Thank you, Dr. Davis. And I have the next session, and it's going to be speaking on the diagnostic trail. And here, too, we are going to start with some of those audience response questions. OK. Which of these is the most common CTR mutation encountered in the United States? If you were just listening to Dr. Davis' talk, you're going to get this right. It's a V30M, V50M, T60A, or V122I. OK. OK, it looks like they were listening. 70% of V122I. And Jay, still in the lead, but Meg, I believe in you. All right, let's move forward. This is question two. Which of these is normal in a patient with advanced chronic kidney disease? Is it a trace M spike on serum protein electrophoresis? Is it a trace lambda light chain band on urine protein electrophoresis with immunofixation? Is it a serum capital lambda free light chain ratio of 2, or a serum capital lambda free light chain ratio of 0.3? »» Okay. So the plurality got it right. You weren't fooled by my normal range there. So we'll get to that in a moment of why the answer is the free light chain ratio of 2. And Nicholas pulled into the lead. OK. Let's go back to the next question, please. What is the gold standard test to determine amyloid subtype on biopsy? Is it mass spectrometry, immunohistochemistry, Congo red staining, or H&E staining? Okay. So this time the plurality said Congo red, but no, the answer is mass spectrometry, as we will get into. All right. Which biopsy site has the highest yield for, oh, I'm sorry. There we go. For ACTR cardiomyopathy, is it the abdominal fat pad, the endomyocardium, the bone marrow, or peripheral nerve? OK. And here, most people did get it right, and the answer is the endomyocardium. And if we could go to the next slide, please. Zink has pulled into the lead. And let's go to the final question, please. Which of these tests is not essential to be able to interpret the results of bone scintigraphy? Serum protein electrophoresis with immunofixation, urine protein electrophoresis with immunofixation, the serum-free light chain assay, or the urine-free light chain assay? Which one is not essential? All right. So most people got it right. Urine-free light chain assay. And if we can go to the next slide, please. Which of these tests is not essential to be able to interpret the results of bone scintigraphy? Serum protein electrophoresis with immunofixation, urine protein electrophoresis with immunofixation, the serum-free light chain assay, or the urine-free light chain assay? All right. So most people got it right. Urine-free light chain assay. Urine-free light chain assay. And if we can go to the leaderboard. Zank's still in the lead. All right. Let's go ahead and jump into the talk. So Dr. Davis went over this as well. It's an ATTR session here this evening. But to understand testing for ATTR, you need to understand something about AL amyloidosis. So if you'll bear with me, remember that, of course, plasma cells make antibodies, which are made up of heavy chains and light chains. The light chains, importantly, come in two broad flavors, two categories, kappa and lambda. And so even though there are thousands, maybe tens of thousands of different light chains, you can break them up into these two parts. So now imagine somebody develops a clonal plasma cell population. Three things will happen. One is that that plasma cell clone will take over some percentage of the marrow, maybe a little, maybe a lot. Two is that it will typically produce whatever antibody goes along with that clone, whatever it was destined to produce. In this example, IgG lambda. And three is that it will typically produce an excess of whatever light chain goes along with that antibody. So in this example, that would be lambda light chain. You now have three possible outcomes, the most common of which is that the cells take over just a little bit of the marrow. They don't cause any problems. The immunoglobulins and the light chains come out harmlessly in the urine. And this is what we call MGUS, or monoclonal globopathy of undetermined significance. The second possibility is that it's a more aggressive cell. It takes over more of the marrow. It causes things like anemia, fractures, hypercalcemia. And that's, of course, myeloma. And the third possibility is that regardless of how much of the marrow it takes over, it just produces the wrong kind of light chain. And so you don't need a lot of the light chain. You have to have had the bad luck that the light chain that your clone makes happens to be one that is prone to misfolds and deposits. And that's what is a amyloidosis. Or put another way, if this is the world of clonal plasma cell disorders, most of it is MGUS. A smaller amount is myeloma. We could split it up into what's called smoldering and symptomatic. I'm not going to go into details of that for those purposes here. And AL amyloidosis is a much smaller circle that overlaps all of them, but mostly overlaps what would have otherwise been MGUS. And then we can make it more complicated when we put ATTR amyloidosis, which has nothing to do with AL amyloidosis or even MGUS. But in older populations, both are pretty common. So it's not rare to find an ATTR amyloidosis patient who also has a monoclonal gammopathy that can overlap with any of these, but the most common of which would be MGUS. Now, when you are testing for the monoclonal protein, which we'll see why this is so important here in a little bit, there are three tests to check. The serum protein electrophoresis with immunofixation, that's what I'm going to call S-Pi. The urine protein electrophoresis with immunofixation, what I'm going to call U-Pi. The first one is for looking for the whole immunoglobulins. The second is for looking for the light chains because