Hello Good afternoon, good morning. And good evening for those on the other side, so this is my name is Alejandra Palacio's. M associate medical director of Hematology Medical Affairs at the LPN headquarters and I want to welcome you everyone to these. The first of a series internal medical affairs training in quality Hepatology. Today's today's webinar topic is going to be on diagnosis of acute my Lloyd leukemia and we have the. The Great honor of and pleasure of having doctor Paul Evans. Uh an presenting today as an external expert in this topic. I would just would like to introduce very briefly introduce our speaker doctor. Evans, an document is a principle clinical scientists within the haematological malignancies diagnostic service. HMDS son James University Hospital in Leeds United Kingdom. Doctor Amber has been involved in the development of molecular diagnostics in lit scene, saying this since the inception of the Department in 1987. And he was awarded his pH D working on the topic of molecular methods for the diagnosis of leukemia an. Lean formal also from the University of Leeds and getting his degree in his PhD degree in 1997. It's a great pleasure to have someone with the level of expertise and experience is up for Evans, an. Just want to add that his main research and development interests are currently the about on the integration of next generation sequencing and gene expression technologies. Into a routine dies, not diagnostic practice within the field of. Hematology call malignancy Stan. And I just would like to hand over to the parents and thank you very much. Oh also excuse me just just would like to say we've so excuse me that was just before we start. And to everyone that we will not be able to hear anyone in the audience an you will be hearing us. We hope of course, very well and just wanted to let you know of course, that you will have. A section of your of your webinar display where you will be able to type questions so feel free to type your questions at any moment during the presentation. And also we will we will make enough time at the end of the current presentations to to answer. If not all of them and many of them and also wanted to let you know that the window. Flights from the three events are showing in this slide. The frame that is in the center of your screen. And there's in the upper right corner. There's a little icon with a rectangle if you click on it. It also will increase the size of of the display so you can see better this light San without further ado. The payment thank you very much. Thank you and Joanne. Thank you as well As for inviting me to give this talk today talked about the diagnosis of acute myeloid leukemia. And my my thoughts really about it firstly couple of seconds just to please read this disclaimer are slide. Hum. And then will start. OK will press on South there's wanted to start really about Sir bought normally marquises, which shows the Democratic differentiation pathway or the hemopoietic system. The majority of which occurs in the in the bone marrow as we see right at the Top of this cascade is a multipotential stem cell. Because I fell 3 new in potential and it really decorated out differentiates down to specific pathways my load all employed. And now my load pathway really encompasses all the circulating you know, he went excels you'll see that peripheral blood. Or elbow Mario cell form megakaryocytes, producing platelets to Clock word. Tarifa sites to carry oxygen around the body, etc, etc. Again, the multi pencil stem cell really is triggered by the light of stimuli. All stresses for instance, that VOA bleed was stimulate they make? Megyn Kelly City Pathway generating more platelets. A box here will stimulate more red cells, etc, etc. So arms in AML well in AML. It is this early precursor of what we call precursor mileage cells. Which are really the cells involved in in acute myeloid leukemia? These are blocked in differentiation so they are held at this particular stage. A differentiation and they also proliferate quite profusely and again. The molecular mechanism, which result from this and now well recognized and will be speaking about these later on in the talk. Bro historically historically AML was diagnosed on bone marrow morphology with conventional. Romanowski stains in a variety of sizes chemical stains. Which basically stained the enzymes within uh within a cell and basically it's a process of differentiation with the? Very early mileage felt under a large open chromatin with a very extensive nucleo lie and as these progress down and become more mature. Nucleus Condensors and we start reducing granular site. So I'll grab Daniels within the blast cells and they will eventually progressed down if they were allowed. If that were blocked through a normal neutrophil again passing down that pathway. We will see our usual current suspects. Acute promyelocytic had been at the bottom, which has been the most differentiated of these types of my Lord leukemia. But we also can pass down to the monocytic. Linneage also be referred Lenny Edge on the Mega Kelly City Glen age again. These are usually you know now fairly not used in the model. We always use morphological assessment of a bone aspirate. So you supposed to say a million to context. This is our figures from last year around about 5000 new presentations of humans. Logical on Koleji again, you can say at the Top AML napm allowed group those together constitute less than 10% of all him. Haematological oncology cases and really the case that would actually likely to treat aggressively only represent about 3% of those cases. So these are patients under the age of 60, where we're going to have a curative intent as you can see the vast majority of human social oncology. Malignancy Zara represented by B cell disorder, so we are lymphomas RCL else are my Lomas. To put this into context across the whole of cancer. Hematology oncology represents about 10% of all comfort. So therefore AML across the board represents about 1% of all cancer worldwide. So what do we do currently well since 2003. There's been a push towards a completely integrated. With video of reporting of AML Andover hinge logical malignancies again. This was nice guidance. He massaged links is improving outcomes. Uh, which was published in 2003 and it outlines good practice, both of the treatments of hematology oncology problem. The stocking in the control unit for the clinical environments. The introduction of regular MDT is discussing patients both presentation and throughout the course of their treatment, but also more. More importantly for myself. It actually indicated that specialist integrated hematology unit so our diagnostic units ought to be set up. In in large group there geographical areas and we have one of these we were the 1st in the UK and we're still probably the largest in the UK. And this slide here just shows you that