Webinar 6 - Acute leukaemia cases

Welcome everyone. Welcome to the sixth and final, uh, webinar in this blood cell morphology program. Uh, we've had some really interesting webinars so far, covering a whole range of different topics. I'm delighted to be joined again by Dr. Audi Satardy from Canada. We're going to be discussing three interesting acute leukemia cases. But before we do introduce the cases and go through the cases, and also introduce Aussie, I've just got a couple of things just to go over to remind you about the series.

The first thing is that this webinar series has been kindly supported by the company Urban Mannheim, and that CME certification is available for this webinar. It's been accredited by the Royal College of Pathologists in the UK. You can still register for your CME certificate. I've posted a link to the registration page on the comment section of this video. It's really important that you register for your certificate before the end of the webinar. We hope that the webinars, uh, all of the webinars in the series have been useful, and we hope that you, uh, want to go back and revisit, um, the, uh, the presentations given. And all of them will be available, uh, on the YouTube page for the, the Blood Academy YouTube page. So hopefully, in about a day or so, we'll get this webinar recorded and uploaded onto the Blood Academy YouTube page for you to see again.

The whole point of this webinar series is an introduction to blood cell morphology, to try and cover as many, uh, different topics as possible, but we, we can't be exhaustive. Um, and hence, uh, there is the opportunity to go into a little bit more detail using the Blood Academy on-demand essential blood cell morphology course. It contains video tutorials, online e-modules, and assessments. And again, this whole course is accredited by the Royal College of Pathologists. You can save 20% on the course, uh, using the discount code HIG at checkout. So please feel free to, to have a look, a look at the, the course on the Blood Academy website. Uh, the first lesson is actually for free to give you an idea of what the course entails.

Okay, so, uh, I'm delighted to be joined again today by Dr. Audi Citadier. She is a hematopathologist at British Columbia Children's Hospital in Vancouver, in Canada. She has a very keen interest in lymphoid malignancies, flow cytometry, especially medical education. She's lectured widely in various national and international teaching and academic programs as well. So thank you so much, Audi, for joining us today. Uh, can you hear me okay?

Yes, thank you for the introduction. Thank you. It's great to drag you back again, uh, after such a while. Happy here. Happy to be there. We had a few weeks ago. So let me just, uh, stop sharing my screen and allow you to share your screen there. We go. So you should be able to show your screen, RD. Um, what I will say before you, you do go through the, the first case, uh, is that like all the other webinars that we've had before, we've had really great engagement from everyone who's been involved on Facebook. So, uh, ask questions, comments. I'll try and relay that to Audi as well, uh, and really just try and stimulate some, uh, some interesting, uh, discussions, really. So should we move on to case one?

Sounds good. So case one, we've got is a 68-year-old female who's presented with bruising. She, hemoglobin isn't too bad, although I suspect it might be a little bit lower on the, uh, the slide. Her white cell count is 23.1, so increased, and she's thrombocytopenic with a platelet count, at least from the analyzer, of 77. So I'll, I'll leave things over to you then, nowaday.

Sounds good. Okay, so, um, I will make this screen larger. So, can you see this okay? All right. Yeah, we can see it. Perfect. Okay, so as we cruise around the slide, what we see is that there's not much of, um, any of your normal neutrophils or platelets. Um, but there are, um, these abnormal cells that look about medium and large in size, and they have a moderately abundant basophilic cytoplasm. There's some variability in size and morphology, but in general, these are abnormal looking cells. So, and these cells have, um, finer chromatin, and some of them have prominent nucleoli as well. And that's, uh, that's one dysplastic neutrophil that we see here. It's a pel-ger-huet anomaly. So as we cruise around, we see a lot of these, um, abnormal cell population. All right. So anybody would like to take a guess what, um, these cells are?

So I think we're getting blast cells from a couple of people. Okay, very good. So you recognize that these are abnormal blast population. Um, and there's a, like, and sometimes we would like to take a guess, like, as you know, there's acute leukemia can be divided into myeloid or lymphoid. Um, and of course, you have to perform flow cytometry to diagnose that. But sometimes morphologically, we take cues on what, uh, kind of blast this might be, whether they're myeloid or lymphoid. So I'm looking around for a particular structure, um, when I'm assessing this slide with a lot of blasts. So any idea what, uh, structure I'm looking for? It's a bit of a read my mind question, but something very important, uh, to look for when we're, uh, assessing acute leukemia morphologically.

So because there's a slight delay between our Zoom link and Facebook, there, uh, it may take a couple of seconds for people to register, but we're getting some comments now. So our rods. Very good. Yes. Um, we should be looking for our rods because, oh, there you go. Perfect. So, uh, there's the cell that might be a little bit, um, almost broken, but look at the structure in the cytoplasm with a reddish long rod in the cytoplasm. These are what it's called our rods, and when you see our rods, that's almost diagnostic for. Can people put your answer or diagnosis in the comments? We haven't had any comments as yet, but, um, I'm just to make a comment that, you know, that sometimes they can be really difficult to find. You have to, yes, really spend time doing it. Oh, here we are. So AML, myeloid. Perfect. Exactly. So, um, so something you really have to, like, at least then, uh, cruise around, uh, your slides. Sometimes you have to make multiple slides, um, and when you're lucky, you can find that our rod, like we just saw, and I can't find that cell again now. So, um, so but that's really good that, like, that's diagnostic for acute myeloid leukemia. Um, so when you're assessing a slide, uh, that are full of blasts, first, you have to look for our rods. And the second, second thing that you also want to look for is any evidence of dysplasia, right? So, um, normally these dysplastic cells are more associated with acute myeloid leukemia rather than acute lymphoblastic leukemia, but even in acute leukemia, you can see a little bit of dysplasia here and there. But generally, if you have marked dysplasia, that's associated more with AML rather than ALL. Um, so we saw one pel-ger-huet neutrophil earlier, and other than that, there's not much of, there's not many neutrophils to assess. So that can be very difficult as well, and there are not that many platelets to to assess. But I think we passed by a few that look nice and small and well granulated, like this guy here. So, um, oh, there you go. There's another, um, cell with our rod here that you can appreciate.