they're concentrated in the urine. These tests are directly measuring clonality. The third test, maybe the most important, is actually not directly. It is indirectly measuring clonality, and that is the serum-free light chain assay. So here, remember, I said everybody has kappa and lambda light chains, and there's thousands of different kappa and lambda light chains. But if you just put them in the two categories, you'll tend to have about equal numbers in the bloodstream. So if you look at the ratio, it's usually about 1 to 1. But it's actually important to understand a little something about free light chain production and clearance. So it turns out the bone marrow makes about twice as much, maybe even a little more than that, kappa as lambda. But they are cleared in the body by two different mechanisms, the reticuloendothelial system, which clears them equally, and the kidneys, which clears kappa much, much better. So even though the marrow makes more kappa, they are so well cleared by the kidneys that in people with normal kidney function, you actually tend to have a little more lambda in your bloodstream than kappa. The ratio of kappa to lambda is usually a little bit less than 1. But as you get worse in kidney dysfunction, you end up with the ratio creeping up. And it's not uncommon for that ratio to creep up to about 2 or a little bit more than 2. So that will show outside of the normal range. But as long as it's in somebody with kidney dysfunction, it's still a normal finding as long as their S pi and U pi are negative. So again, this is a study in people with kidney disease. And the long and short of it was that most of the patients who had elevated ratios, these mildly elevated ratios, did not actually have true plasma cell clones. And you'll see this all over the literature. This was a study that was published in Amyloid for crying out loud. You'd think they'd get this right. But they said, we have 197 patients with biopsy-proven ATTR amyloidosis. And we did the testing. And 42% of them had concomitant MGUS. Now look, I said MGUS is common in an older population. It is not 42% of the population. So this comes from, again, these people, most older patients, have some degree of CKD. And it's not abnormal to get these mild elevations of kappa to lambda. Now, Dr. Davis was talking as well about this, the variant or hereditary form of ATTR amyloidosis. The wild type, as we heard, mainly deposits in the heart. The variant can deposit in other places. And the most common place other than the heart is in the nerves. And Dr. Davis talked about the mutations V30M and V122I. I want to talk a little bit about the genetic testing and nomenclature. You heard Dr. Davis saying, well, is it V122I or V142I or V30M or V50M? Causes a lot of confusion. So a couple of points on this. One, if you have a patient who you've diagnosed ATTR amyloidosis and you just want to say, is it hereditary or non-hereditary, you do not need to send a broad cardiomyopathy panel. You only have to check the TTR gene. It's a single gene. That's all you have to check. But if your patient has already had a broad cardiomyopathy panel, it is always included in that. So if they've had a broad cardiomyopathy panel, it's negative. You don't have to repeat TTR genetic testing. Now, if they have a mutation, the nomenclature is important. So I've shown three different versions here that are all referring to the exact same mutation. You could either do it by the nucleotide substitution. You could do it in the amino acid substitution in the whole protein. Or you could do it in the amino acid substitution in the mature protein after you've cleaved the 20 amino acid signal sequence. So you usually won't see that first term used. But you will sometimes see it as, in this example, V142I or V122I. That is the same thing. Technically, if you're using the one with the whole protein, you're supposed to have that P dot in front of it. So just for clarification, because again, this causes confusion. Now, there's a lot of mutations. There's 145 described pathogenic mutations. That's hard to keep track of. So if you see a patient, they have a mutation, you're not sure what to make of it, this is actually a great website, amyloidosismutations.com. And if you pull it up, you can pull up the mutation. And it'll show you the nucleotide substitution. It'll show what the typical clinical manifestations are, what part of the world you'll tend to find it, and some references. I won't belabor this point, because Dr. Davis was mentioning it. But again, the mutations will show a spectrum, more neuropathy, more cardiomyopathy. Now, you want to diagnose ATTR amyloidosis, you have two pathways to do it. So the gold standard is biopsy. And in fact, for AL amyloidosis, the only way is by biopsy. But for ATTR, it's the minority of the time. If you are going to biopsy, I would highly encourage you to just go after the clinically involved organ, which for our purposes is, of course, typically the heart. And the reason for that is that if you do something like an abdominal fat pad biopsy, there are really high false negative rates. And so you'll see rates quoted like 84% in the literature. That is not true. And it's not true for a few reasons. One is that it was derived in a patient population from an era where the only people diagnosed were people with absolutely overt disease. That's different than today's population. Two is that's almost all AL. And AL has a much higher yield from fat pad than does ATTR. And number three is that this was with highly experienced pathologists. So in the real world for ATTR amyloidosis, you're talking about somewhere around a 25% hit rate for the abdominal fat pad biopsy. Whereas unlike other infiltrative