the breadth and spread of technologies and techniques that we used to diagnose. Ola he told you moving sees this particular slide. We use all of these techniques to add to diagnose AML. My regular basis again cited genetics are not will talk about first because this historically with the first thing we started to look at. Again in the 1980s, we were able to identify by magic. They cited in attics whole series of characteristic. Chrome about chromosomal abnormalities a variety of chromosomal translocations also lost in game of the whole chromosomes or parts of chromosomes. The molecular basis of many of these abnormalities was alluded and actually worked out in the 1990s with. The use of molecular biology technologies and again, most of the abnormality involved in AML or most of the ones that. Author prognostic information that actually the result of the translocation. This please trust locations produced chimeric Fusion jeans. I these are a hybrid between 2 jeans. And we have motifs present from both gene partners. This is in stark contrast to the trust locations that we find in. In lymphomas whereby we get deregulation of a particular gene leading to overexpression so that there are 2 distinct molecular mechanisms between these 2 particular. Types of disorder. Again, these optomology provide very important prognostic information, which we can use to guide treatment intensification or even reduce treatment in a certain proportion of patients. This shows the MRC data from MRML 10 again this is from 5000 patients published author bye baby Grimwade. Back in 2010, it really shows you the overall survival in the different cited genetic subgroups of AML. Again, these are this is younger patients. The patients under the age of 60, so this probably represents about 1/3 of all AML the older patients. Not represented in this graph that you can see that are distinct differences in overall survival between different Sacha Dentek subgroups. With the usual place at the Top so light at the Top giving you the best, most favorable outcomes. APM with a graphical 1517 transportation working down to 821 in version 16. I'm sure you will hurt most of these. Then down at the bottom we have. I'm in my life and shout at generally associated with NMDS background and these are loss or gain of. Buy the parts or whole it chromosomes in these clusters aneuploidy inside your genetic terms. So I'm insured by Alejandro that I don't need to go into the very basics of molecular biology but. Back in Italy show you these 2 slides with these are some of the techniques and the technology that we use and. Obviously probably the most current one would be PCR based techniques and this allows you to home in On A. But Ticular regional very specifically in a small region of a gene or part of a gene and amplifier. Many many fold IE many millions of fold, just that area. The genome again. The scheme on the left hand side of the slide shows you what how how jeans are presented normally. Well and you know, thinking thinking text as you probably be aware that are jeans are 30,000 jeans in the human genome. A spread be fairly equally across 23 pairs of chromosomes and again, they usually depicted in this sort of format. With a large blocks representing axons and axons are usually one or between one and 500 base pairs in length, so fairly small compact pieces of genetic material. And they're into space with non coding regions of DNA and these are far larger lamps of DNA maybe representing. 200 Kila bases of DNA so these gene diagrams are certainly not represented to scale what we have here is a typical Fusion gene and what we see are 2 jeans, too very commonly injured AML so we've got lumps one. I wrote T1 on chromosome 8 and what happens as a result of a translocation is these jeans are broken usually almost in. Probably within in front of enlarging forms and again a new Fusion Jeans is created with intronic an excellent material from one chromosome. Going to Internet excellent material on a second chromosome. This conventionally is not applicable or detectable by conventional molecular biology techniques. Uh be cause of the molecular distance. We talking of hundreds of kilobytes is it is very difficult to amplify across these large regions. So what happens in the normal process of transcription and then translations protein is these introns is these large internal. Areas of DNA I removed are excellent brought together this does 2 things it creates a functional Messenger RNA. But also for the molecular biologists beans that you can actually amplify across these these very specific regions from Exxon to Exxon boundaries. And that's what we do. Currently it's on fire many of these very well described chromosome translocation. I was looking up to behold many years ago, 20 years ago in a large pan European standardization study, which was down to designing and. Uh evaluation, the whole set of PCR primers and probes to provide quantitative data in both ALLN AM L and this just shows you a representation of. Some of the chromosome will translocations infusion jeans that we could actually identify these even over 20 years old are still considered the gold standard for MRD monitoring. KML&ALL we still use them routinely and allowed on a daily basis. So. We can also use other techniques based on molecular biology. This is presently situ hybridization, and really in this situation, we're not using small. Uh synthesized oligonucleotide, we're using clone pieces of DNA and in this particular case. We were looking at gene called MLL which used to be called a mixed linear leukemia gene it's now called Care KMT. To a which stands for lysing Meatball transpiration. That's the actual function of this gene it mediate comes to be transferred me filegroups to lysine residues basically. This is involved in many, many leukemia and it has around 50 to 60 translocation partners, so in a genetic code term. It's a very promiscuous gene so we can actually use a similar sort of technology to PPR to actually look for the even again we can approach we specifically labeled. With red and Green Floral Chromes and in this particular case where we don't know Fusion partners, we can actually just. 100 by across all we can actually hybridize across this gene of interest with jeans away with probes which. Founded the normal break point of this gene So what we have in a normal per situation is these 2 jeans. All this genius is kept intact on both chromosome pairs and you get to segregation of both the green and the red fluorochrome. As you can see on the left hand side in these nuclei on the right hand side. We can see a patient who's got A? Rearrangement of the analogy and we can see here we've still got one copy of the gene where the gene is left intact and the. The red and the green are are brought together but we can teach 2 copies are red in the green copy which. Spatially away from one another