I was just going to mention that one person, we've had one, a couple of comments suggesting that this could be, uh, APML, acute promyelocytic leukemia. Just wondering what your thoughts are on that, because it's a medical emergency, like all acute leukemias. But, uh, yeah, just your thoughts on that.

Okay, so we'll talk about it in my lecture slides in a sec, but so I can see why you're considering that because you see some cells or like, for example, this cell with a lot of, I like their one, two, three our rods. But, um, not all AML with our rods are APML. That's the first, um, that's the first rule that you have to remember. So, um, our rods are present in a subset of AML, and if you see that, you diagnose AML, but it doesn't always mean APML. Okay. So, uh, APML cells, they actually, and this can be deceiving, because when I say, uh, there are a lot of blasts here and AML, you like those more than 20 blasts. But in APML, sometimes usually the blasts are, uh, pretty scarce. They're not, they're not as many as in this picture here. Um, they can, they're usually rarer to find, and when you do find them, um, the blasts can be, they're very, very, very granular. So, um, they look, they're a lot of granules classically, and there are multiple stacks of our rods, uh, classically. So that's what APML looks like, and their nuclei can be very convoluted, and it's a butterfly shaped, as we call it, and I'll show you a few pictures on my lecture slide. Um, so yes, we saw a few blasts that look a bit clefted, like this one. But, um, but in general, this doesn't quite, like, bring, um, uh, you know, basically, you cannot jump to APML right away, right away, without, without, uh, doing cytogenetics, uh, or bone marrow, uh, just from this picture alone. So hopefully that answers your question.

No, it does. Thank you. Yeah, sounds good. Okay, and before we go to the lecture slides, I would like to give you the second case so you can compare and contrast less morphology.

Yeah, that's perfect. So should I introduce the second case as well?

Yeah. So the case two, um, is a 61-year-old male. The clinical information that we have is a chest infection. Uh, he's, uh, anemic. The hemoglobin is 92. The white cell count is is increased, as you would expect, 21.6, and a platelet count of 54. Okay. So this patient is quite cytopenic as well. Um, we're seeing a few more neutrophils compared to the last one. And again, as we cruise along, you're noticing that there are, um, these cells that look, that resembles a particular cell in, um, in the blood. What do these cells resemble? Hopefully, we should get some comments coming in now.

Yeah, so I'm talking about these, um, cells with abundant, uh, pale gray cytoplasm and some small cytoplasmic vacuolation, reticular, uh, chromatin, and, uh, convoluted nuclear borders. We've got some very clever people. I think the, the consensus is, uh, monocytic.

So monoblasts. Very good. And it's very good that you recognize that, um, some of these look immature. So they're not, uh, you know, they're not quite your mature monocytes. So these are monoblasts. Um, and there's a whole spectrum, like, for example, this cell here is also immature looking monocyte, but it's more mature than that cell, um, because their nuclei is a little more folded. Um, so this, this could be an immature monocyte or promonocyte. Uh, and I will show you a nice diagram in my lecture slide on monocytic maturation. But in general, monoblasts, so like when we talk about blasts, uh, being always high NC ratio, fine chromatin, it's not always the case, right? So not all blasts, uh, have high nuclear cytoplasmic ratio. So this is a good example of that. This, these are monoblasts. They're large, and they have abundant, usually pale gray cytoplasm. They have some vacuolation. They have this, um, finer, um, net-like or reticular, uh, chromatin pattern, and they have, um, they range from, uh, round to slightly irregular nuclear border, in contrast to the blasts that I just showed you earlier, where they have less abundant cytoplasm.

Okay, so it's getting a little bit blurry. So I'm just going to let it load a bit more. Okay. Um, so as I mentioned previously, when you're assessing a slide with blasts, it's important to look for, uh, so our rods, which may or may not be present in, uh, in AML, and also these plastic features, right? So we're, um, now looking for any dysplastic feature in the other cell lines. So, and then we come across some of the neutrophils, and their lobation is not quite right. Um, they're, they have, um, uh, some of them look a little bit hypolobated. They're not quite as segmented as they should be, and then they also have a hypogranular or pale cytoplasm, right? And you have to be careful as well with your stain. Make sure that the hypogranularity is not because of your stain. This is why it's so important to have a nice correct stain for your peripheral blood. But, um, in general, it looks like, uh, some of these neutrophils are pretty hypogranular and dysplastic. They have poor lobation. Some of them are hypolobated, and there are some pel-ger-huet forms here. So, um, so what is your diagnosis in this case? Maybe some people want to take a guess in the comment whilst we're waiting for the comments about the diagnosis, we've had a couple of interesting questions, um, on roughly the same topic. How do you count, um, how do you differentiate between an abnormal monocyte and a promonocyte as, as part of your slides?

