Transcription
Foreign. [Music] I'm a consultant hematologist. I work in an RM Bevin University Health Board in the UK, in South Wales, and I'm delighted to be your host and presenter for this Blood Academy webinar, where we'll be looking at the historical aspects of acute myeloid leukemia, or AML classification. So, really taking a deep dive into the past classifications, uh, presence, and the potential changes in the future as well. So, welcome. Um, before we do start the webinar, I'm just to remind you about Blood Academy, who we are and what we do. So, we're an online educational platform for a whole range of different people: hematologists, pathologists, and scientists. We run various different courses. Uh, we help people in terms of preparing for various, uh, postgraduate exams, including the Royal College of Pathology exams in the UK. We run a monthly interactive morphology cases, and we have a whole range of other things going on, including a presence on various different social media platforms as well. Uh, we also have resources for institutions, especially laboratory scientists, looking for for training material and resources in blood cell morphology. And as part of that, we're currently running a 50% discount for our lab package. So that expires on the end of this month, 31st of October. So, if you're interested, then please contact us at the email provided here: admin@bloodacademy.com. We've also shared a link to our contact page on the chat. So, if you are interested in in helping your laboratory staff train in terms of blood cell morphology, then please contact us via that link. Excellent. So, let's make a start in terms of this webinar and just want to highlight the main objectives and discussion points that we'll be covering today. So, the, the first thing is really to get in our mind clear what, uh, the morphological definitions of what a blast cell is. Then understand some of the historical aspects related to acute leukemia, and really going into the, the history 200 years ago, if not longer. And then we'll spend some time looking at the, uh, the WHO classification that was published in 2016, uh, covering acute myeloid leukemia, and just highlighting some of the limitations that this revised edition had. Then we look at the new classification. So, that, the most pertinent ones are The WHO and the ICC classifications, and what has changed more recently. And then thinking about what could happen in the next five, ten years, if not longer, with future AML classifications.
So, let's start back in the 1800s, and we'll start with a chap called Alfred Donny, who is a French, uh, physician. He was based in Paris, and he really was the, the father of of, uh, being able to take pictures down the microscope. Uh, he was, uh, also famous for various other things, including, uh, highlighting various different bacteria. But he really started the process in terms of thinking where leukemia actually developed from. And this really spanned from an interesting case that he came across. So, it was a particular case of a 50, a 44-year-old female who presented with splenomegaly and a very raised white cell count. And obviously, they didn't have automated analyzers at the time. Uh, but you can tell from this centrifuge sample here, this, the buffy coat is massively enlarged in comparison to the plasma and the, uh, the red cells at the bottom. But certainly, there was evidence there that the patient had a significant leukocytosis. And unfortunately, due to the limitations of their diagnostics and their therapy, she died, uh, soon after presenting. Now, what the initial thought was, was that patients, uh, with leukemia, um, were essentially, uh, as a result of a proliferation of cells, uh, thought to be due to infection. Because the increase in white cell count in patients with infection, significant inflammatory disease, looks very similar in some cases to patients with leukemia. And they called white cells, white globules. And you can see in this patient, in in A here, that there are a number of of white cells here. So, these are the cells, not the dark ones, but the, uh, the lighter cells, which are significantly increased in number. And these were similar in appearance, as I've mentioned, to patients with active infection, or if you took an aspiration from an area of body that was infected, from pus, then you could also see a number of white cells as well. Uh, he exposed them to acetic acid, and what that did was, um, give them the, the ability to visualize the nuclei better. And what you could see here was that in this, in F, after treatment with acetic acid, there are a number of white cells, number of nucleated cells, and there are various different differentiation steps. So, you've got some larger cells, some slightly smaller cells, you've got some cells which have a slightly, slight indentation to the nuclei. So, this mixture of cells with the splenomegaly and the raised white cell count, we would now probably realize that this is a patient in chronic phase, chronic myeloid leukemia. But he postulated, and really moved away from this idea that this was all due to infection, that Luke, the process that was going on was due to a maturation arrest of the white cells. So, he wasn't the only one in terms of, um, developing different theories about leukemia, and there were a number of different people involved. Um, so, Donny, who I've already mentioned, uh, really allowed us to think about, uh, the maturation arrest, the differentiation that happens, uh, in patients with acute leukemia and also chronic leukemia, but also be giving us the ability to visualize them and record that. And then we had other people as well, such as Virchow, who made the distinction between splenic and lymphatic leukemia. And if you're interested in the, uh, some of the history regarding, uh, various different leukemias, then this is a very good, uh, very good paper to to read.