and this is a disruption of this gene. We don't know what the Fusion partner is between OMLL. Is rearranged? When we know the 2 Fusion partner copy numbers. We can make pros, which is actually far easier to evaluate. In this situation, we got 821 complication. We've got probes red probes, which attracted looks T1. And we've got green publish with their captors chromosome partner, which is wrong, Steaua Trunks launch, one T1. I didn't see here with a normal cell these 4 signals are separated 'cause they 2 copies of 1821. Separated so we have no coat segregation, but in the presence of a translocation we can see on the. Right hand side of the screen. We can see probably have been brought together because of the influence of the translocation so. Gay to another metal to Armory again fish based technology. Technology is quite rapid we can do fish based testing within a. The space of a couple of hours and we don't need metaphase spreads as you can see with these. Which is here, he's only interface cells? Are these cells haven't gone into into metaphase we could just do it on normal ball mouse, Smith. So our cells in suspension. We don't need to get these Holdings in metaphase. Other technology which is useful, but not for translocations, but it's useful for copy number variation is a sniper A and a sniper a bit is based on a similar sort of. Strategy whereby a piece of genomic DNA and we have genomic DNA from our leukemia sample in solution we then. Attach that to an array in this array contains millions of individual dotted oligonucleotides and these other nucleotides. Basically pretend to 2 copies of a particular polymorphic region. An becaus we acquire a copy of got 2 copies of every chromosome. One from from the from the maternal chromosome on that Channel, chromosome, the chances are laughing at a different. A base at this position is quite high if we choose the appropriate jeans and we can use this and we can do a genome wide sniper eh. Basically, what we've seen before. Here we can look for abnormalities. A copy number after mounting a caustic chromosome again. This has far better resolution than? Metaphase spreads I we can look very sensitively at very small areas of copy number gain and losses. We can do this we don't need metaphase. Material we could do some DNA from the patient is ready lately applicable to crystal blood and again when. I meant to say is chromosome X Rays spreads fail. We can, we can revert to this technology, the schema on the right hand side shows you. Chromosome 4 we can see the centromere is about 3/4 away up that chromosome. There is a small the red line in the middle. He's looking at copying them again. And So what we have right at the TI. Let me write the Top of that chromosome. Is a small area of chromosome Los but more importantly here and this is a very important for flip free abnormalities? I've been loyalist chromosone. We've got what is called lots of history, guys exercised autofix or uniparental die, so mean this in this particular system? Situation each one of these dots represent a snake for that smell it represents a polymorphism across the length of this chromosome. We can actually pick up areas where by a chromatid exchange occur Dan. We've got 2 copies of the same long arm and so it obviously when. Mutation is involved on that long arm, you get 2 copies of the mutated allele, which is possibly what. It is most definitely what happens when flat foot pain patients have higher League burdens. So if we move on somewhat where we're going to move onto to flow cytometry flow cytometry is the workhorse. I'm on cloud again virtually every sample goes through a flow cytometry and it's like the DNA to DNA specific interaction between. A strand of DNA is complementary partner a similar sort of very, very specific interaction occurs between an antibody. Alice Antigen again molecule antibodies came to the fore really in the late 70s early 80s and we now do you use on a routine basis? Basically cells are stained with everybody. These antibodies can be attached to a number of different flora cones, so we can look. At a number of different antibody staining at the same time? What happens is we passed the cells in A. A single cell order through uh, she fluid by a process called hydrodynamic focusing these cells pass through and add cited by labor. Like citation is given off and detected by a whole number that detectors and mirrors, depending on the floor a comb. All the light scattered at that we generate. This shows you the flow cytometry pattern or by a number of different things that you can see we're doing things into my 2 dimensions here. The Top left scheme. I've a dot plot and each one of those little dots represents a cell passing proven flow cytometer. Passing through the album laser we the first scheme gives you forward and light scatter forward scatter measures the size of the cell so the further down. The access we are going the larger those cells are the higher of the Axis were going there. Most the most sites scattered. We have the size matters related the granularity or the number of granules in a cell so the big Gray area that we see that their normal. Normal gun outside so normal mature neutrophils. The green area at a monocyte an again CD 45, which is the next one down after Pound Mayer. Hemopoietic markers that should stay in the majority of hemopoietic cells if we saw a walk down the very. Bright light blue are T cells and we can see the 4th one on the on the Top row. 356 CD 34 CD 34 is a marker of early precursor cells, so not surprisingly, are T cells are negative for both. The Orange Salad on that same plot their natural killer cells and then above which are both cells are CD 34 positive we have some purple. And we have some red felt at least purple red cells are the purple cells RB cell precursor cell. Early pre cut pieces pictures of B cells and the red cells are cells were really interested in. They are Milo precursors and they are but. The pictures of what would become AML if we had a malignant process occuring so as you can also see that these are very much in the minority if we certainly take into consideration. Grammar sites as well, we've gated out the grammar sites on most of these plots to get rid of them and we also get it out the monocytes. But we can see that the Milo precursors really do form a very, very small percentage of the cells. In a normal bone marrow and you be less than 1% so I thought that gives you A. A background of what normal build my looks like if we compare this to somebody with AML and the red cells. Well, the red events are my load because as you can see the ones I've circled are really abnormally then so these are not. What a normal Milo precursor would staying with so we've got the on the very right hand side the Top? An species so she chose CD34 which is are my Lloyd precursor cell marker so we are both strongly positive for that. So