That is a very good question. I have a few more pictures to, uh, help answer that question. But in general, so abnormal monocytes often, or atypical monocytes, often I use that term, uh, to describe mature monocytes that are that are dysplastic. So, we don't really see it here, but, um, sometimes in monocytic leukemias, I see mature monocytes where their their nuclei looks like a flowery pattern. So it's very convoluted. It has this flower pattern nuclei. It looks, it looks abnormal. You don't see normal monocytes like that. That's what I call atypical or abnormal monocytes. So I'm referring to the mature subset of monocytes that look abnormal. Uh, whereas immature monocytes, I usually reserve that for, um, these guys here where they, um, their chromatin is more open, uh, their nuclei, uh, is not as, as, uh, as irregular, um, because they're more immature cells. So more like the, the, the monoblast and, uh, promonocytes, uh, uh, is collectively, I sort of call them immature monocytes. I always find it very subjective. If you ask hematopathologists to do a differential count, you probably get ten different answers. Yeah. And monocytic leukemia is one of the most difficult, um, diagnosis to make, or morphologically very challenging, um, in in hematopathology. So a lot of texts and even hematopathologists, uh, you know, struggle with this entity because of the, the more monocytic morphology. It is a real challenge. Yes.

So we're getting some answers now. So we've got, I think the, the consensus is that this is monoblastic leukemia. One comment, interesting, is for MDS/AML, acute monomonocytic leukemia as well. Sir, sounds good. Yeah. So both are very fair. And really, in the whole classification, acute monoblastic and monocytic, they don't separate them into different categories. So they actually lump them together. They call it acute monoblastic/monocytic leukemia. Um, and really, it's, it's all semantics. It doesn't really, um, it makes a difference in patient treatment. They're treated the same. It's purely morphological. So if, if you have more than 80% of your, um, of your blasts in the, in the bone marrow, usually, uh, look more monocytic, then you call it acute monocytic leukemia. And I would, I would argue in this case, it looks like it's the case that looks a lot of these, um, um, these blasts look, uh, more, uh, monocytic, uh, than monoblastic. Um, but if you have like, uh, more, more of them, more than 80% of them are monoblasts, uh, and usually they have like less, uh, cytoplasm, less irregular nuclei, then you call that acute monoblastic leukemia. And again, that's purely morphological. It doesn't make a difference in patient treatment. And, and I like that the answer that is possibly myelodysplastic related because they're quite a profound dysplasia in in the neutrophils. Um, and we'll discuss how you make that diagnosis as well, um, in, in my lecture slides.

Okay. What, one comment I was going to make out before you move on was that people are using the, um, the FAB classification, M5, M4, etc. Are you guys still using it? Do you find it useful at all, though?

So it's, it's a historical classification, um, and I will discuss that as well. But it's, it's in general, it's, it's a pretty convenient classification and generally understood by clinicians. It's based on morphology, right? And just to some degree, cytochemistry and flow as well. Um, we still, it's an older term, and now largely, uh, the official classification is based on WHO, but we still sort of throw that term, um, uh, just because it's, it's generally understood what, what I mean by M5 and M4 and M3, by morphology, right? So I think that term is still going around nowadays, and I, I personally find it useful to know what, what type of, uh, uh, blast they are, um, but it's not very prognostic, right? And, and, um, that's why, uh, WHO classification is now more widely used because it has more prognostic meaning to the patient. Yeah.

Okay. Yes. So, um, moving on. So case one and two are both, uh, acute leukemia, in particular, acute myeloid leukemia. And, uh, we touched on this a little bit, but, um, there are some morphological cues that we, we use to differentiate between myeloid blast versus lymphoid blast. Um, and of course, you, we have a saying, you gotta flow it to know it. So you have to perform flow cytometry to know for sure. Definitely, uh, you can, you cannot just, um, diagnose them by morphology. But there are some clues that we use. So myeloid blasts are generally larger in size. They have, um, more abundant cytoplasm, and sometimes can be granular as well, and they have more prominent nucleoli. Um, and finer, more open chromatin pattern as well. Um, in comparison, the lymphoid blasts, they're generally smaller, um, they have very scant cytoplasm, so very high nuclear cytoplasmic ratio, and usually they're, uh, agranular, um, and they have less distinct nucleoli. And as you see here, the chromatin pattern, um, is more condensed than the myeloid blast. So they, uh, they can be quite difficult, actually, to distinguish from hematogones or mature small lymphocytes. Now, I work at a busy children's hospital where we see a lot of acute lymphoblastic leukemia, and I have a huge respect on ALL, because they can be very, very tough to, um, to distinguish from mature lymphocytes or reactive lymphocytes. So, and they're always exceptions to these rules. In real life, it's not always clear-cut, but these are, uh, in general, what we look for. And as we talk about, is this one structure that when present, then you can actually diagnose acute myeloid leukemia morphologically, and that's our rods, right? It's not always present in every case, but when they're present, it's diagnostic of AML, and you shouldn't see that in acute lymphoblastic leukemia.

