Hi, my name is Vatsal Patel. I am moderating the session this evening. And today we have Dr. Cresak this evening. She's gonna talk to us about the diagnosis of glioma in 2021 and the updates that are coming in the new WHO Blue Book. Dr. Cresak, she completed medical school and her residency at the University of South Florida. She later attended the University of Florida for her neuropathology fellowship where she remains as faculty. She's currently the Director of Anatomic Pathology at UF and the Associate Residency Program Director. Dr. Cresak's comprehensive training included emphasis on surgical neuropathology, neuromuscular pathology and ophthalmic pathology. She's a co-investigator on several brain tumor studies and her funded research at the Preston A. Wells Jr. Center for Brain Therapy focuses on the host immunologic interaction with primary brain tumors. She has several book chapters and numerous publications related to her field. Dr. Cresak also is an attending within the GI pathology division and she has a passion for resident and fellow education. That's something that I can attest to as she wasn't attending when I had the privilege of being a resident at the University of Florida. And let's see some interesting things about Dr. Cresak. She is a huge baseball fan and I think the Rays are her team, if I remember correctly. That's right. She also teaches or used to teach an undergraduate class in her spare time at the University of Florida. She loves fishing and she used to race cars and work in a mechanic shop and would love to restore an old car. So she leads a very interesting life and I'll turn it over to Dr. Cresak. Hello everyone. I'm so glad y'all are here. Thank you, Dr. Patel for all that. He makes me sound way cooler than I am. I am just a simple pathologist. So we're gonna talk about gliomas and not so much like how to make the diagnosis, it's what's changing between now and the next who that is coming out soon. There is a lot changing. Here we go. So we're gonna start with a historical context, move on to the C-impact updates and I'll let you know what those are and how they translate into the new who and then talk about some emerging entities. So brain tumor classification really began in the 1920s with Harvey Cushing and Percival Bailey. Percival Bailey. Cushing was a neurosurgeon and Percival Bailey came to train under him. He'd already had actually a pretty illustrious career but wanted to come and work with Cushing in Boston and Cushing put him in charge of looking at all these brain tumors under the microscope and trying to classify them because Cushing said, we need a way to know how these are going to behave. Some of our patients are not doing well, some live for a long time. How can we predict after we take the tumor out? And so based on what he saw, he gave it his best shot. What the cells looked like to him, he gave the name to. So you'll see that many of the names that he gave are the names that we still use. So astrocytoma, oligodendroglioma, ependymoma, glioblastoma. These are original terminologies from 1926. Then it wasn't until 1940 that Dr. Scherer proposed, there's gotta be two different types of glioblastomas. So they look the same under the microscope, but some of these patients have a prolonged prognosis. They do quite well for numerous years while others present as this high grade tumor and die rather quickly. And so there must be a difference and he called them primary versus secondary, realizing that the secondaries were developing from a lower grade and that's all we knew. So we didn't give it a whole lot more thought after the 1940s, other than to say, yep, there's primary and there's secondary. So primary GBM being the more common type, this is their older patient that presents as a grade four, just de novo, it's already a GBM. These are prognosis, 18 to 24 months. Versus secondary GBM, these are our younger patients. They usually start out as a lower grade and progress on to the GBM as a grade four. So in our 2007 WHO classification, you'll notice that it's all descriptive names and many of them are the same exact names from 80 years prior when they were first described. So gliomas is an umbrella term that encompasses our astrocytomas, our oligodendrogliomas and our ependymomas. And we're going to focus mostly on the astrocytomas today because that's the bulk of our tumors and that's where the biggest changes are occurring. We will briefly mention oligos and ependymomas at the end. And of the astrocytomas, we have two categories, right, we have two main categories, the infiltrative type and the focal type. Focal astrocytomas are, your prototype would be pilocytic astrocytoma. So these are your WHO grade ones. They're, as the name says, they're focal, they're circumscribed. The surgeon, the neurosurgeon can go in, remove the tumor without follow-up. There doesn't need to be radiation or chemotherapy most of the time. These patients are cured with surgical resection. This is vastly different from the infiltrative type that starts at WHO grade two historically and progresses on. And so that's where we're going to be starting. So back in the day, like, you know, five years ago, and when I started as a neuropath fellow, this is how we would grade them. This is probably what you're used to as far as grading astrocytomas. So infiltrating astrocytoma start at WHO grade two. And the