we all can also can see that these blocks, although I proportion of these glasses actually staying up for a CD 56. Which is a number of marker and that's what we're looking for when we're using flow cytometry to diagnose AML. A more importantly, it's these abnormal expressions that would actually looking for and using other marker when we want to monitor. Residual disease, we following these patients through treatment. So we can see here as a whole host of abnormal expression, which has been highlighted by the? The circles on the dot plots. And again not every animals. I think this is an AML mistake time we've we've kept the monocytes in in green. On my Lord Blackstone precursors are in red and you can see here that we have probably got Milo Monocytic. Case of AML circled here in particular. This particular bar cell population is CD 33 negative so there's none. No CDs 33 positive staining cells and this would be the norm for both my load an monocytic components so. This is already abnormal we got nothing in there so. Robin gaining or just gaining a antibody expression. We can also lose antibody expression both of my Lloyd and. Monocytic component or all sorts of CD123 positive so again there's many ambulances in this particular. Don't plot again we can actually also look at specific animal specific flobots linked to particular mutation patterns this is. Very common mutation in AML that you will be aware of MPM one and you can see that these cells because they are going down to monocytic linneage. Are actually CD 34 negative and CD 34 as we've already discussed is a marker or they? Milo precursor cell so most MLB city, 34 negative. These and these are negative because they are progressing down the monocytic. Pathway light waves they RCD 133123 positive again, something that's particularly abnormal for normal Milo precursors. And we can look at Monotype so again normal monocytes are represented in the right and we can see in this bill manner. We still have some normal monocytes again to the right hand side of the plot, but we have some. We have modified the negative so their negative. 4C D 300 they are negative 4C D 11. Be both of these are modified markers and they and hence these would be abnormal populations, but we could potentially monitor. You know in MRD sort of Aesthetic. I showed you everything in 2 dimensions, but flow cytometry is going down the path now where things can be looked at. In 3 dimensions and again the left hand side upper quadrant here is number of BCD. These 2 dimensional plots, but over later these are. Looking at these things in multi dimensions and this is this shows you the normal differentiation pathway of my load precursor and the arrows follow. Going from immature to mature, but you can see we've got 3 separate different AML cases here and they all have strikingly different. Differentiation patterns, which sweetener venues again at a marker of MRD. So we're going to move away from flow cytometry now, so obviously there is a big portion that has been a big push for. In the last 1015 years to look at mutations in AML and they are the drivers that give you there. Be appropriate muna phenotype, but will give you access to treatment, etc. So they are very important players and again. In very early studies would suggest that we need at least 2 independent cooperating molecular masses and these are split into 2 classes so class Walmart. Opthamologist, which mediated through Parations. These were using mutations within housing kinase jeans, leading to uncontrolled activation so that proliferation side of our. Yeah, a 2 step process and +2 mutations. These were interfering transcription and so in this situation these are blocked. Differentiation and these generally which applications and these years, 2 office is particular pertinent in younger patients 'cause when we look at. The mutational spectrum of younger patients with AML there. Mutational landscape is very sparse. They have very few driver mutations worn or potentially too. In some patients, all they will require is a single chromosome translocation and using a large mutational panel. We can't find anything else so. Younger patients it requires very few genetic drivers to actually drive a leukemia. Mister shows you a few examples of cooperating mutations, and we see these all the time again click free. Is represented quite regularly in our class one mutation Odyssey kick? I can see the Top we've got a PML&RPML Laura Fusion gene this is. They found a link to flip vid or TKD mutations in a high proportion of patients, but unlike other. Some types of AML it doesn't appear to have a prognostic civic significance, but only in APML. Additions obviously you'll be all aware that flicks free how they die a prognostic significance again. Likewise, for what would currently be. Now would be considered to be good with conditions so R8 twenty one associated with lunch when Roxie One. I don't know in version 16 associated with CBPF Alpha Fusions if you acquire that a kick. PKD mutation this shifts you're very good risk AML to a fairly porous came out so the acquisition of A. Extra mutation actually gives you a portal risk phenotype for your leukemia. So we can't have a talk about AML without talking bout high throughput sequencing and I've been involved in high throughput sequencing. 1015 years and in this allows you to look at all the information. We just talked about in the last couple of slides. But in massive parallel so we can look at many jeans at the same time, and we can look for the identification of numerous driving mutations. Not only in a MLB equal that my log malignancy. Zeshan lymphoid allegiances as well so it has become. A little like flow cytometry was 10 years ago, he had become one of our main workhorses that we use. Routine basis every day in the lab. So how can we apply this well, he could be used for diagnosis all subclassification as you probably wear now there are? Numerous new WHL classifications, which involve the identification of a molecular abnormality in a particular gene to sub classify. That particular type of AML it can be used prognostically and obviously we're aware of 3. I TD they poorest feature. We come, although it's in its infancy, we can use high throughput sequencing to monitor disease and to look for. MRD and again. Quite obvious this audience, we can identify targets therapy again what we do here and what we are trying to do is we're trying to use this as an objective marker. But I want him if we see so we can actually identify and hopefully identify patients with. A isolated site appear or multilinear cycle Pedia And if we've got appropriate clonal himo priestess with appropriate clonal markers. Then that could infer that this is more likely to be a malignant process than a just a bowl standard cytopenia. Which is based on a sort of nutritional deficiency? So we have 2 real techniques are used routinely in diagnostic laboratories for doing high throughput sequencing and they are. Hampton Bay City out of PCR based system. We have 2 panels. We use, we use a panel frameless MDF and we have a lymphoid panel, which released for CLL. Modules only film at these are very rapid we can have a Libra Prep performed an on the sequencer within a working day. Again, the number of jeans you can actually look at is fairly limited our particular panel allows us to look at 48 patients at anyone time with 28 jeans. And this represents 3 wasn't 61 ounce becomes. So he can take on his company name usually covered in a single Exxon again we can do the Mass. Am I think when will give you 20,000,000 reads and you know if we've got 360 one hundred? 