Let's talk a little bit about myeloid leukemia in terms of clinical, uh, and classification. So by definition, um, this is a textbook definition. You should have more than 20 blasts in blood or bone marrow. Um, and it can be deceiving because, um, there could be only few blasts in the blood, right? It doesn't, it's not always that it's like our two cases where they're everywhere. You can see pretty rare, uh, or only occasional blasts in the blood, but when you do a bone marrow on the patient, it's packed with blasts, right? So it's very important for for us to diligently look for this abnormal cell. Sometimes you have to make multiple slides to increase your sense of sensitivity in finding this blast. Um, and, uh, you know, at first, that's, uh, that's all I know. And more than 20 blasts, that's the definition of AML. However, um, as I went through my path residency and I learned more about this condition, and WHO actually, uh, says that not only that, but you can also have, uh, blast infiltration in other tissues in the body, like in the skin, for example, let's call it sarcoma, and that's by definition, is also acute myeloid leukemia, or they're treated the same way. Or you can also have less than 20% blasts in in blood or bone marrow, as long as you have these, uh, cytogenetic abnormalities. And there are a few, uh, cytogenetic abnormalities like translocation 8;21, inversion 16, uh, translocation 15;17, this is PML-RARA, or that you see in APML, acute promyelocytic leukemia. And if you see less than 20 blasts, you can still diagnose that as AML if you have these chromosomal translocations. Um, so it is more common in the adult population, with a median age of diagnosis, uh, 65 years old, but we also see this in kids as well, not infrequently. And, and it is, unfortunately, a disease that has still a poor prognosis, and even in younger adults, five-year overall survival is only half or 50%, and it's worse in older patients, or AML that arise from previous chemotherapy, or AML that arises from MDS, and those with relapse or refractory disease really have dismal prognosis. And stem cell transplant can prolong survival, but even with that, um, it's, it's generally not curable. And hopefully, with the advancement of targeted novel therapies that are emerging nowadays for AML, we will see a better survival improvement for this disease.

Let's talk about AML classification. So this is what we discussed, um, about FAB classification. FAB is historical and it's developed back in the 1980s. Um, it's widely used and generally well understood, but a lot of distinctions have little prognostic significance, and it's mostly done on the basis of morphology and cytochemistry, and to a certain degree, flow as well. Um, so, um, uh, based on the FAB classification, AML is classified by asking two questions: Is it purely granulocytic versus other cell types like erythroid or megakaryocytic? And if it's purely granulocytic, how mature is it? So, um, this is, um, uh, the FAB classification of AML, M0 to M7. M0, minimally differentiated AML, and generally lacking [Music] cytochemical or flow cytometry evidence of myeloid maturation, like it's usually lacking MPO or myeloperoxidase. And then you also have M1, AML without maturation, if you have less than 10% of your, um, granulocytes being mature. And an M2, with maturation, if you have more than 10% granulocytes have maturation to the, uh, neutrophils. And then you have M3, which is your APML. M4 is myelomonocytic leukemia. M5, acute monocytic leukemia. M6 is your acute erythroid leukemia, and M7, acute megakaryocytic leukemia. So, um, now, anything that has myeloid morphology, our rods, or MPO positive can be M1, M2, or M3, right? Because we talk about M0 usually lacking MPO. So now, uh, the distinction of M1, two, or three is the key is really the morphologic maturity. So this is, um, this diagram is showing you the granulocytic maturation. M1, um, in general, you have very few or less than 10% of, um, mature granulocytes. And M2, you have more than 10% of mature cells of the granulocytes are mature cell components. And M3, usually they're arrested in the promyelocytic stage. So M5, this is what we, this is like an example of monoblast with abundant cytoplasm but very round nuclei. It can be monocytic as well. Myelomonocytic M4, I don't show you a picture here, but it's a mixture of myeloid blast and monocytic blasts. M6 is your erythroleukemia. So, um, you have classically, that's if you throw proerythroblasts that have abundant cytoplasmic vacuoles. In WHO, the current WHO classification, um, um, unless you have a pure erythroid leukemia, which is like more than 90% are erythroid blasts, they are now lumped together in the in the MDS category. So there has been a change in classification now. And M7 is your acute megakaryoblastic leukemia, and the megakaryoblasts classically have this, uh, cytoplasmic blebbing, almost like Mickey Mouse ears. So when you see blasts that look like this morphologically, you have to consider M7.

Okay, like I mentioned, now, uh, the classification of AML is mainly based on WHO, um, and it is more meaningful in terms of prognostication and therapy for clinicians. So, so under that category, we have AML with recurrent genetic abnormalities, AML with myelodysplasia-related changes, therapy-related myeloid neoplasms, usually arising after cytotoxic chemotherapy or radiation from another malignancy. For example, someone has breast cancer 10 years ago that's treated with chemo and radiation therapy, and then 10 years later, they develop AML, and that's associated with the therapeutic toxicity. So that's an example of that. Um, and then there's a sort of a garbage can category that's AML not otherwise specified. Um, and, and under that, then that's where FAB classification is useful because then that's, um, it's still, um, classified morphologically under AML and OS. Um, and, and myelosarcoma, where you have blast infiltration in other tissues other than the bone marrow. And then there's also myeloid proliferation associated with Down syndrome, which I deal with not infrequently at children's hospital. This Down syndrome kids are at higher risk of developing, uh, myeloid malignancies. Um, so with the advancement of genetic techniques and next-generation sequencing, it is now recognized that cytogenetics and molecular abnormalities like gene mutations play a very important role in AML prognosis. So, there are certain types of cytogenetic abnormality that, uh, that can help classify patients into favorable risk, intermediate risk, or poor risk category, and, and the clinicians will then decide their treatment based on this, uh, risk classification. For example, they will decide whether or not to transplant the patient if they're poor risk, as opposed to just a standard chemotherapy regimen if they're more favorable, uh, risk, for example. Um, so, uh, cytogenetic abnormalities such as inversion 16, translocation 8;21, 15;17, just APML. APML is a medical emergency, and, but it has a very effective therapy, uh, using retinoic acid. So it actually, when it responds to that treatment, it's actually, in general, favorable. But there are other, uh, cytogenetic abnormalities like, uh, 9;11, that's more intermediate, and poor risk, like complex karyotype, chromosomal 5 or 7 abnormalities, translocation 6;9, 9, 9;22, which is BCR-ABL. It's a new category in the current WHO classification. So these are all chromosomal abnormalities that constitute poor risk. But not only that, but now it's recognized that a certain genetic mutation can also affect this prognosis. So, for example, if you have favorable risk cytogenetics, but you have a KIT mutation on top of that, so you've got to be classified into intermediate risk category. So you're no longer favorable. And third, for a huge subset of AML, is actually they actually have normal cytogenetics. They don't have any of the cytogenetic abnormalities. And in this, um, um, in this subset, then if you have additional gene mutations like NPM1, they get reclassified into favorable risk categories. So they, they tend to do better than patients who do not have any of these mutations. And conversely, they have mutations in the genes that are recognized to have, um, um, adverse prognosis, like FLT3, TP53, RUNX1. So all these bad players, then they get reclassified into poor risk category, and they will be treated accordingly. So, um, this is, um, the category of AML with recurrent cytogenetic abnormalities, and that's why WHO will classify them this way. So, uh, here are a lot of the cytogenetic abnormalities, what we talk about, and their corresponding prognosis. And then AML with gene mutations also falls under there, and it depends, depends on which gene is mutated, they may have better or poor prognosis.