WHO grade twos are defined by just a mild increased cellularity and a little bit of nuclear atypia. These are the hardest to diagnose because they can look so near normal. Give it a little bit of time and they will progress to an anaplastic astrocytoma, WHO grade three, which you need mitoses to make that diagnosis. Then a little bit more time and it will progress to the glioblastoma, which you need microvascular proliferation and or pseudopalisading necrosis. And so here is going through that journey. This would be a diffuse astrocytoma. So obviously the coronal section of an autopsy specimen here, and it's hard to even identify where the tumor is. If you notice on the left basal ganglia that you can identify all the areas of the basal ganglia. Globus pallidus, internal, external. Whereas on the right side, it's very effaced and wide. So this is involved by a diffuse astrocytoma. And on imaging, what this could look like would be a non-enhancing diffuse tumor. And on histology, just mild increased cellularity, nuclear atypia. So that would be a good grade two. Fast forward a few years, five to 10 years. And now you have the tiniest bit of enhancement on imaging. On histology, you have mitoses and obviously the cellularity is increased and the pleomorphism, the atypia is easier to appreciate. This is clearly tumor. And then give it a little bit more time and you're at Q1 grade four glioblastoma. So the classic ring-enhancing lesion here. And on histology, you have your micro-rascular proliferation and or your pseudopalysating necrosis to make the diagnosis. The gross image is the classic butterfly glioma crossing the corpus callosum. So then all the way from 1940 from Scherer, staying primary to secondary, you don't really think about it for another 60 years. So 2009, when we finally figure out what exactly that difference is. So it is the IDH mutation. Once we were to the molecular era and we can sequence everything, it's found that the lower grade gliomas, so the diffuse astro, anaplastic astro and the secondary GBMs that arise from them all harbor IDH mutations. So do oligodendrogliomas. Whereas your primary GBM does not, your pediatric tumors, medulloblastomas, the others do not. So it's a mutation of this pathway of lower grade gliomas onto secondary glioblastomas as well as oligos. Also notice that the vast majority of the IDH mutations here are IDH1 and specifically the R132H mutation. There are others and IDH2 encompasses the rest as well as a few other point mutations for IDH1. And then with the Cancer Genome Atlas Project, it confirmed IDH as a defining mutation in secondary GBMs. As well as we looked at the ones that are not harboring IDH, the IDH wild types, the primary de novo GBMs and we realized what they harbor. So they're defined by these EGFR amplifications, TERT promoter mutations and alterations in chromosomes seven and 10, usually a gain of seven and a loss of 10. So we realized that they are molecularly very distinct tumor types. So now we have kind of a reason behind the primary and secondary GBMs. And if we look at the survival curves for IDH mutated versus wild type glioblastomas, we see that there is a difference. So mutated IDH gliomas have a better prognosis than the wild types, though they are still fatal tumors. And we have an immunohistochemical stain for this. So the stain that we have is specific to the point mutation to the R132H mutation. So it's going to pick up the vast majority of these for you. However, if you have a lower glioma and your IDH mutation by immunohistochemistry is negative, you do have to go seek it out by other means. So molecular analysis, NGS, looking for an IDH mutation. So then we get to our 2016 WHO classification. And now we have the same descriptive terms, diffuse astrocytoma and the plastic gastro GBM, but we also have IDH. So for each of the tumor types, it's, well, is it IDH mutant or is it IDH wild type? And our oligos are not defined by IDH mutation plus 1p19q co-deletion. And we have a new kid on the block, this diffuse midline glioma with its own mutation. So we've added molecular to our tumor classifications in 2016. A lot happened between the 2007 WHO and the 2016 WHO. So we went nine years between updated WHO and so much changed. And it's difficult to keep up with it. And it can be really tough when the new one comes out if you aren't following the literature all the way along, if you aren't a neuropathologist attending all the conferences and whatnot. So this is where C-ImpACT comes in. What is C-ImpACT? This is the Consortium to Inform Molecular and Practical Approaches to CNS Tumor Taxonomy, Not Official WHO. So it's a group of people who get together and review the literature and all the changes that are going on and put out these guidelines, these papers that are basically going to guide the next WHO. And even though they're not the official WHO, if you look at the authors and the people who comprise this consortium, it's the same authors as the WHO. So a pretty good idea that these C-ImpACTS are probably going to make it to the WHO. So following these between 2016 and 2021, when the new WHO comes out, has given us a good idea of where the field is going. So the first thing that the C-ImpACTS have said, and that's like the easiest rule to follow is we're going away from Roman numerals and moving on to Arabic. Just so there's less possibility of