48 patients, we get an average read get free champion for each patient of around about 1000. So it is very cost effective. It's relatively cheap again. The main costs are sequencing costs and again we're running this. 2 or 3 times a week, so that gives you an idea of the turn around time so I'm just going based systems. We are also now working up a larger hybrid capture panel this differs from Amber come based systems by. The fact that we actually fragment are DNA. We use an Amazon Magic fragmentation system. We then attach are. Indexes and these are indexes are what we use to identify what sequences come from? What patients we will then wash off. Everything else we then had local hybrid capture bees and these are less of RNA or DNA in a very similar manner to fish based probes. We are homing in on particular parts. Of the genome we designed which jeans we want to look at and we can look at hundreds of jeans are large. Can he won't panel actually contains 238 jeans and most of them are the full gene? Uh what he also allows you to do, if you design your probes appropriately is you can get a snipped based backbone, but like the sniper a that we talked about so. We can contact we can actually combine 2 technologies, there, we can actually look at stick based backbone by sequencing. Now we can look at all the mutations, etc that we want to that, we would do by an Amber Code base system. Again, the turn around time for something like this is longer. So we're probably talking that we couldn't do this on a daily basis. And the sequencing would also take longer than that, so you know you have advantage of worn and disadvantages. It's also far more expensive to do hybrid capture based the route that most NHL clubs in the UK. Taking. So this is just a little bit about the sequencing process again at the Columbia Sniper, a we have an array, which has got lots of times. Which are the complementary partners for our art pieces of DNA are pieces of DNA are arcandor terminated with exactly the same sequence? Which we We add by either PCR based technology or buying Corporation again. We flood are numerous millions of PCR products or fragmented DNA. Across this and they'd find their analogous partner. We then Co amplify this so we create lots of thermal problems in about 1000 products. His response required for the machinery to actually see that sequence and then we carry out sequence in again like all of these things. Frequency is based on the hypothesis that we have a primer, which bisects analogous partner and then we sequence. From that primer incorporating one-at-a-time a nucleotide nucleotide is presently tag we could pick that stuff, pleasant tag up. I put it in Corporated in a cycle by cycle of situation and this little. Picture down the bottom shows a magnified version of this so you can see each of these little dogs. Is actually a cluster and each one of those clusters for laughing with one of 4 different nucleotide in anyone cycle? So to go with all that massive data. We we require by fanatics support and you cannot carry out. High throughput sequencing out without a substantial bio for Mattix Report, an we have some excellent by informaticians here. In the Department and it's not surprising really that you are generating when I'm I think when you're generating 20,000,000, two hundred base pair. Read and obviously they contain tundra based solutions. Actually, 4 billion pieces of individual information 4 billion billion nucleotides. Which need interrogating so we have lots and lots of quality steps we have to align all those sequences? Against the human genome, we have to annotate everything we find. We have them more quality steps so it's a very, very. Long process, but thankfully once you've written the script. It's a case of hitting a button. Things my Viper. Magicians would kill me for saying that. But in practice, it's actually wants a scripts are written. It is a very fast process to actually annotate all of these sequences. How many kisses are small panel are my load panels was really designed for MDS but it's? Is totally and completely applicable to AML as well. 'cause the jeans are very, very similar again we tend to split? These things into different pathways, so we have jeans involved in DNA methylation, modifying chroma teen, splicing RNA transcription factors, which had been a bind. Unchange expression of genes signaling pathway, which are involved in the preparation. So are flip 3 is in there, Jack to Maple all of which will hurt. A few of those which have different functions. So we run this on a say we run this panel at least twice a week, so we're running about 100. My location is a week in our Department. Again, the spectrum of mutations is subtly different between the different primary. My Lord disorder that we look at and we can look at AML here. MDS in that the MD SMPM overlap, the same jeans are represented but you can see that. Let's see I TD then we can't do that. CIT testing by amplicon based high throughput sequencing because of the size of the internal tangent you play. Duplications, it quite large. The buyer tomatic pathways on the actual process itself is really quite difficult to pick up quickly. I TDs by high throughput sequencing. Silly amplicon base type high throughput sequencing and the same is also true for porn very difficult time provide jeans. GC rich jeans, such a CBP Alpha so we have problems with hyper sequencing. But as long as you're aware of those problems. You can actually elicit different technologies to actually pick up these optimality's you can see that we have jeans are specific for AML fits the ICD. And we also have jeans are over represented for instance, in MDF and F3B. One is one of the slices on jeans. And it's highly represented in a particular subtype of MDF with ring cinder blocks in the bone marrow. So the last part my talk is about Companion Diagnostics and company and diagnostics is really quite knew and this is the FDA's. Thinking Wanna Companion Diagnostic is again if you look at the bottom makes to identify patients who are likely to. You bet. It did actually benefit from a particular paraplegic product. Also on the opposite flip side of