Many thanks, Anthony. Great to be here as always. So, this isn't, this whole idea of doing this virtual kind of lecturing has actually become very real for me because, the vet school here at Colorado State has moved their entire curriculum to being online and that even includes the the clinicals for the 3rd and 4th year or final year vet students and that's been very challenging, to say the least, but we haven't been getting too much complaints from the students.

I think they're very grateful for any content that we can provide, so. I've been spending an awful lot of time talking to a screen in front of my computer for the past few weeks and that's probably not gonna let up anytime soon. So, yeah, very pleased to be here.

I love doing these webinars for you guys. It's a great pleasure. And some of you who've who've kind of been clients of the webinar that for a while may have noticed that.

I have done a mast cell tumour update before. I think we did one maybe 4 years ago or so that sort of covered the basics. And really there's been enough that's, that's changed in the last few years that I really do intend this to be more of an update.

So I'll sort of very, very briefly touch on some basic concepts of mast cell tumour management. That of course remain important, but a lot of this hour is really gonna be, as the name implies there. So what's new, what's changed, some of it's gonna be what do I think that kind of thing.

So, and again, please happy to save some time for questions at the end and as always, if in fact you don't have your questions answered, because we run out of time or if there's something that comes to you later. Don't ever hesitate to reach out, reach out to me by email. Communicating with vets around the world is one of my favourite things to do, so I'm always, always happy, to respond to any queries, via email.

So, just my conflict of interest statement, for this particular hour, the only thing that I should mention is I have served as a, paid consultant for Qbiotics, which is an Australian company that's about to launch a mass cell tumour drug both in the EU and in the US. So, this is some of the things that we're gonna talk about in this hour. So a brief introduction covering some basic concepts.

Some things about diagnosis and staging that might be a bit new, surgery issues, some things about pathology and molecular diagnostics, when to use them, what they're good for, etc. And then an update on non-surgical therapies as well. So, we really think that this is an important hour to have people consider because mast cell tumours are so frequent, so frequently seen.

Anyone who's practising in a small animal practise is going to encounter them in dogs. People say they account for about 20% of the skin tumours that dogs get. And as Anthony mentioned, they can be a bit tricky because of their potential to have quite a, quite a variety of different types of behaviour ranging from being really effectively benign, so they can sit there for months or years.

Little tiny surgery is all it takes to get them off of there and then you're done to on the other side of the spectrum, these tumours that grow before your eyes and they're open oozing, bleeding and they spread all over the place and the dogs feel terrible. But the good news is that the majority really fall into this category of lower intermediate grade, depending on the grading schema that you're using. And these are the ones that are primarily local problems and with effective local therapy, essentially you're done.

One of the other things that's really a complicating factor with mast cell tumours is the fact that they can really look and feel like anything. So they can be soft or firm, haired or hairless, painful, non-painful, ulcerated, not ulcerated. I could go on and on with the adjectives really to say that any, any mass that you find on a dog could theoretically be a mast cell tumour and that sort of allows us to have this pretty firm recommendation with our students when we're teaching them to say, really.

You should offer an aspirate for any mass that a dog has just to really get a better handle on what it is. . The other thing that's a little bit unusual about mast cell tumours compared to a lot of other, the tumours that we deal with is the fact that we do have the ability to have these kind of odd unusual effects from mast cell tumours that are not seen with many other kinds of cancer.

So they're obviously notorious for sort of doing this local swelling and erythema type relation or type reaction which is due to the vasoactive constitu constituents. In the granules, the one that sort of gets the most press is histamine, but there's actually quite a few others. And this is why sometimes we can see these reports of mast cells, shrinking and growing over time, things like that.

But in patients with Heavy disease burdens, we can also have enough systemic histamine release that you can actually see hypersecretion of acid from the gastric parietal cells which can result in gastric and, and upper intestinal ulcers and clinical signs related to that. And a little fun fact, we just do not see this happen in dogs with small focal mast cell tumours. So this is a very strong indicator that there's probably disseminated disease.

So if I see a dog that has a mass cell tumour and also has these kinds of GI signs, either, they do have a high likelihood of having disease elsewhere or they may have some completely unrelated problem going on that you'd want to get sorted before considering surgery for the mast cell disease. So how do we diagnose, how do we stage dogs with mast cell tumour? So, a good physical examination that includes a thorough abdominal palpation and a fine needle aspirate is really usually the first line of defence.

And thankfully, this is a tumour type that is actually fairly easy to diagnose cytologically. About 90% of mast cell tumours can be diagnosed quite readily with the conventional diffquik type stains that you're likely to have in your practise. There are about 10%, however, where those granules may not be very apparent and those can be a bit more challenging to diagnose.

The special stains that, that referral pathology labs have, with the right games of stains are a little bit better at bringing out some of those granules, but there's still a few percent that really cannot be where those granules cannot be visualised with cytology, and that's where histopathology and sometimes even some special stains may be necessary in order to firm up that diagnosis. So if in fact I, I aspirate a mass on a dog and I come back with a mass cell tumour, the very next thing I will do right away while the owner is still in the room with the dog, is try and aspirate the regional lymph node. Whether it's enlarged or not.

So we do have data suggesting that a fair number of dogs who have normal sized lymph nodes may still have mast cell disease in those nodes. So if we can make a good approximation about what we think the regional lymph node should be, and we're able to feel it, we do try to aspirate it whenever we can. And just a little fun example, this is one of those very, very, very poor poorly granulated mass cell tumours.

That's within a lymph node and again, you really can't see any granules in this particular sample at all. And then again, there may be additional diagnostics that are recommended prior to surgery, really kind of depending on the clinical presentation of the patient. But one of the things I want to digress and say, talk about just for a second, this sort of like news flash thing that's relatively new is there are actually 2 or maybe even 3 papers that have come out recently.

That are actually looked at cytological grading schemes for mast cell tumours. So can you classify mast cell tumours as high grade or low grade using conventional fine needle aspirates that might give you similar information to that which you obtained from a biopsy. So there are a couple of early papers that suggest that the answer may be yes.