Let's talk about APML, because this is a special subset and is very, very important for for you guys to recognize this subset because it is a medical emergency. As Ali says, and, and why is because these patients are at higher risk of DIC, or disseminated intravascular coagulation, that can lead to, to bleeding, and I can have bleed in the head as well, which, and the patients can die pretty quickly. And, and it is the characteristic finding is translocation 15;17, or PML-RARA translocation. So this is a diagnostic, uh, test for this entity is using a cytogenetic technique or FISH for translocation 15;17. Um, and if, if it's diagnosed in time, there is a very effective therapy for this condition. It's called all-trans retinoic acid, and sometimes arsenic is used as well. So, so we have to diagnose this quickly, and we have to recognize this quickly, and let the clinicians know about this. So, uh, classically, uh, APML is characterized by abundance of promyelocytes. These are very granular looking immature cells, and they have abundant, uh, our rods as well, and they sometimes have they're stacking on each other, and there is an old term for that, it's called Fagot cells, because they they resemble, it's an old term for, uh, stacks of cigarettes, or stacks of, uh, wood sticks, or something like that, um, um, and that's where the Fagot cells get its name. And MPO is very, very positive in in APML or M3. Having said that, you also have to recognize that APML don't always look like that, right? So, so I told you, like the one that's usually, um, uh, classic with classic morphology, they're not that, that abundant in peripheral blood. You can only see probably two or three circulating in peripheral blood. You have to really look hard for these. And whenever you see cells that look like this, you have to raise that suspicion. Uh, but there are some subsets of APML where they're not as granulated, and they're not that many, uh, our rods as you see here, and it's, it's called hypogranular APML. Not as common, but I've seen it. And, so these cells actually had the clue, some of the morphological clues, that, uh, these cells have, they have this butterfly-shaped nuclei or dumbbell-shaped, whichever you want to use. So if you see like a cell that have like this dumbbell shape or butterfly shape, even though they're not as granular, you might have to be suspicious for APML or hypogranular APML as well. And so you can see that this is like overlapping, some of these nuclei by loop and overlapping with each other. Okay.

Any questions about APML so far?

Uh, we've got a couple of questions. Um, I think the main question really, there's a very interesting one about how you, um, do differential counts in them, in cases of acute myeloid leukemia, and I think this probably relates to the FAB classification. The, the comment and question are, uh, to differentiate AML without maturation with maturation, the cutoff blast percentage is 90%, and it's at what point do we start counting mature granulocytes in terms of differentiating the, um, the maturation pathway? Do we include promyelocytes or only just mature neutrophils, basophils, and eosinophils? Um, I don't know if that question makes sense or not.

I would say, yeah, no, I think I know what, what you're getting at. So when you're diffing a bone marrow, um, you usually, you separate, uh, so granulocytes, and you're diffing pro, and you're diffing myelocytes, and you're giving, um, mature neutrophils separately, right? And then you're giving blasts separately. So you have to list your blasts, um, first, and then, uh, get the percentage of the blasts, and then separately diff the other granulocytic cells. Um, and usually, if, if I see, um, more than 10% of your granulocytic cells are [Music] myelocytes onwards, so like myelocytes, metamyelocytes, neutrophils, band cells, that, that, that to me constitutes some maturation. So, so if I don't see any of those components at all, like less than 10% of those are mature neutrophils or bands or, you know, then, then I would be, I would likely call it, uh, without maturation. Does that make sense?

No, it does. Yeah. So it depends on the sort of context as well of what are the, if you've got a case of APML, they're all going to be promyelocytes, and then the category when M1 and M2 don't, doesn't really apply, right?

Yeah, definitely. Um, one, another interesting comment is that, and this, what you've highlighted is that hypergranular APML is difficult to diagnose morphologically, but if, what I always say is, if you think about it, treat the patient, and just get your PCR and or FISH, whatever you do, it's better on the side of caution.

Then yes, yes. So that's a really good point, Ali. So often our clinicians will treat the patient anyway, preemptively with ATRA. Say it comes in the middle of the night, and we don't have FISH until the next morning. They, uh, they put the patient on ATRA first until they get a FISH confirmation. Um, but it is very important as a hematopathologist or a bench technologist to raise the suspicion because then the clinician, if there is any, any possibility of APML at all based on morphology, they will start ATRA, right, until they get that FISH confirmation. Exactly. Yeah. Thank you. Yeah.