So, let's go back to basics. Um, we all know that our blood and bone marrow cells are derived from a common hematopoietic stem cell, which resides in the bone marrow. And we've got our two main types of cells: myeloid cells and lymphoid cells. Where myeloid cells will eventually, uh, differentiate into megakaryocytes and platelets, all the way down to our red cells, monocytes, and our granulocytes, namely eosinophils, basophils, and neutrophils. A monocyte can also terminally differentiate into dendritic cells and macrophages. And then we have our lymphoid cells, which differentiates into T, B, and natural killer cells. And the B cells will terminally differentiate into plasma cells, producing our immunoglobulins, as well as memory B cells.
So, the first thing to think about is what is a blast? What is a, what are these cells? This term which is commonly used. So, generally, acute leukemia is an uncontrolled malignant proliferation of hematic, hematopoietic precursors. And this is what we often described as clonal. They share some sort of, uh, genetic abnormality and are relatively homogeneous, uh, in terms of appearance. Um, interestingly, the term blast is rarely used outside the world of hematopathology. Um, that there are some diseases which have an association with the term blast, such as neuroblastoma, a nephroblastoma. But generally speaking, blast cells, the term, um, is reserved for the, the hematologists and people who work around them to define the blast. It's always important to understand what happens, uh, during normal maturation of cells. So, uh, blast cells are essentially very immature cells, and they are normally found at low numbers, and they will mature into to more mature cells. And during that maturation process, there are various different processes that occur to that cell. So, we have a reduction in the cell size. There's a loss of nucleoli, so these pale centers found within the nucleus. There's a reduction in the amount of nucleus to cytoplasm. The chromatin of the nucleus becomes more condensed, it becomes darker, and we, in in some cells, such as the red cell, the nucleus is actually lost. Um, in some other cells, particularly the monocytes or the granulocytes, there's a change in the nuclear shape. So, the polymorphonuclear cells, um, they will develop multiple lobes to the nuclei. And in some cells, there may be a change with the maturation process in the color, the, um, and the appearance of various different cytoplasmic granules.
So, based on those definitions, we can define the general characteristics of blast cells based on these appearances. So, generally speaking, a blast cell will be large, and if you use the red cell as your reference, they're often two to six times the size of a mature red cell. They usually have these immature clumps of of genetic material, nucleoli, as we've highlighted just here. Uh, there's a very high nuclear cytoplasmic ratio. There's not that much cytoplasm in most of these cells, and the chromatin pattern to the nucleus, uh, is often described as open, delicate, and diffuse. They're not dark and pyknotic as mature cells are. And in a lot of cases, blast cells, if you take them into account with the other cells that you see, they're often homogeneously appearing. They all look the same. Um, based on the expression of intracellular and cellular proteins and markers, you can distinguish blast cells based on the lineage going back to the, uh, the hematopoietic stem cell and the myeloid and lymphoid compartments that we see during differentiation. So, we could potentially have a myeloid blast, we could have a lymphoid blast, and then we have something in between where you may have a blast cell which expresses either both myeloid and lymphoid associated antigens, or there may be a blast cell which doesn't express either of them. And we would generally cast these, uh, as acute leukemias of ambiguous lineage. So, we could be confident based on these definitions that the cell to, to my left here is certainly a blast. It's large in comparison to the background red cells. It's, doesn't have much in terms of cytoplasm. The nuclear appearance is very open. It's not dark and pyknotic, and there are some nucleoli within the nucleus. Uh, but there are, and there are some cells which are clearly different. And this is a pre-erythroblast, the patient with acute parvovirus infection, very large, immature erythroid cell with these very distinct nucleoli. And then here we have a, um, we have a, uh, a dysplastic nucleated red cell. We've got very dark, pyknotic nucleus, and then we've got the cytoplasm, uh, of, uh, of a cell between a reticulocyte and a, um, a reticular site as well. So, these are relatively easy to say, well, this is a blast. This may be related to something else, could be a blast in terms of erythroid leukemia, and this is a dysplastic erythroid cell. Uh, but then, they don't always fit the textbook definition. So, here we have a case of acute erythroid leukemia, uh, but they can be easily just, um, easily, uh, recognized as megakaryoblasts, based on this very distinct blebbing around the cytoplasm. Some of the cells also show some distinct vacuolation as well, and we've also have the, the blebbing, the cytoplasmic fragments, and some dysplastic features as well. So, this certainly doesn't always fit the textbook definition of what a blast cell is. And then to make things even more confusing, when you have patients with malignant monocytic disease, what do we call a blast? Do we call this cell a blast? Is this a dysplastic monocyte? Is this a pro-monocyte? You know, it's not always that straightforward. You can see in the background, there are some dysplastic mature neutrophils as well. So, things in, in real life don't always come up with the, uh, the textbook definitions. So, this just gives us an idea of the different morphologies that we can see based on where that maturation arrest occurs. So, if we're, we're concentrating in this, in this talk, purely on the myeloid lineage, um, but depending on where that maturation arrest occurs and that proliferation of those malignant cells, you can get different cells with regards to morphology. So, very early on, you'll get this typical blast cell of a myeloblast, although that's pretty much impossible to distinguish between a lymphoblast without flow cytometry. And then later on, during the maturation process, you can have things such as pro-myeloblasts with these heavily granulated cells within the cytoplasm and multiple Auer rods, monoblasts, erythroblasts, basophil blasts, megakaryoblasts as well. Eosinophil, eosinophilic blasts are not generally recognized by the most famous classifications. It's, you usually, if you have a proliferation of the eosinophilic cells, it tends to be a chronic eosinophilic leukemia. But you can see, based on this diagram here, that you have various different morphologies associated with these cells based on their lineage.