So in this particular grading scheme, the factors that were used include the number of mitotic figures. The presence or absence of multinucleated cells, the presence or absence of bizarre nuclei. And the presence or absence of carriomegaly, which means very, very large nuclei.

And actually when this schema was built that uses these three factors, so greater than 1 mitosis, observed. Multinucleation and carriomegaly, there is, you know, mid to high 90s sensitivity and specificity versus the gold standard of histopathology. So there are, there are a couple of flaws in this that you guys might be able to pick out from thinking about it.

So a lot of this has to do with the density of mast cells that are present on, on the slides that you submit. So if you've got a sample that's got just sheets and sheets and sheets of nothing but mass cells on it. And you happen to see a mitotic figure, you know, or a multinucleated cell, that might be a lot more common in that situation than if you've got a sample that's, you know, mostly blood but has enough scattered mass cells that you can still make the call of mass cell tumours.

Just statistically, you might be less likely to pick up a random mitotic figure or things like that. So it's not a foolproof system. And it really does require some additional validation, but it certainly is the kind of thing that might give you a hint that you could be dealing with one of those more aggressive mass cell tumours and when one where you might wanna take a step back and do a bit more poking around in the dog before contemplating surgery, or again, maybe going for those, you know, quite substantial margins versus using a sort of more conservative margin for your tumour.

So, this, this cytlogic appearance can be one of the factors that we would use to make that decision about whether to stage or not to stage. But really there are two situations in which I really think that being thorough in my staging is probably worthwhile. And I'm actually gonna skip to the third bullet point here first and say one of them is, can I do an appropriately large surgery, you know, so wide margin excision.

Without having to do something heroic or expensive or disfiguring. So if I have to take a leg off, if I have to do a full thickness thoracic wall resection, and a reconstruction, you know, something really, really aggressive, then I think that being very thorough in my staging is just really a good insurance policy. So the likelihood of finding something.

Might still be very, very low, but wouldn't I feel terrible if I didn't check and that dog happened to be the unlucky one who did have disease beyond that local site that we weren't aware of. The other situation in which I'm gonna be more thorough about my staging is that there's something about this dog that makes me think, it's got a higher than, than average likelihood of having disease somewhere else. So one of them is obviously the nodal status.

So if I find disease in the node, I certainly wanna make sure that it hasn't gone beyond the node. But then there are another series of kind of historical or physical exam, presentation type of prognostic factors that might increase my index of suspicion that this is gonna be a bad actor and that's kind of some of the things that you'll see on this slide. So breed is one of them that's actually quite interesting.

So there's data out there that contrary to most breeds where the likelihood of a, of a high grade mast cell tumour occurring is maybe 20 or 30%. In Shar Pei, it appears that about 70% of mast cell tumours are high grade. So if I encounter a mass cell tumour in a Shar Pei, I am going to assume it is a high grade until proven otherwise.

So this is a dog that I would want to stage very thoroughly. Presence of clinical signs we talked about already, that can be an indicator of either a separate problem that you need to get sorted or an indicator that there's a much higher likelihood of, of systemic disease. Noal status, we talked about already.

Location. So there's some tumour locations, specifically mucous membranes and mucocutaneous junctions that are generally associated with, with a, a much worse outcome. So this includes the oral mucous membranes, the nail bed, which is technically a mucous membrane, the prep use, the perineal region, the possible exception to this rule may be the conjunctiva.

So when we see mast cell tumours on the conjunctiva, they're often actually relatively well behaved. Growth and proliferation rate, the presence of ulceration. So, ulcerated tumours, some people think that that actually may be a surrogate for recent rapid growth.

And then a lot of it just has to do with history. So if you have an owner who comes in who says, there's, it's been there about 6 months or so. He was just due for his yearly exam, so I thought you had to check it out.

I'm not obviously gonna be as concerned about that mast cell tumour as the one where the owner says, you know, I swear, doc, it wasn't there 2 weeks ago and now it's the size of my fist. So those kind of, those kind of, historical, not very scientific measures of proliferation can actually be very informative and then we'll talk about some more sophisticated ways that we can measure that in a second. Local recurrence.

So there are some studies that suggest that if a tumour has been cut and has grown back locally where it was before, that may be associated with actually a more aggressive behaviour. So we're not talking here about the de novo mast cell tumour. So a dog that gets one on its front leg and then it gets one on its lateral abdomen later.

Those do not seem to necessarily be more aggressive, but the true locally recurrent tumours. And then grade, of course, but we don't know that information until after we get the tumour off. I guess I would add cytologic grade to this schema as well if you're working with a pathology lab that, that routinely reports that.

So again, it's not perfect, but it certainly might raise a red flags. So quick information about surgery for mast cell tumours. The good news is, as I mentioned at the very, very beginning, that for most lower intermediate grade mast cell tumours, complete excision is really all you need to do, and, and problem is solved.

And that constitutes the majority of the mast cell tumours that we see in practise, which is great. So the party line, the, the gold standard is to shoot for 3 centimetre margins in all directions around the mass and at least one uninvolved fascial plane deep. So there's accumulating evidence and we'll talk about it in just a second, that in certain circumstances, smaller margins than this may be a sufficient if necessary.

So, we're still big proponents of the, you know, if I can get 3, I take 3. But if I'm in a situ situation where I can't get 3, you know, I certainly take what I can get, and there are certain tumours where those smaller margins might be adequate. So, take what you can.

Maybe forewarn the owner that there's a little higher likelihood that a resection might be incomplete, and we may come back at them recommending additional treatment. But but again, you may, you're, you're often gonna be OK with smaller margins depending on the exact tumour that you're treating, which we'll go through in a second. And again, for a lower intermediate grade mass cell tumour where local treatment is sufficient, I won't hesitate to recommend, you know, really ruthless procedures like amputation and body wall resection.

So this is a situation where sometimes I will offer an incisional biopsy of the tumour for a, for a, a histologic grade if that's gonna change what the owner is choosing to do. So if the owner says, oh, if this is a low grade tumour, we can cure it with amputation, sure, I'm gonna amputate my dog's leg. But if this is a high grade tumour, where, hey, we're really worried that we're gonna see spread at some point in the future, even with amputation, I might think twice about that and I might opt for some other kinds of treatment modalities.