So having said that, uh, there is this entity, um, uh, AML with NPM1 mutation, and it's very interesting morphologically. It's recognized, and there are some case reports about this, where, um, the, uh, the blast morphology have this cup-like nuclei, like you have to use your imagination, but if you're looking at the blast from, uh, from the top, it looks like there's this indentation that resembles the lip of the cup, or it has like, uh, it is indentation, and they can sometimes look a little bit, um, uh, irregular as well. And, not only that, monocyte leukemia sometimes they can resemble this, right? So there are a few times when I'm looking at and blasts, and they have like very irregular looking nuclei. Are they monoblasts? Or are they hypogranular APML? This is another one that can be confused with hypogranular APML due to the blast morphology, but also especially the flow cytometry findings. So in APML, usually you have absence of CD34 and HLA-DR. That's classic, uh, findings in, um, in APML by flow cytometry. But AML with NPM1 mutation also has that. So, just something to keep in mind that there are some overlaps that may be confused, and you have to have FISH for confirmation to diagnose APML.

Okay, so we talk about blast, high NC ratio, not always. And a great example of, uh, case number two, which is acute monocytic leukemia. And this is another picture I took, um, from, from a child with acute monoblastic/monocytic leukemia, very poor prognosis. And this is a, hopefully, this diagram somewhat helpful for you to look at the mono, monocytic maturation. So monoblasts, they generally have more rounded nuclei, um, and more regular nuclei. Um, they can still have some vacuolation. It's very open chromatin. As they mature, the chromatin gets more condensed, and they get more convoluted in promonocytes and immature monocytes, and monocytes. Mature monocytes, they have, uh, very, very irregular looking nuclei, um, as you see here. All right. And then there's another entity that is interesting, inversion 16. And, and this is, I often put this as a, as a spot diagnosis on a resident exam. If you see this picture, what's your diagnosis? So you recognize this by this hallmark eosinophil precursor that shows dense basophilic granulation, or we call them actually harlequin cells, kind of resembles the costume of harlequin, and monocytic blasts as well. So when you see eosinophil precursors that look like this, you have to, uh, think about this, um, entity.

Okay. Uh, and for the interest of time, I won't go into details, but, uh, as we talked about, there are some gene mutations like NPM-1, or whereas when when it's mutated, the the patient do better than those who who have NPM1 unmutated in terms of survival. And CEBP alpha is another gene. If you have biallelic mutations of CEBP alpha, you tend to do better than those without or with only, uh, one allele mutated. And RUNX1, uh, is the reverse. So patients who have RUNX1 mutation, they tend to do poorly, compared to patients with wild-type RUNX1. So this is what, uh, we touched on, uh, AML with myelodysplasia-related changes, right? So, um, so the, how you diagnose this is a bit different than just diagnosing straightforward MDS. So, um, in MDS, the cutoff for, uh, dysplasia is more than 10% of your cell lines have to be dysplastic, uh, to call it significant. But in, uh, AML with MDS-related changes, because you, like I said, you can see a little bit of dysplasia here and there, always with leukemia, the threshold is much higher, right? So, to diagnose this, you need to have at least two, two lines, so erythroid, granulocytic, or megakaryocytic, being dysplastic, and it has to be more than 50% of the cell lines being dysplastic, greater than 10. So I have two cell lines with more than 50% of them being dysplastic morphologically to suspect this, or if you have prior history of MDS diagnosis, or if you have cytogenetic abnormality that is MDS-related, you can also diagnose this condition. But also, you have to exclude prior cytotoxic or radiation therapy, which will then qualify this as therapy-related AML instead, is a separate category. And if you have cytogenetic abnormalities that's listed in the other category of recurrent cytogenetic abnormalities, then that that trumps the diagnosis. So you have to instead classify that under the appropriate WHO classification rather than MRC, even though they might be dysplastic morphologically.

Okay, so, uh, yeah, and this is what we talked about, Down syndrome. Um, and newborns with Down syndrome often present with increased blasts, and usually they are megakaryoblastic, uh, uh, feature in the blood. This occurs in 10% of Down syndrome newborns and is associated with GATA1 mutation, which is a gene required for erythroid and megakaryocytic development. And there's a rule of three I teach my residents. So most cases spontaneously resolve within three months, but 30% of them can develop AML within three years. Um, and usually this, uh, leukemia associated with Down syndrome is better in prognosis if present at a younger age compared to a child with AML without Down syndrome. Okay. So this is a hallmark morphologically. You have, I usually have thrombocytopenia with large hypogranular platelets, as you see here, and also circulating blasts. And they don't always look like a classic blebby megakaryoblast. They can also look pretty bland, and some of the blasts can look like they have some cytoplasmic blebs as well. So this is an example of myeloid proliferation due to Down syndrome.

So, how do we diagnose AML? So in the old days, and I wonder if some sites in the world still use this technique, cytochemical stains. It's a column colorimetric reactions using enzymatic dye or and non-enzymatic dye that can tell you whether it's myeloid or lymphoid in origin. So this is, uh, myeloperoxidase, examples like, this is a brown granules and MPO positive myeloblast. And periodic acid-Schiff, where it's, uh, like if it's positive, that's more diagnosis of lymphoid blast. They have these red dots, that's so-called block PAS positivity in lymphoid blast. And monocytic blasts stained with non-specific esterase or NSE.

Okay. So we have to do flow cytometry to classify our myeloid or lymphoid blast appropriately. And, uh, for those who don't really have much basic and flow cytometry bases, in flow cytometry, basically, like you have an antigen on your cell surface, and you conjugate that with an antibody that's, uh, that has conjugated fluorochromes. That antibody will bind, uh, to the antigen of, uh, of interest, and it will, uh, emit fluorescence, right? And when the cells pass through the column one by one, uh, it will be interrogated by a laser, then that gives you information what cell, uh, surface antigen the cell expresses. And their combination of CD markers, uh, that we use to diagnose AML versus ALL. Examples, there's a ball. Um, so in AML, you're usually positive for a myeloid antigen such as CD117, CD13, CD33, CD64, MPO or myeloperoxidase. And in ALL, usually positive for CD19. So I highlight in red, the ones that are antigen, the antigens that are lineage specific. So if you see that, that's essentially, uh, myeloid. And if you're seeing CD19, that's, that's, uh, for sure B-cell.

