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Introduction

Hello and welcome. My name is Sean Pittock. I'm a professor of Neurology and director of the Neuroimmunology Laboratory at the Mayo Clinic. And it's my pleasure to speak to you today to provide an update, 2021, on Aquaporin-4 IgG flow cytometry assay. 

Disclosures

These are my disclosures. 

Outline

Over the next 20 minutes, I'm hopefully going to give you an update on neuromyelitis optica spectrum disorder. I'm going to give you an update on how we detect aquaporin-4 antibodies. I'm going to talk to you about the discovery of the antibody, and how Mayo Clinic has spent essentially the last 16 years working on optimizing the assays that we use to detect this antibody so that we can provide you with the best, most sensitive and specific assay available. We'll talk a little bit about the pros and cons of different methodologies. And then we'll talk about what the optimal specimen is to send or submit for testing. In addition, I will also provide you with some information as to why testing for aquaporin-4 antibody is so important, and why early diagnosis and treatment is important. And I'll give you a brief update on the recent advances made in terms of therapy. 

NMO: Devic’s disease traditional definition (1894)

So let's take it from the start. Devic, in 1894, described patients that had bilateral optic neuritis and myelitis. These generally occurred simultaneously or in quick succession. Patients had a severe course, and this was considered quite an extremely rare condition, and it was termed Devic’s disease. Other terms have been used for this condition: neuromyelitis optica, which remains, and optic spinal multiple sclerosis is the term that was used in Asia prior to the discovery of the biomarker. 

Pathological Hallmarks

Devic’s disease, or neuromyelitis optica, has a characteristic immunopathology. And this was actually a study published in 2002 by Claudia Lucchinetti from Mayo Clinic, where she looked at patients that fulfilled the clinical and radiological criteria — remember this was prior to the discovery of the antibody. And she found that patients with NMO had pathology that looked different to multiple sclerosis. They had very destructive, long lesions in their spinal cords. They had eosinophilic infiltration; they had the deposition of immunoglobulins around vessels. And also there was evidence of complement deposition around vessels in a rim and rosette pattern. And based on this pathology, Dr. Lucchinetti suggested that this disease was antibody mediated. 

NMO-IgG Autoantibody (IF)

It was around this time that discussions occurred between Dr. Claudia Lucchinetti and Dr. Brian Weinshenker, who had shown that plasmapheresis was a benefit in patients with this type of disorder, that Dr. Vanda Lennon actually began to request samples be sent to our Neuroimmunology Laboratory for testing on tissue IF. And what was found was that about 70% of patients with the phenotype of NMO actually had immunoglobulins that were binding to mouse tissue in this pattern. For example, the antibodies seem to be binding to the pia around the cerebellum and midbrain. But also, interestingly, around blood vessels in a very similar distribution to what Claudia Lucchinetti had described as the immunoglobulin deposition around vessels. This pattern became known as NMO-IgG. In other words, it was an antibody pattern that was specific to NMO. 

NMO-IgG targets AQP4 — Where in the brain is AQP4?^2,3

Within one year, Dr. Lennon and her team discovered what the target of that antibody was and discovered that it was the water channel, aquaporin-4, that was expressed on astrocytes. Aquaporin-4 is expressed in the CNS perivascular and peripial end-feet that are in direct contact with the basal lamina of the endothelium and the pia mater. And this is exactly what we saw when we looked at patient IgG binding to the mouse tissue. It's also highly expressed in the ependymal cells and subependymal layers lining the ventricles and in the hypothalamus. Interestingly, it's not found in neurons or oligodendrocytes. And in fact, this discovery really resulted in a seismic shift in our thinking about central nervous system inflammatory demyelinating diseases. Because most of the focus up until then had been on oligodendrocytes. 

NMO Spectrum Defined by AQP4-IgG

The discovery of the aquaporin-4 IgG allowed us to redefine the spectrum of disorders seen in patients with neuromyelitis optica. Prior to the discovery of the aquaporin-4 antibody, patients had to have had optic nerve involvement and spinal cord involvement and no lesions in the brain. However, after the discovery of the aquaporin-4 IgG, it became evident that patients could have brain lesions. In fact, 60% of patients with aquaporin-4 antibody positive NMOSD have brain lesions. But patients could also have limited forms of the disease. For example, a patient with a single episode of optic neuritis, who's aquaporin-4 antibody positive, has a 50% chance of having another optic neuritis or a transverse myelitis in the following year. Thus, this spectrum evolved, and thus the term NMO spectrum disorder resulted. 

