Hot Topic: Tissue-based biomarkers in Parkinson's disease

[00:00:39] Dr. Thomas Beach: Thank you very much, Eduardo.

[00:00:41] Dr. Eduardo Fernandez: So despite significant efforts trying to get reliable biomarkers for Parkinson's disease, unfortunately this has not resulted in a clinically applicable test to make a confident diagnosis and monitor the progression of the disease. So in your opinion, what are the needs of [00:01:00] an ideal biomarker on what would be the characteristics of the ideal biomarker for Parkinson's disease.

[00:01:05] Dr. Thomas Beach: It would be dependent on the stage of disease you want to diagnose, but we all know that the clinical diagnosis of Parkinson's is actually highly accurate, sensitive and specific against neuropathology when the disease duration is longer than about five years. But still, even for that stage of Parkinson's which I'll call, say, advanced, you still could use biomarkers as a progression marker during a therapeutic clinical trial to see if the synuclein, and I'm assuming here that most of the biomarkers we're going to be talking about today would be synuclein, but there could be others, of course.

But... , for advanced Parkinson in a therapeutic trial, you'd want a progression biomarker. So, that could be, of course, right now the most promising seem to be CSF seeding assays. But also I [00:02:00] think the tissue biopsy biomarkers either treated with immunohistochemistry or, a seeding assay like RT QuIC are also quite possible.

And, and those I think are, are really On the verge of being available, especially the CSF markers. The biopsy based RT QuIC biomarkers are lagging a little behind because we haven't done as much work on those. But I expect perhaps a skin punch biopsy done by RT QuIC to be quite well established through the literature anyway over the next year or two.

There's quite a few labs working on that. Of course, we all want to not just treat Parkinson's and other neurodegenerative diseases after they've already settled in to the brain and already impaired the patient's lives. We want to detect it before that. So early stage symptomatic disease and also preclinical disease are [00:03:00] areas where we need biomarkers, but those are a little out of range, but of course I think early symptomatic could be within range with the same type of biomarkers within the next few years, but the preclinical biomarkers are still going to be lacking for a while unless there's new breakthroughs in sensitivity and specificity of the available biomarkers, including RT QuIC, which from a couple recent studies, it looks like it, CSF RT QuIC can't pick up preclinical or early brain stages. So we need some more sensitive and specific biomarkers for those stages.

[00:03:34] Dr. Eduardo Fernandez: Specifically for tissue biomarkers, obviously as you mentioned, they are going to mainly focus on the demonstration of a synuclein, the position in the tissue. We will discuss a bit at the different tissues that have been evaluated but to start with what we know about synuclein deposition in tissues outside central nervous system.

What are the main tissue targets and what do we know specifically in terms of, what happens or the [00:04:00] proportion of people in the normal population that may show synuclein in different tissues and also the incidental Lewy body disease in different tissues. Can you comment on that?

[00:04:09] Dr. Thomas Beach: Yeah, we've been working on this for quite a while. Initially with a good grant from Michael J. Fox Foundation back in 2007. They specifically came to us and asked us to look for Parkinson's disease markers in the body outside of the brain. That was because they knew we were one of the few centers in the world that has not only a comprehensive clinical annual research characterization of patients and volunteer normal aging subjects, but also because we have been doing whole body autopsies along with the brain.

So with that initial grant, we published in 2010 a survey of phosphorylated alpha synuclein, P serine 129 alpha synuclein, which is been shown by several groups to be extremely specific for pathological synuclein. We published the distribution of [00:05:00] phosphorylated synuclein in the body across I think it was 45 different body sites.

Throughout the study, we kept focusing in on what we call the hotspots. To do 45 different body sites as well as the comprehensive brain investigation is quite a lot of work. But we started focusing on the areas that were lighting up the most, and it came to be obvious that of course the spinal cord, but that central nervous system, so, that was expected that that would be amongst the most frequent spots to harbor synuclein deposits. But also the sympathetic nervous system was second in frequency. So sympathetic ganglia and sympathetic nerves for instance, to the heart, but then thirdly, the, the GI tract was the next most frequent area and there was a proximal to distal gradient going from the esophagus and submandibular gland all the way down to the sigmoid colon and rectum.

There was a gradient of ever reducing alpha [00:06:00] synuclein pathology density as you went proximal to distal. And in fact, we found that the submandibular gland and the lower esophagus had the highest densities. So we embarked on a couple small clinical studies of needle biopsy of the submandibular gland, which turned out to show in both early and later Parkinson's about a 70 sensitivity again, and about a pretty high specificity, more like 90%.

Unfortunately it seemed like the neurology community was reluctant to accept needle biopsy of submandibular gland, even though we argued that it was actually less invasive and with fewer risks than lumbar puncture. So to this day, it is still not being widely adopted, but we turn to other sites then especially the skin.

But I have to say that a number of sites have continued to pursue the GI tract lower down and early on it was hoped that we might be able to piggyback on colon cancer screening in older [00:07:00] people as a way for also screening for early preclinical Parkinson's. And unfortunately that didn't turn out.