So, why are classifications important? Well, they're very important because it allows us to standardize various different, uh, different aspects of the diagnostic process. Um, if we diagnose a patient in the UK or in the US, then really, we need to have some sort of standardized process to ensure that the therapy that is delivered to that patient is effective for that disease. Classification is also important in terms of the fact that they should be dynamic. So, with time, more and more information is gained through research, and incorporating that information is really important to help in terms of the diagnostic process, but more importantly, to provide the right treatment for the patient.
So, I'm going to spend some time just looking at the, uh, the historical classification of acute myeloid leukemia, concentrating on the FAB classification. So, that's the French American British classification, and we'll just look at the various different time points, uh, from 1976 all the way up to the 1990s, and see how, uh, the recognition of new data and new information, uh, really changed in terms of, uh, building the foundations for classifications like The WHO and ICC. So, right at the beginning, 1976, um, we didn't have a huge amount of diagnostic tools with acute leukemia. There's a, there was a heavy reliance on morphology and cytochemistry. And importantly, and as we'll realize later, that the blast percentage, uh, that was given in terms of the cutoff at that point to define acute leukemias, 30% or more, just within the bone marrow. The FAB classification recognized two main types of blasts based on their morphology. They had type 1 blast, which were generally agranular, and then they had type 2 blast, which recognized that some blasts could have a few primary or azurophilic granules. They had a slightly lower nuclear cytoplasmic ratio. There's slightly more cytoplasm with these cells, and they may also contain Auer rods. So, these are primary or azurophilic granules stacked on top of each other to give a rod shape, or what we also call pseudo, uh, Auer inclusions. So, you could have, uh, clumps of primary granules stacked together to give these very, very coarse, large granules seen in patients with the Auer-rod syndrome. There was a type 3 blast, which was recognized later on, and these gave the definition of having 20 or more azurophilic granules. How easy it was to count those number of granules, get using this, this picture as an example, probably has a very high variation in terms of the, the subjective nature of the counting process. And then, just for a completeness, they also recognized lymphoblasts as three groups. So, L1, and the patients generally who tend to be pediatric patients. So, these were blasts which were small to medium in size. They had scanty cytoplasm and didn't have much in terms of nucleoli present within the nucleus, and there were some chromatin condensation, so some darkness associated with the nucleus. And then in slightly older patients, they were often lymphoblasts were described as L2, where they were slightly larger, they had more of a diffuse chromatin pattern, prominent nucleoli, more abundant cytoplasm. And then they also recognized patients with an L3 phenotype, which would now often be recognized as patients with, uh, lymphoma or leukemia with these very prominent cytoplasmic vacuoles as well. And originally, the FAB classification recognized, uh, M1 all the way to M6 based on the morphology. And with time, M0 and M7 were introduced into the classification, and we'll cover that later on. So, as I mentioned, there was a heavier reliance on morphology, but also cytochemistry, and then later on, the ultrastructural examination of these blast cells. And their cutoff with regards to cytochemistry was 3% or more of the blast cells had to be positive, generally for MPO or monoperoxidase or Sudan black. It was thought to be that Sudan black is was more sensitive than MPO for detecting myeloid differentiation. But other cytochemical stains, various different esterases, toluidine blue, were later introduced as markers of myeloid versus lymphoid differentiation. Lymphoblasts, generally at the beginning of the, the classification process, um, they were recognized as they were, they lacked any staining for these key cytochemical stains. And MDS, homologous syndrome, was not recognized as a term. Rather, there was a recognition of this other term called myelodysplastic syndromes.