So, but again, in those tumours where we think we can cure them with local treatment, I certainly will recommend aggressive or ruthless local treatment. I don't do those treatments. I'm a medical oncologist.

I can't even spay a cat, but, but when it's necessary, again, I will definitely refer patients to our surgeons for those kinds of procedures. Should we be using perioperative H1 and H2 blockers? They're certainly harmless.

but there's an, accumulating evidence that maybe actually they don't do a lot of good. And again, theoretically, if we're doing our surgery correctly, we shouldn't be touching the tumour and the, the risk of sort of excessive surgical stimulation resulting in, in excessive histamine release might actually be fairly small. So there is this paper that came out a few years ago now, that actually looks at this alternate approach for figuring out how large your mast cell tumour margins should be.

So this proportional margins approach. So it's a small number of cases, but basically what they did was they wanted their lateral margins to be equivalent to the, to the tumour diameter. So if we had a 1 centimetre tumour, we took, I should say they.

So if they had a 1 centimetre tumour, they took 1 centimetre margins. If they had a 2 centimetre tumour, they took 2 centimetre margins. Anything that was 3 centimetres or above, they took 3 centimetre margins.

And at least one fascia plane deep. And in this small study, 85% of the tumours, were completely resected using this, this approach, and there were no local recurrences during the follow-up period. However, one huge caveat to this was that there were actually very, very few high-grade tumours in this case series and we certainly would expect high grade tumours to be more locally infiltrative and maybe less likely to be completely excised using this.

Strategy. So again, that's why whenever possible, we still want to go for those large margins. But again, this is some information that suggests that especially for the littler tumours, you certainly may be able to get away with smaller margins if you have to.

So what about pre-treatment with steroids? This is something that certainly is favoured by some practitioners. Oh, you know, I put all dogs on Pred for a week before I do the surgery for mast cell tumours, just because it shrinks the tumour and that way my margins don't have to be as big.

So, you know, I, I think a lot of us sort of have, have a big issue with that approach, because of the way it changes the margins that you shoot for. And I have a little cartoon here. That I'm hoping can kind of explain this a little bit better.

So there is a paper that looked at this, it was published almost a little bit more than 10 years ago now actually. So it's been around for a while. But here's the thing that I think really is a problem when utilising this kind of approach.

So if in fact, here's our tumour before we give prednisone, We're actually, so the part that you can see in the middle there, the light blue part is the part that we can see and feel and these dark blue things are the little microscopic tendrils or fingers that are extending out into the surrounding tissue that we can't see or feel. So if we're planning our surgery to actually incorporate this this region, where there's this microscopic disease, we're making those measurements from the edge of the tumour. So if we give prednisone and we're actually able to shrink that tumour some, whether it's a direct anti-tumor effect or whether it's just a reduction in peritumoral swelling and edoema, you may very well kill some tumour cells that are left out kind of in these tendrils as well, but these tendrils aren't gonna magically retract.

They're not gonna shrink back and Result in, in smaller margins. So you're gonna kill probably an equal number of tumour cells all along this little tendril. But way out here, even at the very tip, there's still gonna be viable tumour cells.

But if you're now planning your surgical approach and mapping out your margins based on this tumour that's reduced in size in the centre, you have a higher likelihood of skimping on those surgical margins. And one of the things that kind of lends support for, for the problem with this approach is the fact that in this paper, there was actually a 17.5% recurrence rate in tumours that were resected and that's considerably higher than the recurrence rate without prednisone, according to the literature, which is about 5%.

And that may be because of getting, again, we're getting fooled into skimping on our surgical margins. As a, as a result of this corticosteroid pre-treatment. So now, don't get me wrong, if if, if we need some kind of pre-treatment to even be able to do a marginal excision and get primary closure, we'll definitely do that.

But sort of as a routine matter, of course, I'm gonna get some pred to see if I can shrink that tumour so I can take less tissue. That really probably does not make a lot of sense biologically and I hope that's sort of my explanation here makes sense for that. So, let's say we got our tumour off, dog goes home, does great.

A few days later, we get back, our pathology report. So one of the things that many of you are probably familiar with is the fact that we now have two, I mean, there used to be two competing gra grading schemes, the BOS doc and the Patnik scheme. Everybody kind of graduated to the Patick scheme eventually, which was a 123.

Grading scheme that you see here. And one of the reasons that it was used a lot is because it works really well. So these are are grade 1 tumours, these are grade 2 tumours, these are grade 3 tumours.

You can see a very, very big difference in outcome between these three groups. And this is actually manifested in multiple, multiple papers that have looked at the outcome with surgery alone. One of the things that's a little bit confusing though is that if you look at the top two papers there, the outcomes are actually surprisingly poor.

So in that original Bostock paper, which I think dates to the late 70s, only 37% of dogs with grade 2 mast cell tumours made it to 7 months. Like, what's up with that? And ditto for the, for the patneck paper.

45% of dogs alive at 4 years with a, with an intermediate grade mast cell tumour. That doesn't seem to make a lot of sense. And then we have these three papers down here, which are like, all right, I think I understand that.

So Sue Murphy's paper saying 90% of dogs with grade 2 mast cell tumours make it to a year, 70% are long-term survivors in this follow-up study by Bostock, etc. So what's the difference between these two studies and these 3 studies? These 2 studies are really old.

These 3 studies are much more contemporary and I think the big thing that has changed between these two, these two time points is surgical dose. So I think in these early studies, one of the big problems that, that these patients were experiencing was local recurrence because their tumours were resected very conservatively. And again, these 3 studies, I think, I think are really occurring in the era where appropriately large surgeries are being performed.

And I do think that that is probably the reason for the, the discrepancy or dichotomy that you see here in these studies. But one of the things that we also have learned relatively recently, I guess in the last 15 years or so, is that mitotic index has a more a more scientific way to look at proliferation rate. Actually has a lot of prognostic value.

So not unsurprisingly, low grade tumours have very low proliferation rates. Intermediate grade tumours have intermediate proliferation rates, and then high grade tumours have high proliferation rates. And when you take all the tumours and put them together, these are dogs treated with surgery alone, dogs whose tumours are highly proliferative have a much worse outcome.