Okay. And, and this is, this is what I find really nice. There's a really, it is a diagram from a really nice flow cytometry book that compares and contrasts different patterns of flow cytometry in AML. And I'm going to ask you, um, uh, what is A and what is B? Okay. So in A here, you have a blast that is, um, dim for CD45, but bright for CD64 and HLA-DR express, CD33 and CD15 negative, CD117 and negative, CD34. And in B panel here, you have a blast that is dim 45, but higher side scatter, CD64 positive, but HLA-DR negative, CD33 positive, CD117 bright are positive, but CD34 is also negative. So we talk about blasts that are CD34 negative and HLA-DR negative. Let's do B first. What's your diagnosis in B?

We haven't had any comments as us yet, RD. So, okay. Not sure whether it would be better just to go through it all with them.

Yeah, okay. Thank you for your interest of time. So this is actually a classic, uh, example of promyelocytic leukemia flow cytometry, lacking HLA-DR and CD34 expression. And the first one, although it's lacking CD34, which you can see that in acute monocytic leukemia, it is pretty bright for HLA-DR. So, and it's bright for CD64 as well. So that's really the hallmark. And CD117 is usually negative, uh, for, uh, in acute monocytic leukemia, and it's very positive in APML. So it's, uh, important to distinguish, uh, this entity morphologically and by flow cytometry.

Okay. Yeah, I think I just, uh, interjected and said that was a fantastic overview of AML, which we could probably spend weeks talking about. It's not just one disease, but it's so heterogeneous. Yes. I, I don't want to offend anyone who's interested in lymphoid disorders, but I think the diagnosis of AML is a lot more interesting than the, the opposite of acute lymphoblastic leukemia. Yes.

I think this is a good point to move on to case three, if that's okay with the audience.

Yes, excellent. So, yeah, uh, case three, which is not straightforward, I don't think, uh, is a 19-year-old female who's presenting with shortness of breath. Hemoglobin's 110, white count is increased, uh, 69.5, and she's, uh, slightly thrombocytopenic, 112 by the analyzer. Okay. As we cruise around, um, it's not, it's not easy to find a cell. So I'm not going to move it yet, but, uh, we, we see occasional, um, cells that resemble lymphoid cells, but they have this very, very basophilic cytoplasm with some cytoplasmic vacuolation, and a little bit of immature chromatin, open dispersed chromatin, perhaps some impression of nucleoli here. Um, so they're a bit larger, like medium in size, compared to these small mature lymphocytes around it. And let's see if we can find some more. It's really to highlight that some of these abnormal cells, uh, they don't always, uh, they're not always easy to find in peripheral blood. Sometimes you have to cruise around quite a bit. There is lagging a little bit. I'm not sure if it's my internet. Probably okay. Okay. This is, this might be another cell that's a very basophilic cytoplasm. Anybody would like to start taking a guess what, what this case might be heading to? And I don't expect you to nail the diagnosis right away from just morphology, but maybe some people can take a guess or differential diagnosis at least. It's a tough case, isn't it, out there?

I think it, it is always straightforward. Yes. And it's quite hard to find cells. We've got, um, some interesting, uh, suggestions that this could be multiple myeloma. This could be prolymphocytic leukemia.

Okay, okay. Oh, so this is another one. Maybe some people will change their mind after seeing this cell here. So there's a really nice cell again with cytoplasmic vacuolation, prominent nucleoli, and immature chromatin. We've got some interest. We've had some other comments now after looking at that cell, possibly lymphoid, Burkitt lymphoma or leukemia, lymphoblast as well.

Very good. And that's a very good differential diagnosis. So whenever you see cells like this, you have to think about Burkitt leukemia, especially with vacuoles. Um, but you cannot, you also cannot rule out lymphoblastic leukemia. So although Ali says, like, you know, AML is more heterogeneous, actually, working at a children's hospital, I, you know, there's no ALL that looks the same to me. Sometimes they can actually look very deceiving and very, um, they can have some vacuoles, they can resemble it, um, so, so definitely, uh, sorry, they can resemble like a mature, uh, aggressive, uh, lymphoma. That's what I meant. Um, so that's why we have to always keep the differential as well, especially in a, in a child or a teenager where that entity is more common. Okay. So this is actually a Burkitt lymphoma leukemia. So it is a highly aggressive mature B-cell lymphoma, that's that can be circulating in the blood. And it's characterized by these medium-sized cells with abundant, uh, vacuolation, basophilic cytoplasm. This is characterized by MYC translocation in chromosome 8 to immunoglobulin gene locus, that's driving, uh, this, uh, MYC is an oncogene that drives the proliferation of these malignant cells. So it's like in a tissue, sometimes you can see interspersed histiocytes that imparts a very starry sky appearance.