Michael J. Fox again sponsored us for two multicenter studies where we looked at how effective colon biopsies might be. We found, unfortunately, that clinically performed colon biopsies only barely pick up the mucosa and a little bit of the sub mucosa, and there's just not enough synuclein nerves in there, not enough nerve innervation where the synuclein pathology is.

I think because they pinch the mucosa and they pull it up, that the nerves remain bound down against the muscular layer. So colon biopsies, and this was I think four center, multi center study. It was Pascal de Kinneran in France, contributing the colon biopsies. So we repeated that with autopsy.

We found that autopsy, if you get the full thickness of colon, is actually not too bad a diagnostic method. Unfortunately, we just can't get the full thickness of colon in a[00:08:00] clinical biopsy because of the fear of perforation. So the colon has kind of remained dormant some groups are still pursuing it as a site, and especially with RT QuIC that increases the sensitivity now.

But Pascal's group has published on comparing RT QuIC with immunohistochemistry. And there is about a 15 percent increase in sensitivity if you do RT QuIC versus immunohistochemistry of colon. So, I'm looking for perhaps more studies of colon biopsy treated with RT QuIC to see if those can bring up sensitivity and specificity.

We're not doing those ourselves, but I think that's a promising area. There's also the possibility for biopsies further up. And some groups including a group in South Korea has been looking at archival surgical procedures on the esophagus and stomach to see if those might have a greater yield for synuclein pathology so those studies could continue, but of course biopsying the esophagus or stomach would also [00:09:00] have an invasive aspect and may not be as attractive as simply doing punch biopsies of the skin.

So we've moved on to doing punch biopsies of the skin treated with RT QuIC and we currently have a funded project where two RT QuIC labs are processing our skin biopsies. So far we've biopsied about 150 people. And seeing whether for one, can the two assay labs agree? Because there's still some substantial problems with RT QuIC and seeding assays in general with interlaboratory, reliability or agreement.

But we are pursuing that and we hope to have some results published within the next year. We're recruiting not only PD cases, but PDD, Parkinson's dementia, Dementia with Lewy bodies and also normal controls, of course. And so we hope to have some results there within the next year or so.

Right now it's really difficult to see, there's so many groups around the world doing RT QuIC and the [00:10:00] results are quite variable. And one of the issues has always been with biomarkers of all types, including in Alzheimer's and Parkinson's. Throughout medicine really, is that laboratory developed tests are not as rigorously regulated as FDA.

I'm talking about the United States, I guess in Europe it's the EMA. So Laboratory developed tests kind of get a pass on whether they're actually accurate for a disease diagnosis. They're only required to show that if you perform them over and over again in the same lab that you get the same result.

So they're only required to show reliability. And that's a problem. So we've got actually a lot of tests out in medicine today, lab tests, biofluids tests mainly, that have never really been adequately scrutinized and therefore their true accuracy is never actually known. But generally when I give lectures I like to emphasize that not all biomarkers should be considered equal and the reader needs to be critical, needs to see are they a single center study, [00:11:00] are they a multi center study, was there effective blinding and the best blinding is a third party blinding where none of the groups doing the work holds the blind, but a third party holds the blind.

There's considerable wealth to be made from establishing a new biomarker. And we need to be on our guard against tests getting into the medicine's clinical domain that really have not been adequately vetted. 

[00:11:25] Dr. Eduardo Fernandez: So you mentioned, a bit on the colon and the gut in general, the GI tract. So you mentioned a lot about the variability of the results. And I think probably the GI tract is one of the main areas of this problem. How would you interpret the variability in the results?

I guess there is challenges you mentioned about the sampling not just the area of the GI tract, but also the thickness of the sample. You mentioned briefly about the type of immunostaining that the antibodies used, so there's a lot of technicalities to bear in mind. Could you comment on that?

[00:11:55] Dr. Thomas Beach: I think there's a remaining interest of course in the colon and I think a lot of it is due [00:12:00] to Brock's original theory that parkinson's starts in the body, and a lot of groups have published on varying forms of synuclein in the colon that might serve as an initial pool to stimulate synuclein aggregation into a more conventional pathologic form.

I think it's pretty clear that the colon and the GI tract are really affected later in the Parkinson's process. We've published on the fact that over a couple hundred or more autopsies, . We never see synuclein pathology in the body when it's not already in the brain.

So, I think when synuclein pathology progresses into the body anywhere, it's generally at a later stage in the brain. It could be still at the brainstem stage, so it could be still a relatively early and preclinical sign that we can detect. But unfortunately, there's been a lot of studies published where, for [00:13:00] instance they just find that a large amount of synuclein in the gut is perhaps indicative of an initial pool that could spread to the brain. But, we all know that synuclein is a very common, it's one of the most abundant proteins in the brain and in nervous tissue. So just to see synuclein doesn't really mean much, it's got to be a aggregated or phosphorylated form.

A lot of the work that's being done in the colon is interesting, but I think we need to show that it's a pathologic form of synuclein that is found in the colon not a speculative pre infectious form.