So, again, you may say, well, big whoop, that's not a big surprise because these are probably mostly all the high grade tumours anyway. But if you split out, just the grade 2 tumours into those with a high mitotic index and those with a low mitotic index, you can actually see a far worse outcome in those dogs with high mitotic grade 2 tumours than in dogs with low mitotic grade 2 tumours. So, one of the take homes is, again, really, we like to see mitotic indices or now what's called mitotic counts reported on every mast cell tumour pathology report that we get because it does really seem to have quite a bit of important information.

But this piece of information and some other criteria were actually used to develop again, sort of a, a competitor pathologic grading schema. That's called the two-tier scheme. It's also called the scheme after Maddie Couple, who's a pathologist at Michigan State University here in the US.

And, you know, this wasn't really designed to be like a better grading scheme or to be more predictive or anything else like that. It was really designed to try and minimise the amount of disagreement between pathologists, when they looked at a mast cell tumour. So using the old three-tier scheme, this particular paper showed again, like 95 mast cell tumours to a whole bunch of different pathologists, and a third of the time, they disagreed about The grade of the tumour because a lot of the criteria are actually quite subjective.

So these guys actually set out to try and come up with some more objective criteria, which include mitotic index, the number of multi-nucleated cells, pretend high power fields, the number of bizarre nuclei pretend high power fields, and the presence or absence of significant carriomegaly. A lot of the things that we talked about when we were talking about the cytologic grading. And when they use that, actually you could see the agreement shot up to almost 99%.

So much, much, much less into pathologist variability when these criteria were were applied strenuously, which is the main, I think advantage to this. And again, in this, in this case series, it was also quite predictive of outcome. So, is there one that's better than the other?

Should I be using only the new one and not the old one? Should I be angry with my pathologist if they're doing one but not the other? I think the answer of all of those is no, but really more and more these days, I like to see both grading schemes reported on my pathology report whenever possible cause I do think that that gives us the most information about potential outcome.

So, the other thing I'll mention very briefly is there is a, a study now that has sort of established a grading schema for how bad lymph node metastasis is for mast cell tumours. So not all metastatic lymph nodes are created equal. So if we take out a node that we're worried cytologically, might have mass cell disease in it.

Actually, there are, again, gradations of sort of severity of colonisation of that lymph node that range from again HN0 up, there's just like one mass cell that's kind of sitting there to like sort of scattered mass cells, to little clusters of mass cells to a lymph node that's sort of completely effaced with sheets of mast cells, and you can see the accompanying numbers there. And then this does appear to have some prognostic significance where the HN12 HN01 tumours appear to be relatively better behaved. Than the HN23 tumours.

And this does affect my sort of clinical decision making. So, if I do submit a lymph node for histopathology, I do ask for an HN grade according to this YR paper. And if I have it as 01, and there's nothing about the primary tumour that, gives me any pause or suggests that there's gonna be aggressive behaviour.

I'm probably not going to treat this animal aggressively systemically. If I've got an animal that's HN2 or 3, however, sort of this is gonna trump the primary tumour histopathology for me and I am gonna recommend postoperative medical therapy for that patient, which we'll talk about in a second. So, what about all this special stuff that we can do?

So there are a bunch of more sensitive tests that have been done looking at proliferation and mass cell tumours. So you'll see terms like PCNA, AOR, P67, and then A 67 and APCNA are sort of combinations of the things above, and they all appear to have some prognostic value. So here's just AgNOR and K67 as a couple of examples.

But what's not clear from this paper is whether they actually add anything to your typical old grade. So if I just get a grade mitotic index, is that gonna give me exactly the same information as what I'm getting for, for these complicated and expensive special stains. That was not addressed in this paper, so we really didn't have any correlations between what was observed with all this stuff and what the plain old histologic grade and mitotic index are, which you're gonna get from your routine histopathology.

I'll come back to this in a second. So, let's start talking about kit-related diagnostics at this point. So kit is what's called a receptor tyrosine kinase.

This is a little protein that sits on the surface of the cell and its job is to listen to what's going on outside the cell and send signals into the cell to influence that cell's behaviour. And I'll give you a little bit more of a cartoon here to explain that. So normally, let's say this is the outside of the cell, this is the inside of the cell.

These receptor tyrosine kinases like a kit kind of sit on the cell surface, and if, if there's none of their, their growth factor ligand around, they just kinda sit there. But when ligand is encountered actually they dimerize and that dimer then induces some phosphorylation events that occur inside the cell. That signal through a bunch of second messengers and it's not really important to know what these are, what their names are, but those result in a whole bunch of things like enhanced proliferation, enhanced survival under circumstances where those cells might normally die, enhanced migration and invasion, enhanced new blood vessel production that are all associated obviously with worse behaviour and potentially a higher risk of metastasis.

So there are a number of different ways that this can be deregulated in cancer, but the one that's most common in in dogs with mast cell tumours is actually the presence of mutations in this little section of the protein here that's pretty close to the membrane. It's called the juxta membrane domain. And really, these little mutations make it so that this, this molecule, this kit receptor tyrosine kinase is now on all the time.

It doesn't care whether there's any of its ligand floating around or not. It's just gonna be constantly signalling into the cell, telling that cell to do bad things. And again, about a third of dogs with mast cell disease actually have this mutation that renders kit constitutively active.

So, thankfully, there are ways that we can interrupt that process. So there's one paper that actually looks at a monoclonal antibody targeting kit for the treatment of mast cell tumours. So this is a human protein or human.

Antibody, so it's not something that's clinically available right now for dogs, but actually they saw some really interesting anti-tumor effects using this antibody. And then the drugs that we're more familiar with are the drugs that actually work in here. These are called tyrosine kinase inhibitors.

So your Gleevec, which is used a little bit but not that much in veterinary medicine, and your palladia and I guess in the, in the UK it's massive that are, are sort of drugs in this class. And we're gonna talk a little bit more about them in a minute. So, again, going back all the way to like 1996, people have known that kit was present on mast cell tumours and, and mutated in a subset of them.