that is to actually identify patients who could be done serious harm. By a particular treatment and then Lastly and what we do a lot of here is actually monitor responsive a particular treatment so. These situations where really looking at the effects of the durability and the death of response by MRD type technologies. So what we have here are the examples of Companion Diagnostics applied to AML so we can look for the. And I DH one and 2 driver mutations, we can look for TP 53 mutations and again. These are do, particularly poorly when. Given conventional conventional combination chemotherapy and we can look for MRD by a number of techniques to monitor patients. Once they going down the treatment pathway services. Ellie plan use pivotal study innate ganak shows using a panel of 70 jeans. That we have a very heterogeneous spread in AML again flip three of the communist mutation in AML but. What this paper also suggested that we could actually go get away from you know conventional classification systems and we can uh. Classification system for AML based purely on the molecular landscape again. The usual players are in there, so the Fusion jeans associated with in version 16. 820 one 1517R, one of the 11th of groups that they speculated should be should be made. But there are new subgroups, so a very poor little group, which has P53 abnormalities, either by Deletions. Or by Mutation in Association or in isolation, with unemployed thing, so losing whole or parts of chromosomes is a very poor risk thing. Associated with more elderly AML patients and then we have things that are actually quite good so MPM one is in there. It's the occurs in 2 or three of these knew classifications as well so again this is very well represented and probably not. Not surprisingly, it is the communist abnormality that we find in AML. I want to just talk a little bit about 6 weeks obviously. Maxfield primarily what you're interested in flip 3 have 2 separate types about normality. Ities, which are called Internal Tandem Duplications. Occured in the Dukes of membrane position of the gene again. They are in frame and So what happens is. At the end of the gene is a little left intact but what happens is that a whole series of. Knew incorporated amino acids are placed as they extend the length of the protein, but leaves the 3 prime end of a protein. Count this as an effect on expression and it also leads to abrogated expression of that particular protein. Again, we know absolutely positive patients don't do very well on conventional treatments. Whether it's relapse rates treatment or overall survival in this graph. Exemplifies that again we have a second set perfectly abnormalities, which occur in the transparent membrane portion of a gene and these attempts. Beer at these represent between 20 and 30% of quickly as a whole so I TDs represent about 60% of. Patients will flip the Malathi TKD's represents about 30%. Both had a poor but notice, although the indication is that. Patients with a flip through it D do particularly badly. Services some early data from Rose Miguel's lab and again. It's sort of Labor. The groundwork that interactions with flip 3 it dies. Particularly important and also the elite Vern of how much lately. I TD we actually have enough in a patient at anyone time. He's also predicted a proper notice so if we look at the left hand side Neograft Capital Markets, and relapses. The survival codes, we can see patients have an isolated MPM one with no evidence of a flip. CIT do very well and I have a very low level of relapsed risk. With patients who have flipped the ID in isolation allowed no MPM mutations. Do very badly so the group in the middle of the intermediate risk. Our patients who are positive for both. Next to both idea while type above and again. These have an intermediate risk again. Rosemead was also alter shown. Current airline guidelines, I've also showed the same thing that the level of the ITD allelic ratio is also quite important with. Patients are the highly gratiot, Mrs implicated in that these patients probably have 2 copies of the city ID not 2 new copies. It is the same copy as we talked about with the sniper raise this is probably uniparental die, so me, I. About 50 Chromated has actually produced 2 copies of exactly the same chromated and therefore you've got 2 abnormal copies of the gene. Leading to a very high perfectly atd allelic ratio, so patient with low ratios do more do better than patients with slippery I TD high ratios. Thought techniques do we use so this is just exemplifying that so they they ELN guidelines rather than just looking at searching ethics? Which you had done before. Now takes into consideration various molecular genetics abnormalities flip 3, being one and again. It recommends selective testing for these because there are now drugs available inhibitors. They also looks at the prognostic significance of a variety of other gene abnormalities and it spits out the mountains and again based on? The level of the absence or presence in the flip 3 it. Dark D into various risk group, so patients with no evidence of like TV or a low level. Allelic ratio in combination with NPR one app favorable patients who are negative negative or mutated an alert at a high risk by TD but empty one mutator intermediate. In patients who wouldn't MPM one negative on how to fix the high ratio perverse again this also brings into. Mutation analysis, so mutated drunks mutated ASL Warren. Not surprisingly, TP 53 are also all pull this creatures. So how do we actually look for these well? We used to look from by conventional grows electrician summer sequencing. This is a very insensitive resolution is very poor and almost exclusively now people use front by analysis run on a coupler electrophoresis system, we tag. The PCR product and you can see on the right hand side of Christine TDs slide that we have a wild type. Friedman and this particular case at 328 and in various schemes. We can see the presence of low level. Let's see I could be in the Top portion patient who's got an early ratio almost 5% sorry .5%. We've got patient there's no evidence of the Wild Type 3 in the 3rd pain and then patients with multiple it dies in the 4th plain? Which is how we do it in HMDS and we have been for the last 1015 years we look for both? MPM one flip 3 it dies at the same time again, the MPM mutations old we are quite varied. They all have the same net effect. They all result in A4 base pair insertion of that which is what we can see? In 2 of those paint the bottom the Top pain, but we can also look in the same analysis. Netflix DVD that we can see here patients who are touching patients after 3. I TD nag. But when we can see 2 patients with positive and we can also. Based on the area under the curve workout the allelic ratio of these patients. How they don't in the UK and further afield and this shows you the different