[00:13:35] Dr. Eduardo Fernandez: You mentioned also the skin biopsies. Again, I would like you to comment on what is the best area to sample and what are the at the moment, the, the sorts of sensitivities and specificities of the tissue biomarkers in the skin at the moment.

[00:13:51] Dr. Thomas Beach: That's still a rapidly evolving field with a number of labs around the world working on it. And first it was done in skin and I'm [00:14:00] thinking of groups in, Italy, and Spain, and Barcelona, Harvard, and the U. S., as well as ourselves that have done immunohistochemical studies of skin.

 It seemed like there was a consensus between groups, a relative consensus, that the cervical region, i. e. the neck most people biopsy the back of the neck around C6 to C8 had the highest yield or sensitivity for synuclein aggregation versus arm, leg, thigh, but more recently I believe that another group, I think maybe the Doppler group found that the arm or leg was also sensitive, but formerly I think there was three or more groups that seemed to agree that the back of the neck was the most sensitive. They're all more specific than they are sensitive.

And the sensitivity has been the main issue. We would like to see that, of course, get up to 80 percent or more.

[00:14:59] Dr. Eduardo Fernandez: You [00:15:00] mentioned as opposed to the more, immunohistochemically based tissue biomarkers you mentioned, RT QuIC or like a seeding amplification a ssays have rapidly developed over the last few years. And they have shown better sensitivity to pick up synuclein and in peripheral tissues, and we have seen that as you said, tissue samples have not been tested as much as CSF, but we have started to see publications on this. What do you think is the future of tissue biomarkers? Do you think that we are moving from the sorts of more traditional immunohistochemistry to this sort of seeding amplification assays or?

[00:15:37] Dr. Thomas Beach: I think it looks that way now. It looks to me that from a couple studies that have done both immunohistochemistry and, and RT QuIC or seeding assays as I mentioned, I think the RT QuIC is maybe 20 percent more sensitive without having lost much specificity, but again, it's a rapidly developing field.

I think skin [00:16:00] punch obviously would be the best way to go in terms of cost and availability can be done in anybody's office. So I'm really hoping that skin punch biopsies will turn out to be as sensitive and specific as skin. Tissue biopsies have a possible advantage over CSF when it comes to being a progression marker in a therapeutic trial.

We know from Alzheimer's disease, monoclonal therapies that if you give monoclonal therapy to an Alzheimer's person, of course it drops the blood and CSF levels of A beta. But that doesn't necessarily translate into engaging targets in the tissue. So we can't really use that. There's been a few trials where the monoclonal drop, the circulating A beta level and CSF level, but yet there was no effect on amyloid PET in the brain.

The advantage again of tissue sites is that, in a [00:17:00] progression maybe an early stage, say a stage two clinical trial, you could look at a tissue deposit to see if the therapeutic dropped the tissue level of alpha synuclein pathology. And you might assume then that that's a better indicator of whether or not that therapy is going to drop the tissue level of synuclein pathology in the brain. I think it can act as a stepping stone to a brain targeting therapy and give drug companies maybe a little more confidence about whether or not to go to a stage three clinical trial.

[00:17:38] Dr. Eduardo Fernandez: That was my next question. So I guess we need to balance accessibility of the samples with being a reliable biomarker of the process that we need to measure So we have seen recently a publication where RT QuIC in in blood samples has shown promising results. Do you think still that tissue biomarkers will [00:18:00] have a room to assess disease progression as opposed to, CSF is still quite invasive, but blood biomarkers that may develop in the future, do you think there is room for tissue based biomarkers?

[00:18:11] Dr. Thomas Beach: Yes. I think, the target really is tissue based synuclein deposits, not circulating synuclein. So I think it can act as a test for drug companies in their early stages before they go to a fully fledged stage three trial. And I'm not sure about, blood is still very early days for evaluating whether RT QuIC or seeding assays will work in blood.

We know that red blood cells are full of synuclein and so there's potential problems with using blood. Of course, there's many more complicating plasma proteins in blood compared to CSF. So it's a much more difficult medium in which to act. But we've seen encouraging results in Alzheimer's.

The P-tau 181 [00:19:00] and 217, 231 assays have proven surprisingly... accurate.

[00:19:05] Dr. Eduardo Fernandez: Thank you very much for this overview of the state of tissue biomarkers for Parkinson's. I think we have covered everything that I had planned. I don't know if you want to add anything else.

[00:19:15] Dr. Thomas Beach: I appreciate you offering me this chance to comment and I often liken the whole biomarker story to the fog of war. We're always trying to understand what's going on and what seems to be a valid result, what's not a valid result as it's occurring and meanwhile, new assays are being developed all the time, so.

It's a constantly moving field and difficult to keep track of. All we can do is keep trying to read the papers as they come out and try to be critical.

[00:19:46] Dr. Eduardo Fernandez: Try to see across the fog. Definitely. Thank you very much for your time today and thank you very much for the listeners. I hope you enjoy the episode. [00:20:00]