So, let's move forward to 1980. Um, as well as the standard M3, acute promonocytic leukemia, there was a recognition that a hypergranular variant also existed, and this was generally associated with patients with a high white cell count. So, we've got some typical morphological features of hypergranular APML or M3 variant. You have these bilobed nuclei, but with an absence of any granules or Auer rods within the cytoplasm. Then, in 1982, there was the formal recognition of myelodysplastic syndrome within the FAB classification, and they based that based on, uh, the lineages that were affected: the red cells, the granulocytes, and more importantly, the percentage of blasts in the, in the blood and bone marrow. And again, the cutoff remained for acute myeloid leukemia at 30%. Um, so, we're starting to, to recognize that there were other conditions that may predispose to transformation to acute myeloid leukemia, a so-called precursor state to AML. Then, M7 was introduced. So, this is acute megakaryoblastic leukemia, and they defined that as a percentage of megakaryoblasts of more than 30% or in the bone marrow. And this was recognized to be often associated with bone marrow fibrosis, and hence, because of the inability and difficulty, uh, to obtain an adequate bone marrow aspirate, there was a heavy reliance on bone marrow biopsy evaluation. Then, in 1985, the definitions were changed slightly with regards to erythroid predominance, and so that was 50% of erythroblasts of the total nucleated cells, and at least 30% of non-erythroid cells which were blasts. But more importantly, there was the increasing recognition of the importance of cytogenetics, and they could tell that based on the morphology, you could predict certain cytogenetic changes. So, M2 was often associated with translocation chromosome 8;21. The acute promonocytic leukemias, M3 and M3 variants, were translocation 15;17. And M4Eo, which is acute monoblastic leukemia with the eosinophilia, was recognized to be associated with inversion of chromosome 16. So, we're starting to, to gather more information and correlation between morphology and genetic changes in these diseases. Then, in 1991, M0 was introduced. So, that again, there was an increasing recognition that there were other stains available, and also flow cytometry was starting to be used more and more in the diagnostic process. So, M0, based on the morphology, they were minimally differentiated, but they could also express other cytochemical stains or show expression using flow cytometry of various different antigens such as CD13.
So, that, that's the, the background in terms of the FAB classification. What happened after that was The WHO came in and really, uh, took over the, the diagnostic, uh, classification with regards to the various different, uh, things that could be used in making this diagnosis. They really stressed the importance of morphology, cytogenetics using G-band karyotyping, uh, and FISH, as well as immunophenotyping using flow cytometry and immunohistochemistry, as well as molecular genetics, looking at the specific genetic abnormalities using techniques like polymerase chain reaction, more recently, next-generation sequencing. And they recognized that these key diagnostic processes had to be used together in order to produce an integrated diagnostic report for the patient. So, The WHO has produced various different editions with regards to, uh, uh, hematopoietic malignancies. Uh, the, uh, the fourth edition was revised in 2016-2017. The book was actually published in 2017, uh, with a precursor, uh, paper that was published the year before. And more recently, we've got the fifth edition, which is available, uh, in beta version online, as well as a recent publication found in Leukemia, which we'll look at later. What The WHO really stressed was the importance of classifying blast cells based on the lineage. So, to do that, the most sensitive technique, if you have an adequate sample, is flow cytometry that we have at present. You could also use immunohistochemistry and cytochemical stains. But by the fact that we could enumerate and and analyze a large number of cells using flow cytometry, that really has revolutionized the ability to, uh, to provide the information regarding lineage. Uh, by morphology alone, we can't really tell whether a blast is myeloid or lymphoid or of ambiguous lineage, unless we have the presence of Auer rods. Auer rods are pathognomonic for patients with, uh, myeloid leukemia, acute myeloid leukemia. They can be seen in very rare instances in patients with lymphoid and plasma cell disorders, but generally speaking, if you see Auer rods, then we're dealing with a, a myeloid