That's classically described on textbooks on tissues. So, uh, there are different classifications of Burkitt lymphoma: endemic, sporadic, or HIV-associated. So, endemic Burkitt's is usually, uh, uh, associated in kids in Africa or any, any, um, area where malaria is endemic. It's pretty interesting that it has some malaria association. Uh, but it's usually, um, 100% of that, uh, is also, uh, positive for EBV. Um, and often it involves jaws and other facial bones and other extranodal sites. Um, whereas sporadic Burkitt's, it can be anywhere in the world. Uh, more at risk for individuals in low socioeconomic status. Still happens in children and young adults. Less associated with EBV infection, and it's usually presenting with abdominal mass, usually in the ileocecal region. Um, whereas the facial and jaw involvement is more rare. Um, and there's also HIV-associated Burkitt lymphomas associated in endemic HIV areas and sometimes with immunosuppressive medication as well, and bone marrow involvement is more frequent in this case.

So, this, this is an example of this unfortunate child with market infiltration of Burkitt lymphoma in, in the jaw or facial tissues and abdominal mass, and sometimes can present with breast mass as well and orbital mass. Um, so this is a bone marrow aspirate and biopsy example in Burkitt. So, as I mentioned, um, in, in bone marrow, you can see this tangible body macrophages or histiocytes, um, that that ingest the apoptotic cells that that looks like their starry, starry sky appearance, um, like the background is the sky and these are the stars, if you use your imagination. Uh, and this Burkitt lymphoma, they're classically very strongly positive for CD20 and CD10 and MYC, and also another clue is that the Ki-67, which is the proliferation marker, is almost, um, always close to 100% in Burkitt, as opposed to other high-grade, uh, B-cell lymphomas where the Ki-67 may be less than that, usually 60 to 70% or so. In Burkitt, it's close to 100%.

And, um, just quickly, I just want to highlight that the immunophenotype in Burkitt is very important to distinguish that from ALL. And there are some, uh, clues that we can use here. So, in Burkitt lymphoma, the, the, they're, they're mature lymphocytes, right? So they should be very close to CD45 positive, um, where the lymphocytes usually sit. So the CD45 should be, um, brighter than blast, for example, where the CD45 is often quite dim and often negative, sitting more in this region. So that's one clue. And, um, they both can be CD19 positive, and they, ALL is less commonly CD20 positive, but can be, right? In Burkitt, it's always positive CD19 and CD20. Um, both ALL and Burkitt can be CD10 positive as well. So CD19, CD10, um, and they're negative for T-cell markers and negative for CD5, uh, which distinguishes that from CD5 positive, um, lymphomas. But in Burkitt, you have light chain restrictions. So in this case, it's, um, clonal for kappa, okay? Whereas in ALL, uh, uh, usually you don't have any light chain expressions, so it should be negative for both because they're immature blasts, they don't have, like, they don't express light chains yet. Okay?

And, um, in Burkitt, you have t(8;14) rearrangement, but can also, more rarely, have a rearrangement of the MYC gene to the light chain, so like kappa in chromosome 2 or lambda in chromosome 22. No translocation involving BCL6 or BCL2. But 10% of Burkitt can also lack MYC rearrangement, which can be due to either sensitivity of techniques or other mechanisms leading to MYC dysregulation. Or more recently, in the new WHO update, it's found to have chromosome 11q aberration. In Burkitt, that's like MYC rearrangement. So you have to examine that as well. So, yeah, it's highly aggressive but potentially curable with intensive chemotherapy that involves, uh, sometimes with taxanes, um, and combined with CNS-directed therapy, and it can have long-term survival in the majority of cases. Okay? So sorry that I'm a bit over time, but, um, I hope you enjoyed the lecture and you find them useful. And if you have any questions that you don't have a chance to ask here, feel free to shoot me an email. Thank you.

I do. That was absolutely fantastic. We've gone through a whole range of different things, and it's all really important, uh, information. These cases are all look very scary, but, um, I don't know if you have any practical advice for for anyone who's new to looking at blood films and seeing these kind of cases? How would you sort of approach it, um, on, in, in the real world? You know, getting other colleagues involved and, you know, the clinicians, all these kind of things, because, you know, you've highlighted beautifully that these are all medical emergencies. Some have good prognosis, treat early, some, you know, not necessarily. Yeah, not sure if you've got any practical, uh, tips in terms of how to deal with it in the real world. Yeah.

And also, absolutely. So we often rely on the bench techs who are, uh, often the first, really the first line, um, [Music] like to recognize these abnormal cells when they're diffing or examining a peripheral blood, right? And so sometimes a machine flags it, and then it goes to the bench tech to screen. And if you recognize any suspicious cells, um, like, you know, and if they're rare, don't be afraid of making more slides, right? And examine more slides. And if you see them more and more, uh, bring it to your attention to your human hematopathologist, first, right? So, like, path will then come and assess the slide, and we determine whether we should worry about a particular cell or not. Um, and then, um, and then after that, then we will call the clinician saying that there's abnormal circulating cells in association with cytopenia, that's even more worrisome, and maybe this patient needs bone marrow, right? So, um, so I think, uh, really, like, seeing, really practicing your morphology, seeing more and more of these cases, be aware of, um, any morphological mimic, um, that we discussed, um, not just like brushing it off as like normal cells, um, I think will be really helpful. And always bring it to your attention of your path, and, um, and we'll talk to the clinician, uh, to correlate it with clinically further.

Excellent. Thank you. Thank you, Audrey, again for, for such an absolute, uh, delightful and really informative session today. Um, we unfortunately, this is our last webinar. We, the past few weeks have actually flown by, and, um, I personally have really enjoyed, uh, hosting all the webinars, and we've had some absolutely fantastic speakers, uh, throughout our time. Again, I'd like to thank Audrey for her participation today, all the speakers during the webinar series. I'd like to thank Urban Mannheim for for sponsoring the series as well. Most importantly, everyone who's been involved as well for really engaging and fascinating discussions about some really interesting cases. Thank you very much.