technologies and techniques absolutely same before a girls gel is probably not the technique to use nowadays. But still groups both in Europe and in the UK use this again when we get poor performance in the class. It is using patients in using these technologies that are missing things and so we would certainly recommend the use. Called you fat butt analysis and couple lottery systems form since the TV testing and as we see here, the majority of. Last book in the UK in Fairfield uses technology already again. This is probably important to in a few years time. Most laboratories, both in Europe and in the UK use any now say it will be based around the same technologies. We've talked about so these will be. Invariably people like to pieces and fiber analysis, but they are something that's been designed in house they are not. Off the shelf kids and this will or potentially could have problems when the European directive comes in whereby. If there is a fee market out there. We really have to use it so lots of lost both. In the UK and in Europe will maybe need to change that approach to testing them to companion diagnostics. So we haven't really talked that victory, PTKD's, but again, Nicklaus sent out a a pilot scheme. An educational sample looking at fleet perfectly TKD testing in general. This is done by Fab Alesis. Although we currently do it by high throughput sequencing as we can see here, the fact that artist. Is based on a restriction enzyme coaching site this restriction enzymes? Are derived from bacteria? They are there natural defense? I guess bacteria farges it affects 2 codons and as we said before codons are generated by 3 base pairs and. This G att ATC is coding for the D85 and I-836 Codons again should there be a mutation in evil these codons. This restriction enz eyesight will be obliterated and that won't be cut by the particular Amazon again. The problem with this and it is only a minor problem. If you will miss a proportion of patients and these are patients that don't have mutations in these 2 codons. About a mutation outside these code and in our in our hands you can see that the Top 3. Mutations in this particular scheme on the right hand side are involving codons 84589841. This would be missed by this restriction enzyme cut inside and so just just to know notice that you actually do. If you if you stick yourself to this technology you will miss a proportion of patients who potentially could have access to your drug. So when I last couple slides on I think I should just about finished correctly about MRV monitoring and. M ID monitoring is really paying off. We do a lot of them out of the monitoring here in HMDS. Not only for AML but very alive portion for CLL and Milo Manheim. Really what particular technology at all 3 technologies here. For particular technology were dumped really depends on the DS disease that you're looking at for CLL in my Loma. We would be very useful cytometry. They had abnormal emuna phenotypes where we can look for these spaces. We talked about. And I'll leukemic cells located in that space so we can easily click up very small numbers of events when they occur? For AOL for acute lymphoblastic leukemia be cause. They have a clonal marker that is related to a rearrange amino goblin Jean and T cell receptor gene. We can establish very, very sensitive RQ PCR fees based on that very, very unique clonal sequence and so LL. Here is almost exclusively RQ PCR based for AML. We have a problem be cause. We don't have a total marker. Or, a million kalamarka in every case, MPM Warner. We see here is a very good marker, Mrs Hannam ivs. Data showing that if you are MPM one positive following treatment on your MRD positive these patients do well have a higher. Risk of relapse and have a lower your higher risk of dying about disease based on the MPM one. Mutation Unfortunately MPM one as we already know is only positive in about 30% of patients and this is. Richards Dillons data this is the current standard care offered by guys and the reason why they have such a. A large repertoire of Fusion jeans that we look at is that they've done this for many years, they've acquired these assays. Over many years because they have looked at virtually every patient who goes onto a clinical trial in the UK with AML. Does she show you? What this current state of play for MRD monitoring is in the UK are red markers are the common. Fusion jeans that we find in AML BCR. Abl is obviously found in a very small proportion of them are patients. These bound very, very frequently in ALL&CML and then we have in Green. These are the common MPM one targets but as you'll see on the next slide. They represent 90%, but we would also miss. Had the fan because this is the MPM one mutational spectrum and he can see like say 90% occuring 8. B or D but we have a whole host of other abnormalities. All of which generate a net for base pair insertion. Uh, which results in a friendship, which is consistent for every patient last night is really how do we used? MRD and these are this is a single patient of Richard. The MRD has been used for trials, but look at. Whole groups of patients and what Richard R and the guys down at the guy that wanting to do is look so. Individually and personalized the use of MRD to actually change treatment in patients with AML and this is a patient with MPM one. Mutated CIT mutated animals. Only when typical induction chemotherapy with DNA and antibody therapy, however, following the first course there was a slight. Rise in their level of residual disease detected by MP M1 RQ PCR as we can see. That they would give him a liposomal daunorubicin. This had no effect and even more aggressive treatment slider again levels. Still, rolls, but as we can see here. This patient is still in hematological remission. Although the levels of disease are going up molecularly. This patient haven't reached molecular sorry human to logical wraps and this is very important things once Patience Achiever. Haematological lost it is very different difficult with salvage value to get them back into molecular emission and so. As we've seen here they they they sort of clue in the Top slide here. This placement had a flip 3 it D. So this patient was offered and given a flip 3 inhibitor, not yours. But I clicked hitter and it worked. Actually, the levels of residual disease. Fell significantly giving an opportunity a window to ask before Manalo BMT. This patient as far as Richard told Maine. He's still incompletely mission with no evidence of disease by MRD based technology and that is the end of my talk. I'd just like to thank. All of my colleagues in the Athens. There's about 50 of us, but these are some key collaborators in many, many fields. Also like but still Scott from the UK need classic Sheffield and Richard both of which have low lonely slide. And let me use them for this tool so I will stop there and I'm quite happy to