negative and the PD peak fluorescence.

[00:12:21] Dr. Tiago Outeiro: And going back to the LRRK-2 cases, I think for me that was very interesting because it also tells us about the biology, and we know that in LRRK-2 cases, usually you don't have Lewy body pathology. And you saw lower SAA positivity. So that seems to fit.

I don't think you had parkin cases, but given what we know about Lewy body pathology in parkin cases at the moment, would you expect also a lower signal in the SAA in parkin cases or can you make any predictions?

[00:12:51] Dr. Andrew Siderowf: Yeah. So, I think there've been a few published series of small numbers of parkin and patients, and I think there's an occasional positive [00:13:00] case, but the majority, maybe 80% are negative. I know we do have a small number of parkin patients in PPMI that were run but were not included in this study.

And my recollection is that one was positive and that the remainder, which is only a handful, five or ten were negative. I think that this would be expected, I think you'd see the same thing for PINK1, where you'd expect to see mostly negative cases. However, alternatively the alphas synuclein variants, like the A53 T mutation, which is the typical one in Greece and Italy.

They produce a lot of synuclein pathology and I don't think we reported those either, and that's something that you'll see coming out, is that they're quite positive and even the asymptomatic, non manifesting carriers frequently are positive.

[00:13:42] Dr. Tiago Outeiro: Okay. Yeah, that's very interesting. So I'll be looking forward to reading more about that. Now I wanted to ask you, so we touched on this briefly before, but as a clinician, do you think we are ready to really start using the SAA tests in the clinical practice. I mean, you mentioned in the beginning this is now used in a research [00:14:00] setting, but when do you think we'll be able to really start using these assays in the clinical practice?

[00:14:05] Dr. Andrew Siderowf: Yeah, so as I was saying before, I think this is a test, which is forward-looking in terms of the clinical context. I'm hopeful that in the future we'll have targeted therapies that aim specifically at synuclein pathology, and at that point it'll be crucially important to know who has clinical Parkinson's disease syndrome based on synucleinopathy and who has an alternative pathology.

I think the situation's really pretty analogous to what you're seeing in Alzheimer's now with the anti-amyloid therapies. I think that confirmation of the appropriate pathology is, I think seen as a necessary first step before you'd imagine treating somebody with these newer emerging amyloid agents.

I think that we'd be looking at sort of a similar kind of scenario for Parkinson's disease. But I think we have to get ready for it now, cause even in my clinic, I get asked about this frequently by patients like, should I get this alpha synuclein test that I read about? And I think that we [00:15:00] need to, as a community, think about what the right answers to these questions are. And I think that it's good for talking to patients now and it'll be increasingly important as disease specific therapies emerge.

[00:15:11] Dr. Tiago Outeiro: Yeah, no, I think that's true and it'll for sure drive forward, and the quest for novel therapeutics because now you have a measure you can use also to hopefully assess progression. So I think this is very exciting. 

And now we're coming to the end. I have only two more questions, but I wanted to ask you also what you consider the major limitations of the study? And what's coming next? I mean, you already told us about things that will come up looking at specific alpha synuclein mutations. But in the particular study, what can you consider is the major limitation, if we wanna call it like that. And what do you still think needs to be addressed in the context of synuclein amplification assays or seed amplification assays?

[00:15:54] Dr. Andrew Siderowf: Yeah, so I think our paper, we were pleased that it was at a very large sample size[00:16:00] and allowed for some subgroup analysis. And I think that was a strength. I guess you asked about weaknesses, but I'm talking about strength. I should get back on topic. But I think that autopsy verification, because we think that we're identifying pathology and I think that capturing autopsy information about the participants over the course of time will be a valuable addition.

I think that progression, this was a cross-sectional study and I think that looking at how the assay potentially changes over time, but also importantly, whether how a positive or negative assay predicts clinical change over time. So how like your assay at baseline, whether it provides prognostic information as well as diagnostic information will be valuable.

And then in terms of assay development, we've really looked at this as a binary. The actual values that you get from the assay vary greatly. But right now it functions sort of like a genetic PCR test does, where it's either positive or it's negative, but we think there's a potential that you could generate quantitative as well as qualitative information from the assay. [00:17:00] And that could provide either information about progression, if the quantitation say goes up or goes down over time. Or potentially prognostic information like more synuclein might indicate something different rather than less synuclein. I mean, paradoxically now lower peak fluorescence are associated with MSA, so they actually indicate worse pathology.

So it's not necessarily the case that higher would be worse, but it is possible that quantitative variations could have meaning. And we don't capture those right now.

[00:17:28] Dr. Tiago Outeiro: Yeah. Great. Okay, so is there anything else you would like to highlight here for our listeners, or do you think you covered everything?

[00:17:37] Dr. Andrew Siderowf: Well, I would like to say that PPMI is definitely a team effort, from Ken Marek, who's the PI of a study and the Fox Foundation who funds it, to our partners at Luis Concha and Claudio Sota, who are collaborators at Amprion, who made the assays possible. And then of course, all the participants in PPMI, over a thousand of them who had spinal taps so that we could do this study, and then had [00:18:00] spinal taps repeatedly and then came back for clinical visits. Without the contribution and the persistence of this large group of people, especially the participants, we couldn't get this information so, I think all of us who participated in the paper are, really grateful to the study participants who contributed samples and came back for visits, and I just wanna make sure I give them the proper credit.

[00:18:21] Dr. Tiago Outeiro: That's very important. Absolutely, we need all the patients to help us in our quest to try to help them back. So thank you so much, Andrew. That was wonderful. I think we really touched upon the important points of your recent publications in Lancet Neurology. And I thank all our listeners and I invite them to join us for upcoming podcasts.

So, goodbye for now.

[00:18:44] Dr. Andrew Siderowf: Yeah. Thanks, Tiago. Nice to be here. [00:19:00] 

Special thank you to:

Dr. Andrew Siderowf
Professor of Neurology
University of Pennsylvania

Tiago Outeiro, PhD 

Director of the Department of Experimental Neurodegeneration 

University Medical Center Goettingen, Germany

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