mistaking a two for a three or a one for a two, the Arabic numerals are easier to identify. Okay. So now if we go back to our umbrella here, and we're looking specifically at the astrocytomas and their gliomas, we've discussed our focal and that we're not going to talk about those, not much changed in the new WHO. Now our infiltrative gliomas, the biggest branch point is IDH mutated and IDH wild type, which we already knew from the last two from 2016. Then we have this other category, our diffuse midline glioma. So we're going to start with these IDH wild type tumors and see how they change in the 2021 too. So do we still categorize these IDH wild type tumors based on their histology? Now that we know that having a lack of this mutation is poor prognosis, or are they all going to behave similarly? So we study and we find out, and the yellow line here is our IDH wild type GBM. And the purple line up here is our oligo, low grade with IDH mutation and 1p19 cupra deletion. So this is an oligo, this is an IDH wild type GBM. The red line is low grade gliomas, so twos and threes, wild type or IDH. And notice how closely they follow the survival curve of a wild type GBM. Your IDH mutated tumors, here's your IDH mutated low grade and IDH mutated GBM, have distinct separate survival curves. So because of this, anything that is an infiltrating, an infiltrating astrocyte tumor that is IDH wild type is bad. They all act like a GBM. And so if we can say that they are a GBM, we can call them as such. So it's infiltrating, if it's IDH wild type. And because of the molecular studies that cancer genome analysis studies, and we know what a primary GBM is made of, tert primary mutation, EGR amplification, or alterations in chromosome seven and 10. If we have one of those three, plus IDH wild type, plus it's infiltrating, diffuse astrocytoma, IDH wild type with molecular features of GBM, food grade four. This was what the C-Impact-3 said in November of 2018. Okay, we were following along. You know, okay, even though it might look low grade, if the molecular is that of a GBM primary and it's IDH wild type, regardless of histology, we can call it this. Almost two years later, this was in July of 2020, the C-Impact-6 came out and said, you know what guys, that's just a mouthful. Just call it glioblastoma IDH wild type food grade four. So no matter what the histology is, if it fits these parameters, you can call it a GBM. So this is very different from how most of us were trained where you have to see the pseudopalisading process and the microvascular proliferation. It's gonna probably look re-enhancing on imaging. That's out the window. If it's this criteria, GBM, food grade four, Arabic. So here's an example that we had. This is T1 post-contrast MRI. So you notice this right frontal temporal lesion that is non-enhancing. It's bright on T2, but it's non-enhancing. On histology, it looks lower grade. This would be diffuse astrocytoma by histology, but it's a 60-year-old woman. And that doesn't really make sense. 60-year-old women at that age, it's very unlikely to be a lower grade tumor. IDH was negative. And next-gen sequencing told us that this had a TURP promoter mutation and EGFR amplification. So this was glioblastoma IDH wild type food grade four. Now, what about the IDH mutated tumors? Should we still grade those based on histology? So let's take a look. And we're just gonna look at the top half of this chart for now. This is overall survival and this is progression-free survival. So the red line is your GBM IDH mutated. The blue line would be an anaplastic astro IDH mutated. And then this would be an anaplastic oligo, the green. So they do still stratify based on histologic grade into different prognoses. But what they did find was a CDKN2A homozygous deletion is a poor prognostic marker. So here would be your GVM or your anaplastic astro with IDH mutation and CDKN2A versus the blue line, anaplastic astro, IDH mutant, CDKN2A wild type. And so now we found a new marker that can stratify the IDH mutant tumors. So histology is still important. We found a new molecular marker for these, and it's looking for a deletion, not a mutation. And so the terminologies have changed here. If it's an astrocytoma, it's IDH mutated, and it looks like a diffuse astrocytoma, you would otherwise have graded it as a grade two, you still will. But the new name is astrocytoma IDH mutant grade two. If it looks like an anaplastic astro, and it has, so it has mitoses, and it has the IDH mutation, astrocytoma IDH mutant grade three. If it looks like a GBM, if there is microvascular proliferation and or pseudopalysating necrosis, and it has an IDH mutation, astrocytoma IDH mutant grade four. So the terminology of GBM is reserved strictly for IDH wild type tumors. So it looks like a GBM, but it's IDH mutated, don't call it a GBM, astrocytoma, grade four. CDKN2A deletions will automatically make it a four. So if it looks lower grade, has the IDH mutation, and has deletion of CDKN2A, automatically it would grade four. So our hand is now forced to do molecular on IDH mutant tumors, looking for the CDKN2A deletion. Here is an example. We have 68 year old female, and notice in her right occipital region, sorry, her left occipital region, we have this heterogeneously enhancing mass. And on histology, we have pseudopalysating necrosis. So any other day we would have called this