NMOSD diagnostic criteria⁴

Initially, the term NMO spectrum disorder was only used for patients that were aquaporin-4 antibody positive. But now, things are a little bit more complicated. It was recognized that there are patients with an NMOSD phenotype who, in fact, are aquaporin-4 antibody negative and they are called seronegative NMOSD. Many of these patients actually have MOG antibodies and are now termed MOGAD. But for the purposes of this presentation, we're going to focus on NMOSD as a disorder that is defined by the presence of aquaporin-4 antibody, outlined in the red box. 

Why is the accurate detection of AQP4-IgG so important?

Now why is it important to accurately detect aquaporin-4 antibodies in patients? Well, first of all, please note that aquaporin-4 antibodies are the first ever antibody biomarker of any form of inflammatory central nervous system demyelinating disease. MOG antibodies are the second, and there is no antibody biomarker. In fact, no definitive biomarker for multiple sclerosis. 

The detection of aquaporin-4 antibodies allows you to make a correct diagnosis of NMOSD, and it avoids the misdiagnosis as multiple sclerosis. Approximately 90% of patients, when they present with NMOSD, are misdiagnosed with MS. Luckily, this percentage is significantly reducing, as more physicians are ordering this test and making the appropriate and correct diagnosis. But if a misdiagnosis of multiple sclerosis is made, then these patients get started on disease-modifying agents that are FDA approved for multiple sclerosis. And many of these can make NMOSD worse. Also, there are now FDA-approved therapies for NMOSD.

Treatment pathway for MS vs NMOSD

So when you make a diagnosis of NMOSD, patients with an acute attack, just as in multiple sclerosis, are generally treated with steroids or plasma exchange. But the treatments to try and prevent attacks occurring — which is extremely important in NMOSD, as NMOSD is associated with a much higher morbidity, or much more disability then multiple sclerosis. Most disability and MS comes from the secondary progressive or the progressive phases of the illness. Whereas in NMOSD, the disability comes from the acute attack, which for many can be devastating. So preventing attacks is of the utmost importance. There have been three novel therapies that have been proven to work at prevention of attacks in NMOSD that are now FDA approved, and I'll be talking about these a little later. 

Six techniques frequently used to detect antibodies in autoimmune neurology

Now there are a variety of different platforms that we use to detect antibodies. But I just want to show you that originally, in 2004 when we discovered NMO IgG or aquaporin-4 IgG staining on mouse tissue, this assay was what was used to detect the aquaporin-4 antibodies back then. We then moved to immunoprecipitation assays to improve sensitivity. We then moved to ELISA. But unfortunately, we noted some significant issues with ELISA, including false positivity as well as lower sensitivity. And we ultimately moved to cell-based assays, which are now the main approach to neural antibody detection currently used in the world. We then optimized our assays by moving to a flow cytometer assay. And we'll talk about that in a moment. 

Immunopathological mechanisms and drug targets in NMOSD

I want to come back to the concept of mechanism of disease and drug targets because this really, I think, makes the point that making the correct diagnosis early is so important. Because now we have three FDA-approved drugs. So aquaporin-4 antibodies are made and produced and released by plasma cells and plasma blasts. And IL6 is markedly increased in the spinal fluid of patients during the attack of NMO. Now satralizumab is an IL6 receptor inhibitor. And that drug has a benefit in NMO by reducing the likelihood of clinical attack. Similarly, anti-CD19, inebilizumab, is a drug that targets plasma blasts and a variety of different lineages of B-cells and also has been shown to be of benefit in terms of preventing attacks in NMO. When the aquaporin-4 antibody binds to its target water channel on the astrocyte, at the astrocytic end-foot, the FC portion of that antibody activates complement, and it is the complement then that can essentially injure the astrocytes through the lesioning of the membrane or through the production of C5a, which is a highly inflammatory protein. Inhibition of activation of complement through at the C5 mechanism also has had a significant benefit for patients. 