malignancy. Outside of that, it's very, very difficult, if not impossible, uh, to specify lineage based on morphology alone. So, The WHO defined myeloid lineage by, uh, saying that you had to have expression of various different stains, in particular MPO, or if you had monocytic differentiation, two or more of these monocytic-related antigens, so CD11c, etc. T lineage with strong expression of cytoplasmic CD3 or surface CD3, and B lineage, you had to have strong expression of CD19 with at least one of the following: strong expression of CD79a, CD22, or CD10. You could have some diseases, so that the classical thing that you may get is, uh, translocation 8;21, where the blast cells may also aberrantly express CD19. Uh, but that has to be differentiated from mixed lineage leukemia. So, what, what did The WHO change with regards to the previous classification by the FAB? Well, the first thing to think about is the blast threshold was reduced to 20%, and that included not just the bone marrow, but also the blood. The other thing that they introduced was the increasing recognition of various different cytogenetic abnormalities, especially in favorable risk acute myeloid leukemia. And when we say favorable risk, we generally say that these patients can be treated by chemotherapy alone, as opposed to having consolidation therapy with an allogeneic stem cell transplant. So, let's have a look at some of the data behind this. So, The WHO recognized the importance of clinical factors. This is data from the UK NCRI trials from a number of years ago, but essentially, what it shows is that if you had intermediate or adverse risk disease, if you had therapy-related acute myeloid leukemia, so if you were exposed to previous radiotherapy or cytotoxic chemotherapy, or your disease arose from MDS or a myeloid proliferative neoplasm, you did worse than those patients who didn't have therapy or secondary related disease. You can see here for intermediate, the dashed line, the two dashed lines represent therapy-related and secondary acute myeloid leukemia. You can see that the survival is pushed down, indicating a poor prognosis. And the same with adverse disease. Again, if you had therapy-related disease or that arising from MDS or an MPN, you did worse. It didn't make that much of a difference for favorable, favorable risk disease, uh, so diseases such as acute promyelocytic leukemia, inversion 16, etc. But certainly, clinical factors are important to take into account. Again, this is really highlighting from the NCRI group data that, uh, genetic abnormalities, uh, play a significant role in terms of defining prognosis. So, we can see that the favorable risk AML, so that the top three lines, have the most favorable prognosis. So, we've got patients with acute promyelocytic leukemia in black, the top line, translocation 8;21, and inversion 16. And then there are some diseases which correlate to very, very poor prognosis, such as inversion 3, in the blue line right at the bottom here. So, again, we're The WHO is taking on the information of, uh, of the genetic changes that can impact patient's prognosis. They also changed the definition of some tricky cells of monocytic lineage, and they recognized that pro-monocytes are blast equivalents, and they defined pro-monocytes. So, those cells having a delicately convoluted or folded nucleus, they may have some dispersed open chromatin, small and indistinct or absent nucleoli, and some finely granulated cytoplasm. Pro-monocytes are important to distinguish from mature monocytes, and that, in my personal opinion, the best thing to distinguish them is that indentation or convolution of the nucleus, possibly the presence of the nucleoli, and the open nature of the chromatin as well. So, The WHO is trying to make our life a little bit easier in terms of defining what a blast is, with regards to conditions such as chronic myelomonocytic leukemia and monoblastic leukemia as well. One of the other things that they changed, so I've just listed a whole range of other things, so myelodysplasia associated with Down syndrome, and this other entity called blastic plasmacytoid dendritic cell neoplasm, were pushed away into separate and specific categories. CMML was recognized as an overlap syndrome between MDS and MPN, and hence taken out of the MDS category. There were a recognition of various MDS-related abnormalities, and there was an increasing recognition of various somatic mutations which are related to the various different types of acute myeloid leukemia: NPM1, core-binding factor, BCR-ABL1, which is from the Philadelphia chromosome, and SF3B1, especially in myelodysplastic syndrome and its association with ring sideroblasts.