GBM. We do some markers, GFAP shows us, yes, it's legal. IDH mutation is positive. And then this is our ATRX mutation, our ATRX stain. And loss of the stain is a positive, positive for mutation. We can talk about this marker a little bit later. So we have a tumor in this older woman that is IDH mutated, looked like a GBM on histology. This would be astrocytoma. IDH mutant, WHO grade four. So this was cut and paste from our report. This was before the WHO was coming out. We now know that this is true. This will be in the WHO, but at the time we really weren't sure. So we put a note saying, according to the most recent consensus criteria defined in C-ImpACT-6, because it's infiltrating, because it's IDH mutation, it's WHO grade four. And now it is confirmed. These are going to be the changes in the WHO at the time we said this is anticipated to be in the next year. So if there's any take-home message from this entire talk, I'm done with that part. I'm done with the biggest take-home message. And that is these, because that's your biggest change in classification and what is really changing from what I think is so ingrained in most pathologists' minds. So the IDH wild-type tumors are GBMs. You have to fit a little bit of criteria. The IDH mutated tumors, you are still going to grade based on histology, but you need to look and see if it has loss of CDKN2A, unless, of course, it already looks like a grade four on histology, but that doesn't matter as far as grading goes. So now we'll talk about diffuse midline glioma, H3K27M mutated. These we had as a definition in the 2016 WHO. But there is one update to it, and that is we now know that the H3K27M mutation can be found in other tumor types and in lower grade tumors. So having that mutation in and of itself does not equate to grade four instantly. It has to be, as the name implies, diffuse and midline. So if you have an ependymoma and it has this mutation, it does not equate to a grade four. But if it's diffuse and midline and harbors this mutation, who grade four? Here's an example that we had. A lot of these are in children, but they don't have to be. So this is in the brainstem of this child. You can see the tumor lighting up in the pontine region. And these do encompass most of the diffuse intrinsic pontine gliomas, what we used to call that. And here's a gross image. So autopsy, you're looking at the base of the brain. And you can see the expansion of the brainstem structures here with a little bit of replacement. And another example. Here is the immunohistochemical stain for this mutation, for H3K27M mutation. Not to be confused with the figuremethylated stain. So what about pediatric tumors? So the adult gliomas are very well classified and it's easy to follow that tree, right? It's an astrocytoma, it's IVH wild type or it's IVH mutated, we grade it accordingly depending on the mutation status. It's easy to follow. Peds, not so much. So the pediatric tumors that haven't changed are the focal astrocytomas. So your pilocytic astrocytoma, the ganglioglioma. These are often found in younger patients. These haven't really changed much, but the infiltrating pediatric tumors have to a degree. So we have genetically defined some of the pediatric diffuse gliomas. So if you have a pediatric tumor, it looks infiltrating on imaging, they biopsy it, it looks infiltrating. And it's IDH wild type. It doesn't follow necessarily the same prognosis and like biological behavior as the adult IDH wild type diffuse gliomas do. So we had to find other means to try and define these tumors. So Nib altered tumors, we have Nib and NibL1 altered gliomas. And then the others fall under diffuse glioma MAPK pathway alterations. So in the next two, although the C-impact breaks them down further, these are clustered under the MAPK pathway alterations in the next two. So we have diffuse glioma Nib, NibL1, and then diffuse glioma MAPK pathway alteration tumors. So what you would have to do if you had a pediatric tumor that looks diffuse on imaging and on histology is molecular studies. So here's an example. We had an eight-year-old girl and notice there is a lesion in her right mesial temporal lobe. It's non-enhancing. So here it is, you just see expansion of the gyri. It's difficult to identify. But on your T2, you see the flare. On histology, you have increased cellularity. You have mild atypia, some irregular nuclear contours here. Very mild atypia to the cells. You can do some immunohistochemical stains. So GFA can tell this, yep, glial. KI-67 in the brain should usually be pretty low to none. So of course, if you have something reactive, it could be elevated KI. So having increased KI does not automatically tell you it's tumor, but it's a good indicator. The neurofilament stain is what we use to say that it is diffuse. So neurofilament is gonna highlight the axons in the background. So we have these tumor cells infiltrating through these axons. It's not that it's displacing them and pushing them away like a focal tumor. So this is one of the ways, if we're unsure, that we can support our thinking that this is a diffuse tumor. So here in this child, we have a diffuse glioma. IDH is wild type, as expected. Kids don't get IDH mutated tumors. ATRX is retained. BRAF V600E, it's mutated. We have an immunohistochemical stain for that. So when it's mutated, that's very helpful. When it's negative, you know, you're