And actually these mutant mass cell tumours are generally the worst ones. They're more likely to be higher grade, higher mitotic and end up killing the patient. And again, here's just some information about that.

So this is a bunch of dogs that were treated with surgery only and you're looking at your kit mutant dogs here compared to your kit wild type dogs. So huge difference. But again, in this particular study, they weren't able, they, they didn't choose to make any correlations between the presence or absence of the kit mutation and the grade of the tumour.

Similarly, you can also look at where kit is within the mast cell tumour cells. So as a receptor tyrosine kinase, you would expect it to be on the cell surface like it is in this example. But in certain circumstances, you can actually see it present in the, in the cytoplasm, either sort of infocal perinnuclear accumulations or diffused cytoplasmic staining.

So this is not where kit belongs and the presence of kit in these locations is not normal. And as a result, we think that that's actually potentially a surrogate for activation of kit. That, that could go along as, as seen here with the mutation.

So dogs with kit mutations are more likely to fall into these categories of aberrant kit, staining. And this is a special stain that most pathology labs can do. And in this particular study here, we're looking at the outcome in dogs who have been treated with both surgery and chemotherapy.

And you can see similarly, the outcome is worse in dogs who have kit mutant disease. And the same thing is true for kit patterns. So the dogs with kit pattern 1, that normal staining pattern, have an awesome outcome, and then dogs with patterns 2 and 3 have significantly worse outcomes.

But however, again, the, the big sort of missing piece from those studies, just like the previous studies about proliferation are that none of them are taking into account the conventional criteria that we get just from our H&E stay that are free. So actually this is a really cool paper from, from a group in, in Italy, I believe, Rodrigo Horta, that actually tried to synthesise all of these factors. So looked at grade, both patent grade grade and coal grade, looked at mitotic index, looked at chi 67, looked at kit receptor pattern, looked at CIT status, and everything correlates with everything.

So really everything can kind of go back to grade here. So what this would imply is that, really, maybe there's not a lot to be gained from doing all these special stains that you can't get just from your plain old H&E. And actually, when they looked at all these factors and mixed them up into a big pot and tried to spit out at the end of the day, what's really important, the only things in this particular paper that were important were stage of disease and the presence of local recurrence.

Now, again, I'm not willing to throw out histopathology in these tumours yet based on this study, but I do think that all of these correlations really do call into question. How useful the routine employment of these additional expensive special stains for, for, sort of characterising these masal tumours are. So there's certainly may be some situations where they're useful, but as a routine thing, I'm not convinced that it adds a whole lot based on the information that we have.

So quick summary of postoperative therapy for mast cell tumours. So if we have a low grade completely excised mass cell tumour, generally no additional therapy is recommended and 85% of the time you're done. So 5% of these can grow back even though they look like they've been completely excised.

5% can metastasize usually to the regional lymph nodes, and 5 to 10% of dogs can get another, presumably unrelated cutaneous mass cell tumour somewhere on their body sometime in their life. So with a complete, sorry, with an incompletely excised low grade mast cell tumour, our biggest concern is local recurrence. Now these are the patients where optimally will do some kind of additional local therapy.

Either more surgery right away if possible, so recut or radiation therapy. Chemotherapy can be considered in this context, although I do really consider it kind of a second tier choice. I like to use local treatments for local diseases whenever possible.

For high grade or high risk tumours as you see defined here that are completely excised, our big concern is that they're going to metastasize. So usually the first thing I'll do is I will go back and thoroughly stage that patient if they haven't been staged already, and I will talk to the owners about following up with chemotherapy. And then the worst of all possible worlds are high grade, incompletely excised mast cell tumour.

So here, we're really worried about everything. We're worried about both local recurrence and metastasis. And in a perfect world, we'll actually do more local treatment and chemotherapy.

If I really have to do one or the other and I'm not able to do both, I'll usually pick the chemotherapy. And my logic there is that I'm a medical oncologist for one, so that's what I do for a living. But I think, you know, theoretically, these drugs have the potential to work everywhere, so they may kill some tumour cells that are left over at the local site as well as at other parts of the body.

But if we only do more local therapy, we could be doing a great job to control that local disease but we won't be doing anything to address the potential for metastasis. So, that's kind of my logic in making that choice. So what drugs should we use for postoperative medical therapy?

So this is a fairly comprehensive list of the drugs that have been looked at or protocols that have been looked at for shrinking big mass cell tumours. So this is not the same as using them postoperatively but for shrinking big mass cell tumours. And you can see there's a lot of drugs that appear to have some activity when they're used in that gross disease setting.

I am generally a fan of prednisone blastin, and the big reasons for this are it's quite simple. Venblastin is very inexpensive and it's extremely well tolerated. And there are now 3 different studies that have looked at the use of postoperative predvinblastin for dogs with mast cell disease that really do strongly suggest that there's an improvement in outcome for those dogs where it's used in, in patients that are at high risk for metastasis.

So the, the summary of those three studies suggests that the average dog that gets pregnoblastin after surgery does about 3 times better than a dog that does not get any medical therapy after surgery. And we simply don't have that postoperative information from most of the other protocols that are listed here. So, in addition to kit being used as a diagnostic test, we do know that there are drugs that target kit.

I mentioned this briefly before, so I'm not gonna sort of belabour the slide particularly, but we'll just jump into the data. So one of them that's available is Palladia. This is just a quick rehash of the registration trial that was done for Palladia that allowed it to be approved and sold.

So dogs is a randomised placebo-controlled trial. Dogs are randomised to either get palladia or placebo for 6 weeks. And then after 6 weeks, the code was broken and those dogs that were getting placebo were eligible to cross over on to the real thing.

So the overall response rate was about 37%, but interestingly, the response rate was about twice as high in the mutant dogs than in the wild type dogs, and I'm gonna get back to this in a second. So notably absent from this paper was actually any temporal measure of outcome. So yup, so tumours were twice as likely to shrink, but how long did they stay small for?

So that was not reported in this paper. Overall, medium time to progression was about 18 weeks. There were adverse events, and we'll talk about that in a second.

So right around the same time, another study was looking at at mait massive. Similar, not identical, but similar entry criteria, also randomised placebo-controlled. The big difference in this study was that the end point was different.