diseases like sarcoid, if you biopsy the clinically involved organs, so the heart in this case, you approach a 100% sensitivity and specificity. Now, you've shown amyloid, that's not enough. Because now you have to say what kind of amyloid it is. And so Congo red staining only says it's amyloid. It's a subtype. It either needs to be immunofluorescence in really good hands, if your pathologist is very experienced, or sends it off for the gold standard mass spectrometry. Okay, but the way that most people today are diagnosed with ATTR amyloidosis is with bone scintigraphy, which in this country is mainly with technetium pyrophosphate or PYP. So it was discovered rather serendipitously that hearts that take up amyloid deposits, excuse me, that, yes, will also take up bone tracers. And the one that was just validated in this country, there's others used around the world, is technetium pyrophosphate. Now the key point is that AL amyloidosis can also cause uptake, but not reliably, and so we'll see the relevance of that in a moment. The other thing to keep in mind is that if you're going to order this test, it absolutely positively needs to be done with specs. It cannot just be planar imaging, because otherwise you can see the tracer in the blood pool, and you wanna make sure it's in the myocardium. So this is the assessment. So again, I'm showing you planar imaging. That's fine to start with, but they've all gotta have specs. There was an old quantitative system that was looking at the ratio of the photons from the left side of the chest versus the right. That's largely discouraged now. We actually use the very simple visual scoring system. It's a bone tracer, so is there no uptake, which is a grade zero? A little uptake, but less than the bone, that's grade one. Same as the bone is grade two, more than the bone is grade three. Positive is considered grade two or three, although most of your patients should really be grade three. Even grade two, you should wanna make sure that you've got it right. So it was validated by this study, over 1,200 patients. They all had suspected or proven cardiac amyloidosis, and they all got bone scintigraphy, and very importantly, they also had comprehensive monoclonal protein testing. This is a very important slide, which is that of 58 biopsy-proven AL amyloidosis patients, 13 of them, or 22%, had positive scans, positive bone scintigraphy. What this tells us is that you cannot use this test to diagnose ATTR amyloidosis if AL amyloidosis is a possibility. So until you've ruled out AL amyloidosis, you can't do it. So this is the way to rule it out, or to make the diagnosis. You first rule out a monoclonal protein, and then you have a positive scan. And if you do that in this study, 391 out of 391 patients, or 100% specificity, you could make the diagnosis. Just to drive home the point further, another study, 292 consecutive patients with AL amyloidosis, and nearly 40% of them had an uptake on their bone scintigraphy, and about a quarter of those had, frankly, positive scans. Okay, so just a couple examples. This is a 66-year-old white man with heart failure, with typical findings of amyloidosis. You see his PYP scan, they've done SPECT, it's positive. Does this patient have ATTR amyloidosis? No, because I have not mentioned any monoclonal protein testing, and this is a patient with AL amyloidosis. Okay, here's another patient. Here is grade two scan, it's about equal to the bone. Is this ATTR amyloidosis? No, no SPECT was performed here, and when you see the SPECT, this is all in the blood pool, okay? So this is a patient with dilated cardiomyopathy. How about this patient? An 80-year-old white man with echocardiographic features consistent with cardiac amyloidosis. Well, we see the uptake, we see it is in the myocardium, not the blood pool. And you see his monoclonal protein testing at the bottom. Negative S pi, negative U pi, a mildly elevated kappa to lambda ratio of 2.1 with some chronic kidney disease. So does this patient have ATTR amyloidosis? Yes, this patient does, doesn't need any further testing. You've ruled out a monoclonal protein testing, and you have a positive scan. Okay, so again, take home message, do not order a PYP scan without a normal S pi, U pi, and serum-free light chain. Okay, so the diagnostic algorithm is really very simple. Once you suspect amyloidosis, you start with the monoclonal protein testing. And if it's normal, now you can go on to your bone scintigraphy, and that's gonna answer your question almost every time. If you have abnormal monoclonal protein testing, now you need to go on to biopsy. And although I would prefer an endomyocardial biopsy, it's okay if you wanna start with an abdominal fatbed biopsy. But if that comes back negative, you've gotta move back and biopsy the heart. So in summary, it's really important, if you're gonna understand ATTR amyloidosis diagnosis, you have to understand monoclonal protein evaluation. And those three tests, the S pi, the U pi, and the serum-free light chain test. And again, be careful about it, properly interpreting the free light chain ratios in CKD. Genetic testing should really be done in any confirmed, or at least offered for every confirmed ATTR amyloidosis patient. And again, understanding the nomenclature is important. If you biopsy, remember, not enough to say there's amyloid. You have to subtype, preferably by mass spectrometry. And finally, a bone scintigraphy, which in this country is mainly PYP scans. Remember, you've gotta perform specs, and you can only perform it if there's no monoclonal protein. And with that, I thank you, and we'll move on to our next speaker.

cardiac amyloidosis

ATTR amyloidosis

AL amyloidosis

diagnostic tests

genetic testing