gonna have to reflex to molecular testing. And so this would fall under, in the next WHO edition, diffuse glioma MAP-K pathway alteration. Here in this paper, in the C-impact, it would technically be this BRAF V600E, but diffuse glioma MAP-K pathway alteration. So two caveats to this classification is that if these harbor a CDKN2A deletion, then they could behave poorly. And the thought is if they have a KIA 1549 BRAF fusion, which is the most common fusion in pylocytic gastrocytomas, the most common alteration in pylocytic gastrocytomas, that they're gonna behave as such and they're gonna behave as a great one. So even if it looks diffuse, if it looks infiltrating, if it has the KIAA, then it should behave well. If any of these have CDKN2A, they're gonna behave more poorly. Otherwise, these diffuse gliomas, even though they're diffuse, if they fall into these categories, they don't behave poorly like the adult diffuse gliomas do. Sometimes they do, especially if it has CDKN2A, but most of the time, they're gonna behave pretty well. Sometimes they will be watched. Sometimes they might offer subsequent radiation. And the grading is hazy, whether we should call them grade two or grade three, or don't grade them at all. And we're gonna wait to see what the actual WHO says when it comes out, but it's possible that it says not to grade. We'll see. Some emerging entities that are coming out that you'll see on the next few. We talked about the first few. Diffuse glioma H3G34 mutation instead of H3K27M. We're gonna have an example of that in the next slide. Astroblastoma, and then one altered mixed zoically neuronal tumor. PLENTY, polymorphous low-grade neuroepithelial tumor of the gung, or PLENTY tumor. So this list, you will see as new entities in the next two. There's a lot of them. A couple of examples. So this tumor, this new entity, of astrocytoma H3G34 mutant. It's mostly in teenagers and young adults, but we've seen it in older adults. It's a better prognosis than H3K27M tumors than the diffuse midline, but it still is a high-grade tumor. One of the reasons that it might be a better prognosis is that the H3K27M mutant tumors that are midline, if they're in the brainstem, then we can't resect and we can't radiate. So our options are very limited as to what we can do, what kind of treatment we can give. It's chemotherapy. So these are not necessarily midline. They can be, but not necessarily. So then your options are bigger, which might be why they have a better prognosis. So here's an example. We have a 23-year-old, a younger person, and they had slurred speech and some facial weakness. MRI finds this tumor crossing the corpus callosum. We normally think of that as being a GBM. We get a biopsy specimen. Obviously, you're not gonna get a resection specimen here. You're gonna get a stereotactic biopsy, and it's very easy to tell we're in a tumor. It's very easy to tell we're in a tumor. High-grade, we have spleomorphic nuclei. We have mitotic figures. There's no necrosis. There's no microvascular proliferation. So on histology, historically, I would have been an anaplastic astrocytoma. So that would be the histologic classification. But molecularly, it harbors this H3G34R, mutation, and therefore, it's a high-grade astrocytoma, H3G34B2. Another example of one of the new tumor types coming out, the emerging entities, CNS neuroblastoma, FOXR2-activated. I put this one here only because I actually, one of my residents presented this as a poster at FSP a couple years back. So predicting the future that this was gonna be a new entity. This falls under the embryonal tumors. So our medulloblastomas is the biggest category for embryonal and then we have other embryonal tumors and there's a big category there. ATRT, embryonal tumor with multilayer rosettes, and this is one of the new kits, CNS neuroblastoma, FOXR2-activation. So just to be aware that there's emerging entities under the astrocytoma category. Okay, moving on to oligodendrogliomas. Not a whole lot has changed except these Arabic numerals. So we still grade it probably as HOOP grade two and HOOP grade three, maybe not. I'll have to wait and read the new HOOP for that. There's been talk about not giving a histologic grade and only looking to see if it has a CDKN2A deletion. CDKN2A deletion, no matter the tumor type, equals for prognosis. So here's your classic oligodendroglioma. You have the fried egg appearance, this very, very round monotonous nuclei with perinuclear halo, little capillaries, chicken wire capillaries, higher magnification. This would be your fish looking for loss of 1p and 19q. And you do your immunostain and you show that it's IDH mutant. And then this would be an example of an anaplastic oligo, the grade three correlate. On imaging, they can look bad. They can also not look bad. The nuclei start to not be quite as monotonous and sometimes you even lose the halos. And you get, in this case, microvascular proliferation. You'll find increased mitotic activity. But not much has changed there. Molecular, we're still the same. It has the IDH mutant and 1p19q co-deleted. Ependymomas, a few things have changed. So just as a refresher, here's a beautiful smear of an ependymoma. The cells are hugging this blood vessel. And then the permanent section correlate of that is this perivascular pseudorosette. And if you're lucky, you'll find true ependymal rosettes