So, rather than looking at how many tumours shrunk, the endpoint in this study was how long were they able to keep the tumours from growing. So that's called progression-free interval. And actually, when you look at progression-free interval, you can see that when you take all the dogs and put them together, the dogs who got sitib had a longer progression for animal than the dogs that got placebo.

But when you actually stratify that by kit mutation status, it's actually quite interesting. So those dogs, that had mutated kit, the dogs getting the sitinib did about 8 times better than the dogs that got placebo, as far as how long their tumours were at least stable for. And another interesting thing, this wasn't looked at statistically, but actually, the dogs that got mesitinib who had kit mutations, who, who got mesitinib, mind you, did about 4 times better than the dogs that were wild type.

And that actually brings up a very important thing to mention, and that is mesitinib and palladia are slightly different in this respect and that mesitinib was designed specifically to be an inhibitor of mutated kit. Whereas palladia was actually designed to be an inhibitor of some other receptor tyrosine kiages, that are important in blood vessel growth and it just happens to be an inhibitor of kit. So the results of this kit mutation testing thing may be different between the two drugs, and we'll get back to that in a sec.

So, these are not benign drugs. They are pills and they can be given at home, which is nice, but we do see fairly frequently side effects that need to be addressed. GI side effects are probably the most common.

Diarrhoea and loss of appetite are probably the more common ones than vomiting, but they can be quite severe. We can see myelosuppression with these agents. We can see protein losing nephropathy and hypertension.

Muscle cramping and depigmentation have been reported with Peladia. Weight loss has been seen with both both agents and then a few other, sort of more uncommon side effects can also be observed with these agents. So we really need an owner who's willing to be very, very vigilant in watching their dog, bringing them in for recheck appointments, monitoring them carefully.

And again, it's not at all, uncommon for us to need to prescribe those holidays or dose reductions in order to be able to mitigate. Some of the adverse events that we observe. And again, we really try to intervene as soon as possible when we see those adverse events, versus kind of waiting and seeing how they go and letting them get potentially quite bad.

One fact that's important to note is actually that the label dose for Pelladia, the 3.25 milligramme per kilo every other day dose, is actually Higher than the dose that most of us are comfortable using. Thanks to some follow-on studies that were done looking at the pharmacokinetics of, of palladia, we really feel like you can get adequate drug levels with a dose of 2.4 to 2.75 milligrammes per kilo, and have equal efficacy with potentially better tolerability.

So Zoetists cannot tell you this, at least in the United States because of drug marketing laws, unless you ask them, but this is something that they're not trying to pull one over on you. They're not trying to mislead you. They can only talk about what's on their label, but yeah, a little fun fact there.

So monitoring these patients. So generally, what I like to do is see him every other week for the 1st 6 weeks. I start off with again the stuff that you see here, so a PE body weight, CBC chemistry, urinalysis, urine protein, creatinine ratio, and a blood pressure.

Then weeks 2 and 4, I do have them come back for a body weight, good physical exam and a CBC. Ask the owner how they're eating, how their stools, etc. And then week 6 and then every 6 weeks beyond, I repeat this basket of tests to look at adverse events.

So again, when we're talking to owners about utilising these drugs, one of the things that we have to factor in that the owner needs to be aware of is the number of visits that we're gonna be asking them to make for safety reasons and the additional costs associated with the monitoring tests that we're gonna recommend. So, what are some unanswered questions about kinase inhibitors that we can consider? So can we use them in other tumour types besides mass cell tumours?

The answer there appears to be yes. That's not the subject of today's talk, but we certainly do see some activity in other tumours. Can we use it in feline mass cell tumours?

The answer there also appears to be yes. But again, not the topic of today's discussion. If anybody's interested in seeing some papers that cover either of those two subjects, let me know and I can fire those off to you.

So what about post-operative use? So to date, we have no data about whether these drugs are effective if they're used postoperatively. So can I use them to, to prevent metastasis in a high-risk tumour?

Can I use them to prevent local recurrence in an incomplete excised tumour? We do not know the answer to that. So there's a lot of reasons why we think they could work, but we simply have no statistics or percentages to quote about whether they do or not.

Nor do we know how long to utilise these drugs for in the post-operative setting. For, so for these reasons, plus again the cost and the risk of side effects, I do not tend to use these medications postoperatively. So I can't tell anybody who's out there who's, who's using them in that way that they're wrong.

But, you know, I think that there's other things that we can reach for that have a track record, that have a finite number of treatments that we do and actually are quite well tolerated, that I tend to reach for first and that allows me to keep these kinase inhibitors kind of in my back pocket for use down the road. So what about use in combination? And can we use the kit-related diagnostics that I mentioned like mutation and localization status to figure out who should get what treatments.

So I'll talk about this just a little bit and the two kind of go together. So yeah, it does appear that you can use palladia at least in combination with some other chemotherapy drugs. Generally, you need to do dose reductions of the chemotherapy in order to be able to do this in a safe fashion.

So, for example, in this study that we published looking at pulse administered palladia, so just 1 week out of every 3 giving palladia along with Lomustine, we actually had to give about 30% less lousine, in order to prevent the, severe neutropenia. And in this particular study, when we looked at the presence or absence of kit mutation on outcome, we actually found that it had no impact on outcome. So, and again, there are other drugs that have been given safely together, so you can give Palaia withlastine, although you need a very substantial dose reduction.

You can give it with doxorubicin and carboplatin for other non-mast cell tumour indications, but again, you need to do a significant dose reduction. We also did a study looking at a combination of palladia with palliative once weekly radiation therapy, very well tolerated radiation therapy, and actually we saw an overall response rate of about 75%. And a median progression free interval of about 10.5 months.

So it actually appeared to be a very effective treatment and this is my favourite treatment for locally advanced unresectable mast cell disease. It works really, really well. But in this study, we also looked at kit mutation status and found the opposite.

So kit mutants actually did worse when You were looking at progression-free animals. So these are our kit mutants, these are our kit wild types. I'm sorry, backwards.