And this just shows you the risk reductions for the four trials that have been so far published. Eculizumab, the complement inhibitor, 94% risk reduction for attack. Satrolizumab, if you look at the water channel antibody positive patients only, 79% risk reduction and 74% risk reduction. And then for inebilizumab, the anti-CD19 drug, you can see a 77% risk reduction for the likelihood of relapse when compared to placebo. So dramatic benefits and great news for patients. But again, you've got to be aquaporin-4 antibody positive to be approved for these medications. So appropriate detection is of paramount importance. 

Sensitivity and specificity of 6 Aquaporin-4-IgG Assays⁵

Now let's talk about sensitivity and specificity. So this was an interesting study that we did with Oxford University, the neuroscience group led there by Patty Waters, and at that time, Angela Vincent and Sarosh Irani — they were another lab that was leading the way in terms of optimization of assays to detect aquaporin-4 antibody. And what we did then was we compared all the assays that we did in our lab and that they did in their lab. And we did it in a blinded fashion. So we sent 35 NMO samples, we sent multiple sclerosis samples, we sent normal and healthy control samples and a variety of different other samples from other neurological disorders. And these were sent to Dr. Amit Bar-Or in Canada. He basically blinded them, he coded them, and then he sent them back blinded to the two different institutions. And we did a study and then we compared our results. And what we found was, very interestingly, obviously specificity was hard to get at because we were only testing a select number of patients, not a lot of patients. But I will talk about specificity when we're talking about testing thousands of patients. But look at the sensitivity. What you can see is that the flow cytometry assay using the live cells is sensitive at 76.7% compared with a low of 48.3% for the indirect immunofluorescence assay, which is not used anymore. But the ELISA also had quite a low sensitivity, and this is still being currently used. And the cell-based assay also had a lower sensitivity than the flow cytometry live cell assay. 

Seroprevalence of Aquaporin-4-IgG in a Northern California Population Representative Cohort of Multiple Sclerosis⁶

This is another study where we looked at the concept of false positivity. So what we did here was we investigated a Californian population of possible MS, about 3,000 patients, and we had serum samples — this was from Lisa Barcellos — we had serum samples on about a third of these patients. We tested them all. And interestingly, we found some were positive for aquaporin-4 antibodies. So we went back and looked at the clinical record of those patients. We found that two patients were positive by ELISA and cell-based assay and the flow cytometry assay. And when we looked at their phenotype, they had NMO. But there was also five patients that were positive by ELISA, and one that was positive by cell-based assay, none positive by flow cytometry assay, that when we looked at their medical records, they had classical multiple sclerosis. So these were false positive tests. This is highly problematic, because this would result in patients with multiple sclerosis being given an incorrect diagnosis of NMOSD, and then potentially being started on drugs that are not FDA approved for that disease. 

AQP4-IgG evaluation: FACS

This just shows you how we actually detect the antibodies using flow cytometry. What we do here is we use HEK 293 cells that are transfected with aquaporin-4. And then we also mix those with HEK 293 cells that are not transfected, or do not express aquaporin-4. The aquaporin-4 is linked to a substance GFP, which gives off a color. Then what we do is we add in the patient antibody. And then we add in a secondary antibody that binds to that patient's antibody and gives off a different color. And then we looked at the median fluorescence that we see on the transfected cells and we divide it by the median fluorescence on the untransfected cells. And if the ratio is greater than two, then that patient is positive for aquaporin-4 antibodies. And then we do dilutions at a dilution of 1 in 5, 1 in 10, 1 in 100. And the dilution, whereby the ratio drops under two, is considered the endpoint dilution. 

Now I want to bring your attention to the issue of what sample one should test. And this is a study we did where we compared the ability of the assay to detect aquaporin-4 antibodies in both serum and spinal fluid. So these were patients where we had both serum and spinal fluid drawn at the same time. And what you can see is that firstly, spinal fluid unfortunately is not the ideal specimen to test because you can see that there are some patients here that are actually negative on spinal fluid, and positive on serum. And the other thing that you can also see from this study is that the live flow cytometry assay that we use currently at Mayo Clinic picked up on more patients both in serum and in spinal fluid when compared to the Euroimmun cell based assay. 

Should serum, cerebrospinal fluid, or both be submitted for testing?