So, I'm going to just ask the question now as to whether our diagnostic tools at the moment are adequate enough, and we're going to look at various different, uh, limitations with regards to our diagnostic tools. And the first thing really is looking at morphology. And morphology is very, is is very subjective. If you gave, uh, 10 borderline cases, uh, to 10 expert hematopathologists, you're likely to get 10 different answers. But the, the art of morphology is very subjective. What you define as a blast may not be defined as a blast by someone else, based on the experience and their level of training as well. You can also have, uh, the scenario where you're unable to aspirate a bone marrow sample, so the cytology is unavailable, and defining, uh, blast cell by, uh, the bone marrow biopsy can be very, very difficult, especially in patients with fibrotic disease. This is especially so in patients with more advanced conditions such as myelofibrosis. Then you have this issue in terms of flow cytometry. And if I give you these examples here, what you define as a blast, uh, is very important and can vary quite significantly based on the different diseases. So, for people who are not, uh, that familiar with flow cytometry plots, to define where a blast generally is, we use a combination of two parameters: CD45, which is a pan-hematopoietic marker, and then side scatter, which is a marker of granularity of different cells. And here, we would all agree that this is a blast cell, and these cells are agranular, so they'd fall down here in terms of the side scatter plot, but they weakly express CD45, and they're often found here. Now, generally speaking, we would say the blast gate, in inverted commas, would be in this area. However, that can change depending on your disease. So, for monocytic disease, the expression of CD45 is slightly more, so the blast gate would be here. For acute promyelocytic leukemia, because they're heavily granulated, uh, heavily granulated, the side scatter will be increased, and hence they'll be pushed up even further. And then you have erythroid leukemia, which may not actually express any CD45 at all. So, the blast gate, and where you draw those lines and these circles, is a moving target and open to some interpretation in correlation with the morphology. Uh, the other problem may be that you may get introduced human dilution. So, the percentage which is enumerated by morphology can vary quite significantly with the flow cytometry, and also you can have different subclones of blasts as well. Some clones may express a certain phenotypic pattern, whilst other clones may express other ones. So, if you have two or three clones, then which one are we following? Is it the predominant one? Is it the, uh, all of them together? It's not that easy to define in some cases. Immunohistochemistry is very limited because we only have a, a very small range of different, uh, immunohistochemistry stains that we can use to identify blasts. Generally, we would use CD117 and CD34 to highlight blasts, but these may not always be expressed in in certain diseases and blast populations. So, having an absence of these immune, immunohistochemistry markers doesn't always mean that we, these cells are not blasts. And then we have to distinguish the blast cells, which can be increased in, in some conditions as well. So, they can be increased in reactive states. They can be increased in number after exposure to various different growth factors, in particular granulocyte colony stimulating factor, or G-CSF. And then you've got hematogones, which can be seen in young patients, in patients after allogeneic stem cell transplantation. That by morphology can be quite difficult to distinguish between malignant cells and hematogones. These immature B cell precursors, flow cytometry can help quite significantly to distinguish them, but by morphology alone, they can be difficult to distinguish.
So, then, the, the WHO pertinent to the more recent edition of The WHO and the ICC really questioned the, uh, the importance of the 20% cutoff, in terms of defining what acute myeloid leukemia is. So, the things that we should really consider is the, the limitations of both morphology, and also the increasing understanding that genetics have a significant role to play in terms of defining tumor burden and the amount of disease that's actually there, as opposed to morphology. So, let's have a look at some of the data behind that. So, the first thing to highlight in terms of the more, the, uh, the morphology is that the agreement between expert hematopathologists when you have an indeterminate percentage of blast cells, so that percentage of between 5 and 9%, is is generally very, very poor. And what, uh, this study actually showed, um, was that if you took a range of a group of hematopathologists, and you asked them to examine blasts and enumerate those blasts in a bone marrow sample, and what they did was they highlighted a certain feature, which was something something called the generalized Kappa statistic. And this is what this number relates to. So, if you have a number which is one, you had complete agreement amongst the group. If you had zero, then there was no agreement at all. So, the higher the number, the higher the, um, the agreement between all the different observers. And what you can see was that if your blast percentage was low, so 2% or low, or if your blast percentage was high, so more than 10%, 10 or more, then that Kappa, the Kappa statistic was generally higher. Whilst if it was into, it was, uh, intermediate in terms of the number, so, uh, greater than 2% to less than 5%, or between 5 and 9%, that correlation was significantly lower. So, that tells us that generally speaking, if you have a low blast percentage or a blast percentage of more than 10%, most people, assuming that we could report to these expert standards, we could agree, uh, whether there is or there isn't any significant disease. In between that, it's very difficult, even even amongst expert hematopathologists, to agree on that percentage. The other thing to