and that's how you can clench the diagnosis. In adults, these are often in the spinal cord and in children, they're the fourth ventricle. Myxopapillary ependymomas. Oh, sorry, that didn't change. Myxopapillary ependymomas are no longer a WHO grade one. These are now a WHO grade two. So that's one change. Of course, these love the phylum of the spinal cord and they have this very classic histology. But we do know that they behave more like a classic ependymoma. So we grade them as such now, WHO grade two. Sub-ependymomas remain the same, WHO grade one. They have this very classic histology. They're in the ventricles. Not much different there. Then for our classic ependymoma, the classifications changed tremendously. The classifications changed tremendously. Before it was just grade one, or I'm sorry, is it grade two or grade three? Is it classic or is it anaplastic? Now we first classify them as, are they supratentorial or posterior fossa or spinal? So the first thing you'll say is location and then you have to say molecular. So is it for supratentorial? Is it a GAP1 fusion tumor? Or is it ZFTA tumor? Posterior fossa, we have two types, a type A and a type B. And spinal cord, of course, is where you'll find your myxocapillary and otherwise it'd be more of a classic type. So in the 2016, we had just names. And in 2021, everything is first location followed by a molecular. The group A and group B for the posterior fossa ependymomas can be separated out on some aminohistochemical stains. More than likely, you're gonna do molecular on all ependymomas unless it falls into these categories of myxopendyl or subependymoma. Take-home points. We have some really great surrogate aminohistochemical markers that are still gonna be incredibly useful. IDH1 is, you know, my best friend is that aminohistain. It's only gonna pick up the R132H mutations, but that is the vast majority of them. And then you can use molecular to pick up the others. ATRX mutation is, it's most useful to tell you the difference between astrocytic and oligo. So 1P19Q co-deletions are mutually exclusive with ATRX mutations. So if you're unsure on a small biopsy and you don't have access to fish, or you might not have enough tissue or whatever it may be, you could do an ATRX to tell you lineage. But it's not a hundred percent. B. brachii 600E, we have a stain for. H3K27M, we have a stain for. So those are helpful. Location and histology are still very, very important. You know, is it midline? Is it not? The histology, you're still going to grade the IDH mutant astrocytomas based on histology. Pediatric tumors are different just because they're IDH wild type doesn't mean they're just the equivalent of the adult IDH wild type. Molecular comes into play there and proving that it's diffuse is important. The C-impact papers are guiding the next WHO. There was recently a paper released by David Lewis on the WHO guidelines on the changes, basically what I talked about in this talk. So if you want a comprehensive list before the WHO comes out, I would recommend reading that paper. And my last take home point is I never said one thing about methylation profiling, and that is in the pipeline for some tumors, probably starting with brain tumors and then it could expand out to other tumor types. Thanks for sticking with me. I am open to any questions. All right. Can you guys hear me? Yes. Thank you so much, Dr. Cresak for that. That's certainly very enlightening. I don't think we have any questions, but I certainly have a few. So my first question is since IDH mutations are so central to classifications of astrocytoma, should we reflex all negative IDH132H astrocytomas to molecular, looking for other IDH mutations or doing molecular sequencing of IDH? So that's a good question. There are times when you could probably get away without doing molecular. If you have a 70 year old patient who presents and it's a GBM by histology, then you're done. There's nothing further you have to do. For everything else, yeah, you pretty much have to do molecular testing. Okay. And in the category of astrocytomas that are IDH wild type, what's the percentage where you actually find when you sequence them, you actually find a TERT, EGFR, or chromosome 7-10 aberration? Very high. Yeah. If it's an adult and it's a diffuse astro and it is IDH wild type, you are probably going to find one of those things. Okay. I guess for this small subset where you don't find those three aberrations, do you still use histology then to grade those gliomas or those astrocytomas as a WHO grade two or three? It would depend on what else the molecular studies showed me. So if they pointed me towards something more specific, then great. If not, then yeah, I'd be stuck in some NOS category, not otherwise specified category. And I would be descriptive and I would go based on my histology and say, you know, this looks like this. This is probably gonna behave like this. Gotcha. So that should be a exceedingly rare diagnosis. It's basically an astrocytoma, which is IDH wild type and is not a grade four. Yes. Got it. Are there any updates in the diagnosis of meningiomas? There's not a whole lot that's changed for meningiomas. We do know that meningiomas harbor different molecular mutations depending on actually location. Some of the base of the brain meningiomas are different than the superior convexity meningiomas as far as