These are our kit wild types, these are our kit mutants. So we kind of go, huh, well, that's a little bit strange. Should we be using these kit diagnostics to figure out who should get palladia or not?

So more recently, we actually published a randomised, a randomised blinded study looking at palladia or blastin for treatment of dogs with, with gross mass cell tumours and measured a bunch of different things to figure out who should get what. And at the end of the day, we really didn't find that measuring either kit mutation status or localization was helpful in deciding who should get which drug, palladia versus Venlastin. But in fact, when we looked at just the palladia dogs and looked at their outcomes based on tumour type, the palladia, the, in this case, the, the kit localization 2 or 3, the aberrant kit localization dogs did way worse.

And the dogs with kit mutations did way worse. So it really calls into the, into question the utility of using these kit-related diagnostics to be able to pick which dogs should get palladia, which dog should get maybe some other conventional cytotoxic agent. I must very strenuously mention that this is not necessarily the situation for the sitinib.

So as I mentioned, meitinib was designed to inhibit kit mut mutant kit. The data from the registration trial does suggest fairly strongly that kit mutant dogs do better with this drug than kit wild type dogs. So that may very well still be a useful test if you're using meitinib.

With palladia, maybe not. And again, unfortunately, in the US that's really our only tool. We cannot get mecitinib.

So I very, very briefly want to mention a drug that's gonna be launched fairly soon in the EU called igilinol tiglate. And this is actually an intralesional treatment. That was discovered from the seed of some jungle plant in Australia.

And again, this is shot into tumours and actually causes the very rapid destruction of these tumours. And again, what you end up being left with is kind of like a hole in the leg, but it's quite remarkable and I'm gonna show you a couple of pictures here. So, the indication in the US, I'm not sure what it's gonna be in the EU is for non-metastatic, cutaneous mast cell tumours, or for subcutaneous mast cell tumours distal to the elbow or hock.

So these are in locations where wide margin excision is obviously quite challenging. So these tumours can be any cytological grade, but they have to be less than 10 cubic centimetres. So these are relatively small tumours that we're treating and you've got to be able to shoot stuff into them.

So this is the data from their registration trial. So after one single intralesional treatment, there an overall response rate of about 80% and 75% of those responses were complete responses versus the control group where 5% of dogs had a partial response, and that was it. So pretty impressive.

Again, so this is looking at short-term outcomes. This is only day 28, but it actually does appear that a surprising number of these dogs who have short-term responses maintain those responses for up to a year or longer. Adverse events, the big one that we see is again wound formation at the treatment site and tumour and injection site pain.

That's not unexpected based, based on the mechanism of action. And here's just an example. So here's a doggy with a mast cell tumour that's injected.

You actually see a full thickness wound at the site where this tumour was injected, but remarkably, it's actually quite tumour specific. So if you take the same drug, In in laboratory settings and administer it subcutaneously to dogs, you don't see this kind of reaction. Unless there's actually a tumour in the site.

But the thing that's quite remarkable is how wonderfully these sites heal really with no intervention. So there's no debridement, there's no, you know, surgical intervention, there's no wet to dry dressings, there's no silver sulfadiazine. These really just heal up remarkably well and they think that there's something to do with the mechanism of action of the drug that actually promotes wound healing.

It's actually very, very remarkable. So, I think there's actually gonna be a webinar, that sort of accompanies the launch of this drug in the EU coming up in the very near future. So, again, I think this has the potential to be an interesting option for those tumours that are unresectable.

So they're down on a location where we simply can't, can't even do marginal excision and achieve primary closures. So the distal limbs, the tail, the muzzle, those kinds of places. I think we, you know, wanna see more information about the durability of these responses.

So we just kicking the can down the road? Are we truly curing these animals? But the one piece of information that really sort of, that I think is the most problematic about this is that if in fact we're injecting these tumours, we're not able to get a biopsy for histologic grading.

And so we're really forced to rely on cytlogic grading, which I think at this point is not as validated as I'd like to really determine whether there's an animal that needs postoperative therapy, like medical therapy, for example. So you might say, well, can't you just get a biopsy of the tumour at the same time, you, you do the injection. The answer there's actually no.

So if you do a biopsy, And you have like a suture line and a hole and then you shoot the stuff in there. It just comes spewing out of your biopsy site. So you need to probably have at least a week between biopsy and intralesional injection in order to do that.

So, super interesting, more to come. I think it will find a place for a very, very select group of mass cell tumours. But in conclusion, we're wrapping up here and that'll give us a little bit of time for questions if there are any.

I think for the vast majority of mast cell tumours, surgery remains the place that we start for these, and again, 3/4 of them, you can probably cure with just surgery. Grade, margins, and mitotic index really remain the factors that we want to look at for making treatment decisions. The utility of these additional stains for proliferation, it's really not clear.

And again, based on the, the one paper that's out there that tried to put everything together, it may not really add a whole lot. I do consider their use in those cases where there's a, it's kind of a, an on the fence case. So let's say it's got a mitotic index that's like right at 5.

Could it below, could it be high, or a tumour where we'll get back a report that says, well, it has some features of high grade disease and some features of low grade disease. You know, these are the situations where I might actually ask for these additional tests, but they're actually very few and far between. Again, both CIT gene mutation status and kit localization status do seem to predict the outcome following surgery and or chemotherapy in dogs, but the missing piece of information is whether they actually add anything above what we get from standard H&E.

And I think again, based on the hoard of paper, the answer to that could be no. Contrary to earlier reports, kit mutation and localization status don't appear in my mind to identify mass cell tumours that are more likely to experience long-term benefit from paladia therapy. So they might be more likely to shrink, but they may grow back faster.

However, again, this may very well not be the case for acitinib, in which case kit testing may still be very useful. That is the end of my talk, and I think that gives us just a few minutes to potentially answer any questions or, or discuss anything that you might like. And again, I'm just gonna go back to my title slide here.

Again, if you wanna reach out to me privately, with any questions, please, please don't hesitate to do it. And again, thanks very much for your attention. Thank you so much, Doug.

That was excellent. And interestingly, we will be helping Qbiotics with their launch into the UK and Europe, so look out for those webinars. Are you going to give one of the talks, Doug?