So the significantly higher titer of water channel antibodies also in serum versus CSF was also consistent with the concept that aquaporin-4 antibodies are generally produced in the peripheral lymphoid tissues rather than intrathecally. And a critical serum CSF gradient is required for the antibodies to penetrate the central nervous system. 

Value of Auqaporin-4 Antibody Live Cell-based Assay in a Tertiary Referral Center Montalvo, Flanagan et al: AAN 2021

Now I'd like to bring your attention to this study, because a lot of people are often asking me which assay is best, which assay should we use? What’s the possibility of getting a false positive result with the water channel antibody test. And so this is a study that was just presented at this year's American Academy of Neurology, it was a virtual meeting. And the first author was Dr. Montalvo, and the senior author was Dr. Flanagan from our group. And what they did was they looked at every single patient that we have tested at Mayo Clinic over the last few years. We have tested 1,704 Mayo Clinic patients. And the great thing about our Mayo Clinic patient data is that we have access to really excellent clinical records. And what we found was, is that 37 of those 1,704 patients was positive for aquaporin-4 antibody, so that's about 2%. And when we looked at the medical record of those 37 patients, all 37, or 100%, fulfill the 2015 diagnostic criteria for NMOSD. So no false positives occurred, and the positive predictive value and specificity of the Mayo Clinic live-cell aquaporin-4 IgG flow cytometry assay is 100%. 

We also found that the median IgG binding index was 11.7. Remember the range is from 2.1 to 71.7. The median end titer was 1,000. So these are robust, generally, titers of antibody. And the median age of aquaporin-4 IgG seropositive patients was 56 years. Range was 4 years to 90 years, and 81% were female, which is consistent with the female predominance that's been recognized in this condition. 

Take-home points

So what are the take-home points? Aquaporin-4 M1, flow cytometry assay, or FACS assay, provides optimal clinical sensitivity and optimal clinical specificity. Aquaporin-4 IgG assessed by live cell-based flow cytometry assay is a highly specific diagnostic biomarker of NMOSD. This is really important, because we now have three FDA-approved drugs that have proven efficacy to reduce the likelihood of a clinical attack, thus helping patients avoid the development of blindness or paraplegia. Having that diagnosis made correctly is of paramount importance. A recent study, as I've just shown you, shows zero false positive results, despite a large testing volume. And serum is the optimal and most cost-effective specimen for aquaporin-4 IgG testing. 

I would like to thank you for your attention. At the Mayo Clinic, we discovered this antibody and we work tirelessly to develop optimal assays to really help clinicians make the correct diagnosis early and get the patients on the appropriate therapy. 

References

These are some references for your review. 

1. Lucchinetti CF, Mandler RN, McGavern D, Bruck W, Gleich G, Ransohoff RM, Trebst C, Weinshenker B, Wingerchuk D, Parisi JE, Lassmann H.Lucchinetti CF, et al. Brain. 2002 Jul;125(Pt 7):1450-61.
2. Badaut J, Verbavatz JM, Freund-Mercier MJ, et al: Presence of aquaporin-4 and muscarinic receptors in astrocytes and ependymal cells in rat brain: a clue to a common function? Neurosci Lett 2000 Oct 6;292(2):75-80
3. Amiry-Moghaddam M, Ottersen OP: The molecular basis of water transport in the brain. Nature Reviews Neuroscience Dec 2003;4:991-1001
4. Wingerchuk, Dean M., et al. "International consensus diagnostic criteria for neuromyelitis optica spectrum disorders." Neurology 85.2 (2015): 177-189.
5. Waters PJ, McKeon A, Leite MI, et al: Serologic diagnosis of NMO: A multicenter comparison of aquaporin-4-IgG assays.
6. Neurology 2012 Feb 28;78(9):665-671 Pittock SJ, Lennon VA, Bakshi N, et al: Seroprevalence of aquaporin-4-IgG in a Northern California population representative cohort of multiple sclerosis. JAMA Neurol 2014;71(11):1433-1436
7. Fryer JP, Lennon VA, Pittock SJ, et al: AQP4 autoantibody assay performance in clinical laboratory service. Neurology: Neuroimmunology & Neuroinflammation 2014 Jun;1(1):e11
8. (Nielsen et al, 1997)

Thank you!

Thank you very much. 

Questions?

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