molecular. And some of the different histologic subtypes have different ones, but it does not change the prognosis or the grade for almost any of them, except there's probably something coming out about the rhabdoid meningiomas. There's actually been papers on it for a long time. It just hasn't made it into the hoop that VAP1 is an important prognostic marker for the rhabdoid meningiomas. If it's VAP1 mutated, then it's a worse prognosis. Gotcha. And I've got several questions that have- I see them popping up. Yeah, yeah. So let's get to them. What would make you work a tumor of, tumor of for the histone alternate histone mutation, the non-K27M? So, great question. If I'm, most of the time I'm going to pick it up anyway because it's going to be IDH wild type. And if it's IDH wild type, then I'm doing other molecular studies because it's probably going to be a younger patient, adolescent, young adult, mid adult. So my IDH comes back wild type. I need to prove that it's either a GBM or else, or something else. So when I do my molecular studies, I'm looking for an EGFR, a TERT, alterations of seven or 10, it'll pick up the H3G34R at the same time. So I'll find it. Got it. Next is, what is the algorithm to work up a brain tumor? You'll start with, am I dealing with a kid or an adult? If you're dealing with an adult, you're going to start with your IDH mutation. And then if it's wild type, you do molecular studies to prove that it's a GBM. Unless the histology is already that of a GBM, you're all done. If it's IDH mutated, then you have to do molecular studies looking for loss of CDK and 2A, unless it looks like a GBM, then you're done. If it's a kid, you're going to do molecular studies. Okay. Are you routinely getting BRFV600E on all high-grade gliomas? Also, are you using any one specific for, send out for three mutations for the de novo glioblastoma cases? So we have an in-house panel that we use. It picks up everything that we need for adult gliomas. It picks up the very vast majority of what I need for pediatric gliomas. There are times when I have to do send outs. Okay. Are you? Sorry, my screen disappeared. There's times when we have to do send outs. I saw one of the questions is where. There's the LeoSeq panel in Pennsylvania. There's the UCSF500 panel that's very comprehensive. There's others. There's numerous that are good. You can also always send them to us. We can do the panel. Do you routinely run all the relevant mutations on all tumors or are there, I mean, I guess a follow-up question for me would be, is there like a ependymol specific panel? Is there a astrocytoma specific panel or it's just kind of a shotgun type approach with molecular? No, so that's a great question. Oh, and to answer the BRAF question, I don't do it routinely on everything because if I have something that's IDH wild type, or I'm sorry, if it's IDH mutated, then it's not BRAF also. So then I don't need to, but my IDH really stratifies me. It's IDH wild type. I can do the BRAF immunostain, but if I'm sending for molecular anyway, then it'll pick it up. I do use it in pediatric cases frequently as a triaging stain, because if it's positive, that helps me tremendously in putting it in a bucket. There are many focal gliomas that are BRAF B600 mutated. So it does not tell me if it's diffuse or if it's focal. It helps me put it in a BRAF mutated bucket and then I can sort it out. The, what was the next question? Sorry, I, let me get to that. That was the question. Oh, you asked about ependymoma specific or- Right. Astro specific. No, but not all next generation sequencing panels, some are mutations only and won't pick up fusions or deletions or mostly fusions. So if you're looking for a fusion, like the KIAA 1549 BRAF of phylocytics or some of the new pediatric tumors have fusions or some of the ependymomas have fusions, you have to make sure that you're using a panel that's going to pick those up. The commercially available panels will tell you right on the website what they have and what they don't. So those are a little bit more specific, but for your kind of standard astrocytomas, the ones we get by and large most of the time, our GBMs and our lower grades, most of these next generation sequencing panels are sufficient. Do you do molecular on grade two spinal cord ependymomas? No. No. Okay. And in communicating with the clinicians, how is the best way to relay the changes in grading or no longer grading without causing confusion? Are they following these changes as well? Question mark. Great question. So we've given two talks to our surgeons here. We gave it once and then like two months later, they said, can you give them again? Sure. So it was very important in our institution because we run a lot of clinical trials and the terminologies for the inclusion criteria might say glioblastoma. And now that we are not calling everything glioblastoma that used to be called a glioblastoma, it was important to make sure for the clinical trials, do we want to expand our inclusion criteria to include grade four astrocytomas? And diffuse midline gliomas, or do we want it specifically for IDH wild type tumors? So it was very important to have these conversations with our oncology teams, our clinical trial teams, our surgeons. We've given them two talks. We give lots of references. We try and put big comments. This is why I'm calling it this. The terminology