No, I'm not giving any of the talks. Right, OK, but we're, we're doing something for that in the next month or so, so it's, it's imminently being launched. What a fantastic looking, extra, extra, .

Tool in your armament. Yeah, absolutely. It was, we're pretty excited about it for that, for, for those occasional tumours that are challenging.

Yeah, no, it looks really, really interesting. So yeah, just waiting for some questions. Dawn, do you want to, see if you can find the, the link for the, for the virtual conference as well.

So just to let people know what virtual conference, we're doing the World Veterinary Association's congress, which unfortunately had to be cancelled because of the coronavirus crisis. And It's going to be turned into a 3-week festival starting on the 25th of April. It's been quite frantic at the webinar that trying to get this all done in, in about a month, when they had, over a year to prepare it, but obviously very happy to help our, our friends at the New Zealand Veterinary Association where the congress was going to take place.

And we managed to negotiate with them that, members of the Webinar vets Platinum Club would get tickets for only 27 pounds plus VAT. So, do go over and have a look at the site and, you know, if you'd like to buy a ticket, then feel free. And I think Julius Liptak is going to be giving some oncology lectures.

Doug, do you know Julius? Oh yeah, I know him very well. So he and I and my colleague David Vale co-authored the new edition of the Withrow textbook.

Oh wow. So yeah, we've known, we've known each other for quite a while. He's a, he's a good guy.

Good. Yeah, I, I haven't heard of him before, so it'll be interesting to, listen to some of his lectures coming up, and there'll be a mixture of live and recorded, which people will have 6 months to look at afterwards, but it's a 3-week event, and some, seminars on, climate change as well, so quite a lot of interesting stuff there. So do go and have a look at the programme, which is.

Getting put on the site as we get it. Arthur said when he was a very young vet, he used to use cyclophosphamide and prednisolone post-op on multicutaneous masses, and these seemed to respond, I presume, . That he diagnosed him as a mast cell tumour, but he's saying seemed to respond, was this look.

You know, that's a, that's a protocol that hasn't been looked at. So, nothing to say that it couldn't be effective, but we really don't have anything in the literature to, to know one way or the other. The closest approximation to that would be one paper that actually looked at cyclophosphamide plus chlorambusil, which is another kind of related oral alkylating agent.

And again, that hasn't been looked at postoperatively, but when you have big tumours that you're treating, not a whole lot of them will shrink, but a surprising number will stop growing. And in some circumstances, the, that disease stabilisation can actually last for a good long time. So, so it does imply that there certainly could be some activity there.

And if some of the other things that have a bit more of a track record are not something that is available, that certainly would be something that I think might be reasonable to consider. Fantastic. I've, you know, and it's a while since I've been practising, but Mazivet also seemed to be very effective.

I remember a dog that had a huge tumour, on its sort of shoulder area, which we couldn't really operate on, and Mazivet did help to bring that tumour down and keep it at a small size and then unfortunately over time, it, it grew back again, but it gave the dog, you know, several months of Really good control and everything, which was very satisfying. It seems, as you're saying, potentially a bit more effective in certain cases than Paadia or just different cases. It's, it's knowing which one to use, but you don't have Mazi that in America.

No, we don't, we did, but they ran into some issues with the, with the Food and Drug Administration, and as a result, it was withdrawn from the market, which is really unfortunate. But you did hit the nail on the head, Anthony, and that I do think we have certainly seen that there are some dogs that seem to respond to one kinase inhibitor, some dogs that seem to respond to the other, and, and, there's really not a good way to be able to determine that. Again, with the possible exception of looking at kid mutation status and, and using that potentially to, to maybe make you more likely to reach for mass event.

Yeah, sure. Patrick's saying, sorry, you may have missed you mentioning it, but do you have any concerns about the fine needle aspirative mast cell tumours and potential spread? So there's absolutely no data suggesting that fine needle aspirate of any tumour hastens metastasis, including mast cell tumours.

So one caveat to that is a transabdominal aspirs of bladder tumours, is contraindicated. But really in that situation, we're getting into a bit of a semantic argument, but there, seeding of the needle tract has actually been documented in multiple cases following that procedure. But I would argue that that doesn't represent metastasis, that's actually direct extension.

So there is no risk of enhanced metastasis from fine needle aspirate of any, of any tumour that I'm aware of. But so aspirate away, we always tell our students. You know, aspirate with alacrity and authority, aspirate everything.

Brilliant. No, that's excellent. So, any other questions?

Oh, we've got something on the chat. Let me just see. So that's actually just Doug's, email is in chat.

So if people just want to pop over and look at that if you want to chat to Doug, which is really kind of you to offer that, Doug, so thank you so much. And then also the URL for the . The subdomain on the webinar vet site, which do go over and have a look at it.

It is evolving. I mean, we literally, Doug, we're given about 2 or 3 weeks to convert this to a 3 week festival. So the website is, we're we're about 10 days away from the launch and the website is growing as we speak.

So the guys, I know Dawn is working very hard on it and Catherine and and some of the rest of the team. We're now having to try and get 100 hours recorded in, in the next 2 to 3 weeks. So, we're, we're, it's gonna be an interesting time, but do go over and have a look at the website and please, you know, those of you listening, do share that out on social media because, it'd be great to see many people at the conference.

It looks a really good programme and very mixed, not just small animal, obviously, . Cattle, sheep, even deer, equine, public health, a bit of veterinary business, so there should be something for everybody there. Right, any more questions?

That seems to be the lot. Doug, I've really enjoyed, today's talk. It was one of my, areas of interest, the mast cell tumour, obviously with dermatology, nice and easy, you could see them and I think what always amazed me was, you know, you could look at a lump, and there is a tendency in, in GP vets not to do, aspirations, and they look at it and they say, oh, it looks benign.

You can really get caught out with that. So thanks so much for just giving us all that extra information, the new stuff that's happening, it's great to be brought back up to date with, what's happening and particularly with the Qbiotic as well. That, that looks a fascinating new drug to try as well.

So thank you so much and enjoy the lockdown. My pleasure as always. Thanks, take care, take care, Doug and thanks everyone for listening.

Bye bye. Cheers.