take. Questions for the last 10 minutes or so. Thank you very much Victoria vans very, very nice very interesting presentation. Thank you. Oh, we have several questions from the from that in these. I would like to read the first one, and if you could one of them be ask if you could tell us. Even brief just in very simple terms. I leave here about turn around times or each one of the techniques and approximately? How long it takes from taking the sample. In practical terms in real life terms for physicians to visual to get the results, yeah again. How long it gets to. The sample gets 38 fraction for Unix here in Leeds art interview is is 5 yards away. So we are almost well the same. Yeah, level. We are through a set of double doors and we're in our outpatient unit so in that situation. It takes no time at all to get our sample, but we are, we color a a massive area. We cover a population of nearly 10,000,000 and so some of our outline hospitals are on the West Coast. Of the UK in Lancashire in a different County, it may most such situations. We will get their sample. Within the same day so we have transport systems laid on so getting the samples not the issue again as we mentioned. Our Main. So of workhorse is flow cytometry, we can get a diagnosis of a of AML within 2 or 3 hours. About sample entry in the lab it will give you all the extra supplementary information about prognosis etc. But it will tell you this patient that got AML. And that'll would need rapid treatment for things like. PCR based technologies and companion diagnostics, particularly in MMP M1. We are performing that now and we've only started doing this very recently. On a daily basis and so we have, we may only do on 2 or 3 patients today, but we are doing it, we have. We have changed our setup and are logistics. Whereby we can do companion diagnostics on a daily basis for high throughput sequencing. As I said, before we performed this currently with an African based system. The turn around time for that piece is quite rapid and we are as rapid as anybody else in the UK. We perform that I say, and we perform sequences with that has a 3 times a week and so I turn around time. Including the bio for Mattix and more importantly generated reports 'cause generating important actually takes quite a bit of time. Um to actually you know. Comment on the mutations that comment on their prognosis that any say so. We we recommend a turn around time for. High throughput sequencing for 10 days again flip CTKD is on there, but we are also investigating. The restriction Amazon we know we are probably going back a retrograde step as we're actually going back which is something that's not going to give us. Only information for the abnormality, but we feel that they turn around time afforded by the restriction after I died. Digestion and running that on a fat analyzer actually is actually beneficial and because we can do at the same time this little. I TDs we can turn those down on it on a daily basis as well. Thank you very much I have another this another question from the audience or a general question is very interesting. One it says what are the challenges? Of molecular testing in the area of personalized medicine from your point of view. The challenges are manyfold and I'm only talking about the NHS in the UK here at the moment, obviously you. Probably be aware we have the NHS England. We are being given a test directory which. Basically tells us what we can and can't test an in what diseases etc. That's a bit of a challenge becaus? Most of these abnormalities and there's a reason why they are wanting to try and get access to these. Test for everybody, irrespective of where they live in the UK. But they the problems that have their own panel so they're on high throughput sequencing panels. Another woman intimated weeks in a row has been quickly, but 10 days is probably not going to be enough so we do need. Running along side panel based testing, which will confirm most of the things we find. We do need a series. Back of companion diagnostic test whether they be identifying critical things diagnosis very quickly or monitoring patients once they've gone to a treatment by MRD. We need to have those also sat side-by-side by the standard of care high throughput sequencing NGS type testing. Great thank you. Another question from the attendees on the audience is what is the utility of 3 three as a marker? Or MRD determination together with the other better known markers in AML in question in simple is it useful. You seem pretty useful as an MRD marker good question. The answer is no and the reason why it's noise. Is split 3 is not stable over the course of the disease patients are in relapse? Can loose but three of the marker so they can lose. The need for that lifted push they can gain foot clear the marker so they can actually gain for 3. Through the course of the disease not having had it at presentation and the click free market can also change so. You might have affected by TD which is a particular size but when you sequence that relapse that size is changed. That rearrangement is changed. I TD is changed so the reason why we can't use it takes a very useful prognostic marker. We can't use it. They marker them RDB 'cause. It is not stable so that's why we use MPM one MPM one is a stable marker. There are also other markers that we can find by hyper sequencing cousin that Hoffman asked why can't use. A lot of the mutations you find aftermarket trap vigil disease. They obviously there in the patient and the problem with that is that some of these markets. 2 sensitive IE beast markers and we are talking about 3 jeans in particular tattoo DNMT 3A NS XR1. Are actually found in remission so they're actually probably found in a proportion of cells? Which are not malignant or not. Want to go on that malignant transformation, so again what we would be seeing in that situation are false positive. MLD analysis if that makes sense. So we have the two scenarios, but please the marker is not stable. But we can't use every mutation that we find in a mutational screen because some of those actually are. Positive still in a patient who is completely in remission morphologically. Excellent thank you very much, though for Evans. I think well, I think we have completed our time and. Time I would like to thank you for your great presentation and would like to thank all all all all the audience. Or taking part of attending these very important internal training and thank you. Again, though pretty much has been a great presentation. I I'm sure that we all have enjoyed it a lot. In an yes. Thank you very much. And, yes, I would just like to remind everyone of that in these that after you. Finish this session, you will be requested to answer some questions as the survey so you can evaluate these by me now. Thank you very thank you to everyone. Thank you bye bye. Thank you thank you. Bye.