has changed in our diagnosis. Our surgeons here are used to this. We've been doing it all along. So this next two is not going to be a huge surprise to them. There really are some big changes with this whole astrocytoma IDH mutant grade four. They say, no, that's a GBM. It's a GBM on imaging and suspicion. And the next question I have is, WHO guidelines mention what mutations slash deletions and et cetera. But don't recommend a particular method for testing. What is your advice to a pathologist in prior practice with no in-house molecular testing in choosing the best, most comprehensive panels for some of the testing, especially when tissue is limited? Am I allowed to say, send it to a neuropathologist? Absolutely, why not? So if really, if your tissue is really limited, then just send it, because then they can triage it and send it to the right panel. It's hard to pick a single panel. I don't want to single any place out. They're all really good panels. So, especially if it's an adult, you're pretty safe in just picking one. But then of course, if it's not somewhere that's going to give you a consultation, then you still have to know how to interpret the results when you get them back. Got it. And are there any microarray tests to streamline the molecular studies? Not that I'm aware of. Okay, and then because the diagnosis rely on molecular findings, what is the minimal information do we need to give to the surgeons during a time of frozen section? Great question. Nothing much changes for them as long as they, what they ever really wanted to know is one, is it an astrocytoma or glioma? Is it a glioma? And is it diffuse and infiltrating? That's their biggest things, you know, is this focal and I'm going to get it right out, which is hard to tell in frozen many times. Pylocytics you sometimes can tell, otherwise it's tough. And is it a glioma, right? So alternatively, if something is ring enhancing on imaging, as an example, it could be a lymphoma in an immunocompromised patient. It could be tumor factor multiple sclerosis, it could be other things. So what they really want to know is, is it a glioma? And if you can just say infiltrating glioma or high-grade glioma, nothing changes for what they're going to do management-wise. So that's what we say. We say infiltrating glioma, high-grade glioma, you know, net. And then how do you test for CDK and 2A deletion? It's on our in-house panel. We can pick it up. It's picked up on most next-generation sequencing tables. Okay. And I think this is probably going to be our last question is we seem to be making quite a bit of progress on the diagnosis front. Are there any advances on the therapeutic side of these gliomas that you're aware of? No, there's targeted treatments for IDH mutations. There's vaccines against IDH mutations down the pipeline. I don't think that's where the solution lies. Brain tumors, you treat one mutation and they're just going to pop up with other mutations. So we can chase mutations all day. It's sort of like the BRAF B600E story with melanomas. You know, you can treat it, you can get a short response, but not very often durable response. We're working hard on immunotherapies. You take the patient's own tumor and you dissect out, you know, total tumor, a whole tumor, RNA, and you can make a vaccine against it and give it back to them. So it's very specific to them. Lots of vaccine trials like that. And, you know, we can get a little bit of prolonged therapy or survival, but it's not a durable survival and it's minimal at that. So no, not yet. Hope so. And just the last question that popped up quickly and sounds like a methylome profiling isn't a commonplace test yet. Is that correct? Yeah, not at all. There's a few tumor types that are actually defined by their methylation profile, even in this next few that's coming out, but it is not done routinely anywhere. It's hard to get done because it's not done routinely anywhere. So right now, if we need methylation profiling to help us on a case that we just, it doesn't fit into any category. So it's like a Hail Mary. Let's see if the methylation profile fits it into a category. Or if we think it's one of these tumors that has the specific methylation profile, we send it to the NIH, but the turnaround time has been about a month. A few places are working to get theirs validated, but right now it certainly is not necessary. But I do know that there's quite a few papers coming out with methylation profiles of different brain tumors, and I know it's starting to spread into other tumor types, much like molecular did. So I imagine seeing more of it in the future, but not commonplace now. Perfect. And I guess we will end it at that. Thank you so much, Dr. Cresak, for giving up your evening to educate us. I certainly found this very enlightening as someone who does not encounter gliomas on a regular basis, but nonetheless, it's still important to keep up with the changes. So again, thank you very much. Thanks to all of our attendees. I think we had over 40 attendees on the Zoom. Yeah, thank you to you all for spending your Wednesday evening with me. I could talk brain tumors all day, so I'm just appreciating the chance to do it. So thank you all.

glioma classification

IDH mutations

molecular testing

WHO updates

astrocytomas

glioblastomas

pediatric gliomas

C-ImpACT papers

molecular profiling

targeted therapies