consider is, is the, uh, the number of blasts that we see down the microscope, or even by enumerating by flow cytometry, um, does that equate to the actual tumor burden within the patients? And this data suggests not. What it did was take a small, uh, group of patients, there was only eight patients that were analyzed, but they looked at the tumor burden by morphology of patients with MDS, and after they progressed to, uh, to acute myeloid leukemia, i.e., develop a blast percentage of more than of 20% or more. And you can see that the tumor burden by sequencing, by genetic profiling, using, uh, next-generation sequencing, by looking at the variant allele frequency of their mutational profile, was pretty much the same before transformation to after transformation. So, this data suggests that the blast percentage is not a good, uh, it's not a good indicator of tumor burden, and that we should be using other techniques, in particular, next-generation sequencing, to potentially predict that change in phenotype from MDS, high-risk MDS, to, uh, to secondary myeloid leukemia. The other thing to, to highlight was, what about clinical outcomes in patients, if you compared secondary acute myeloid leukemia to patients with, uh, myelodysplastic syndrome with excess blasts 2, so a blast percentage of between 10 and 19%. And what this, this group did from Seattle was treat patients using intensive chemotherapy, which is the standard treatment that we would give for patients with acute leukemia, both to acute myeloid leukemia and patients with a blast percentage of between 10 and 19%. And you can see here that overall, the outcomes were slightly different. So, MDS excess blasts 2 in red, and, uh, acute myeloid leukemia, more 20% or more blasts, overall were different statistically. But if you took into account various different other patient factors, including the patient's age, the performance status, the cytogenetics, etc., and whether they received a, a hematopoietic stem cell transplant, there was no difference in terms of outcomes between the two groups. So, as well as saying that from a mutational profile, there's no difference, this retrospective data, and I need to highlight that this is retrospective data, it's not a formal trial that was conducted, suggests that there's there is no significant difference between how patients are treated with intensive chemotherapy based on their blast percentage. Then, there's the recognition that certain other genetic abnormalities are important, and in particular, mutations of TP53. So, this data essentially shows that patients with TP53 mutation, irrespective of your blast percentage, provided it's more than 10%, unfortunately have very, very poor outcomes. So, we can see in patients with the TP53 mutation in red, with acute myeloid leukemia, so blast percentage of 20% or more, or excess blasts, so more than 10%, do equally as bad in terms of prognosis and survival, whilst those patients who do not have the TP53 mutation do significantly better.
There is the, um, the recognition here from this data set, um, that it's not just the TP53 mutation which is important, but also whether the patient also harbors a complex or non-complex karyotype. So, patients overall with a non-complex karyotype with the TP53 mutation, uh, did better in comparison to those patients with a complex karyotype. The outcomes overall were still unfortunately very poor, but there's a suggestion that patients with a non-complex karyotype with a TP53 mutation who underwent an allogeneic stem cell transplant still derive some benefit from that procedure in comparison to those patients with a complex karyotype who unfortunately, uh, can, uh, did did very poorly.
So, that brings us on to the current day. And if you haven't heard of The WHO Fifth Edition and the, uh, the International Consensus Classification, then I would certainly suggest that you put these terms into Google and, uh, get the, uh, these three papers which have recently been published. They have caused a lot of, uh, problems for various different people in terms of which classification we should be, uh, using in in routine practice. My personal opinion is that The WHO continues to be the, the gold standard, and I'll try and highlight some of the, the advantages with The WHO in comparison to the ICC. So, this is a very complex slide, but this just highlights, uh, the current, uh, diagnostic criterion subclassification of, uh, of acute myeloid leukemia based on The WHO Fifth Edition. And what, what they essentially introduced was, uh, various different other, uh, genetic mutations, somatic mutations, which could define acute myeloid leukemia, irrespective of the blast percentage. This includes things such as NPM1, which is a really important mutation, as that's seen in a significant percentage of patients with acute myeloid leukemia. We recognize secondary myeloid malignancies, uh, and also had an increasing awareness of of defining somatic mutations for patients with acute myeloid leukemia, myelodysplasia-related. They highlighted the importance of the four key facets that I've noted here: morphology, previous conditions such as MDS and MPN, whether the patients had previous cytotoxic therapy, and genetic changes as well. But they can, they continue to recognize that acute myeloid leukemia could still be differentiated by morphology alone, which I think is still important in places where unfortunately advanced techniques, especially picking up somatic mutations, and even more detailed flow cytometry is unavailable. So, that, that's a key distinguisher with the ICC. So, I'm just going to highlight the key things between the two classifications, and, um, I mean, there's a whole range of other things, but at least just go over some of the, the most important things. So, the first thing really is the blast thresholds in the absence of genetically defined AML. So, those patients with PML-RARα, or patients with, uh, inversion 16, etc. The WHO continued to have a blast cutoff of 20% or more, whilst the ICC introduced a similar state for patients with a blast percentage of between 10 and 19%, and they call this AML-MDS. And their thinking was, is that based on the data that I've shown you, as well as other data sets, uh, patients with this blast percentage could still be eligible for acute myeloid leukemia treatments, both within a clinical trial setting or an outside the clinical trial setting as well. The blast percentage for genetically defined AML could be any blast percentage. Uh, it didn't have to be a specific cutoff, whilst for The WHO, whilst the ICC gave the cutoff of 10% or more. The AML not otherwise specified category was removed by The WHO because it was thought that you could still apply a classification to, uh, to a patient because of the, the fact that the morphology, morphological differentiation, that list that I showed you, was still there. Whilst the ICC retains the AML-NOS, not otherwise specified category. Uh, the TP53 saga was only recognized in patients with MDS rather than acute myeloid leukemia, but the ICC recognized that those patients with a blast percentage of between 10 and 19% would be called MDS-AML/AML with mutated TP53, and also recognized a separate entity called AML with TP53 mutated TP53. Now, interestingly, as I've mentioned earlier, that morphology by The WHO, they retained their classification by by morphology. They also removed morphology as a diagnostic premise to make a diagnosis of AML with myelodysplasia-related changes, whilst the ICC, if you look in detail, there's still no significant role for morphology, with regards to classification. So, that can be a problem if you have a limited range of diagnostic tools with regards to genetics and flow cytometry for using the ICC. And I've just highlighted the myelodysplasia-related genes which were recognized by The WHO, and just highlighted in red, the ones in the ICC column which are different from those with The WHO. And the last thing was that both groups could agree on the fact that, uh, certain types of core-binding factor mutations also conferred a favorable prognosis, uh, in patients with acute myeloid leukemia.
So, that, that just leaves us to consider what could potentially change in the future, and how can we improve on our current diagnostic tools? So, one of the most important things is that technology does, and development in technology, do not, uh, stay still. There's always advances, there's always an evolution in this process. So, that there are a whole range of new, uh, diagnostic tools that are being developed, and I'm certainly going to be used, uh, in, in maybe not the near future, but in the next five to ten years, or so. Um, and the, the one way that we can use these newer technologies is to try and define our blast population, uh, with greater accuracy. So, this recognition of the heterogeneous population, showing that these cells are truly malignant or neoplastic, as opposed to being normal or reactive, in, in origin. There's an increasing, uh, recognition that artificial intelligence will play a significant role, and that can be done using image analysis, so using various different AI algorithms to define what a blast is. And there are various different things that can be done as well. So, even using tissue samples cut in different layers, you can actually analyze not just the malignant population, but the, uh, the cellular matrix which houses them, which almost certainly have a part to play in, in these, in how these leukemias develop. Again, using artificial intelligence. And I've highlighted some key references if you do want to have a look into further detail as to what the future may look like. The other thing which I think is a very exciting, uh, field of evolution in terms of diagnostics is the role of mass spectrometry and flow cytometry, using these two methods together to highlight more and more of these subpopulations of blasts. The problem with using standard flow cytometry is that you can have overlap in all the different antigens, uh, in terms of their expression. So, if you expose a sample to 20 or more different antigens, you will get an overlap between the expression. It can be difficult to define whether this blast, uh, expresses this antigen or it doesn't. What mass spectrometry with flow cytometry together allows us to differentiate using, uh, to differentiate all the different blast populations, the subpopulations, using a much larger range of antigens, to make that diagnostic, diagnostic process even more accurate.
Okay, so we've covered a lot. What have we covered? Uh, we've covered the, uh, the various different classification systems of AML, and hopefully, I've highlighted the fact that these classification systems are all dynamic. The diagnosis of acute myeloid leukemia, as well as all of the hematopoietic malignancies, the central theme to this is to have an integrated diagnostic approach, not just rely on morphology alone. It's important to be aware of the limitations and the diagnostic techniques used, and hopefully, I've highlighted that with regards to morphology, flow cytometry, and immunohistochemistry. Uh, I've shown some data with regards to the blast percentage not always being a good indicator of tumor burden, as well as disease subtype, and ultimately defining patient's prognosis and their treatments as well. The new Fifth Edition of The WHO and the ICC classifications, they've certainly helped to integrate new knowledge in the field of acute myeloid leukemia. However, they have caused some political issues with regards to which classifications that we use and how that is used in terms of defining therapy for patients. What's really important is that we need further studies, ideally in a prospective manner, with these new neural classifications to see whether various different treatment approaches make a difference based on blast percentage, mutational profile, etc. And I just spent some time highlighting that newer technologies are all around the corner, and I'm sure that if I give this talk in five years' time, it will be completely different, especially the second half of what I've discussed today. [Music]