A Blood Test Before the Scalpel: MicroRNAs and Canine Splenic Masses

A splenic mass is one of those findings that can flip a normal day into a crisis. You may have an older Labrador or Golden Retriever, an ultrasound that shows a splenic tumor, and an owner asking the question you cannot fully answer yet: “Is it cancer?” We sit down with Dr. Janet Grimes to unpack why that gap between suspicion and certainty is so hard in canine medicine and why better preoperative diagnostics for splenic masses could change everything from emergency decisions to long-term screening.

We walk through what veterinarians currently juggle when counseling clients, including the role of hemoabdomen, the wide spread in prognosis between benign lesions and canine hemangiosarcoma, and how rules of thumb like the double two-thirds rule fit (or do not fit) in different clinical scenarios. Then we zoom in on the science of microRNAs: tiny non-coding RNA molecules that regulate gene expression and can be detected in circulation, making them promising minimally invasive biomarkers for veterinary oncology.

Dr. Grimes explains how a multi-marker microRNA panel is built from blood samples and measured with quantitative RT-PCR, why panels can be more specific than single markers, and what it could look like to use this as a send-out test today with the longer-term goal of a cage-side diagnostic. We also discuss the real-world barriers: differentiating hemangiosarcoma from other splenic malignancies, avoiding misleading results in sick dogs, and integrating any new test as an adjunct to physical exam, imaging, and standard lab work.

If you care about earlier cancer detection in dogs, smarter decision-making around splenectomy, and the future of blood-based cancer diagnostics, listen through to the end and share this with a colleague. Subscribe, leave a rating and review, and tell us what question you most want a pre-op splenic mass test to answer.

AJVR articles: https://doi.org/10.2460/ajvr.25.07.0258 and https://doi.org/10.2460/ajvr.25.07.0250

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Transcript

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Welcome And Why Splenic Masses Confuse

Lisa Fortier 0:35

Welcome to Veterinary Vertex, the AV Made Journal's podcast, where we delve into behind-the-scenes looked at manuscript authors. I'm editor-in-chief Lisa Fortier, joined by associate editor Sarah Wright. Today we welcome Janet Grimes to discuss microRNAs in dogs with splenic command juice sarcoma. Janet, thanks again for being here with us on a repeat visit today. Thank you for being here, Janet. Thanks so much for having me. I'm really excited to chat with y'all today. So, Janet, as the editor for JAVMA and AJVR, I have the pleasure of reading a lot about splenic masses in dogs. As you know, I'm an equine surgeon, so I still am marveled at why splenic masses remain such a challenging and diagnostic area for veterinarians today.

Janet Grimes 1:18

Yeah, I think it's it's hard because we just don't know what the mass is until we get the histopath report back. And so the huge problem we struggle with is there's a very big difference in the prognoses that you can have based on that diagnosis. So on one hand, you have benign disease like hematoma, nodular hyperplasia, and that can be cured with surgery. And then on the other hand, you have femantiosarcoma, which is a terrible prognosis with median survival times of like six to nine months, even with chemotherapy. And so it makes it really difficult for us to talk to owners and counsel them on how to proceed, particularly in an emergency situation when the mass is ruptured and they're bleeding into their abdomen. And so there's also an entire spectrum of uh prognoses of different diagnoses between benign and homangeosarcoma, because there's non-homangosarcoma malignancies as well. And so a lot of groups have looked at different ways to kind of solve this challenge. And unfortunately, nothing so far has been kind of the one thing. So you can use a combination of factors to guesstimate. But I'm really hoping that microRNAs kind of become a more accurate way to tell what these masses are before

Why Preop Answers Matter

Janet Grimes 2:35

surgery.

Sarah Wright 2:35

I think I may know the answer to this next one. But Janet, why is a reliable pre-operative test important?

Janet Grimes 2:41

So I think there's kind of two ways to look at it. So the first for me is that it can save the lives of dogs with benign disease, because if the owners know that that prognosis is really good, they might be more likely to invest in surgery and postoperative care, which can be very expensive. Um, and then the second way I think this is important, and this is a goal I'm working towards with my research as a whole, is that if we can diagnose homangiosarcoma earlier, dogs do better. Um, and so studies have shown that lower stage disease dogs have better outcomes. And so if we can find a preoperative test that could even be used for screening dogs that are at risk, that could change outcomes by allowing us to detect that sooner.

Lisa Fortier 3:24

The preoperative diagnosis is important for the client, of course, but I would imagine it's also like a oh moment as a veterinarian when you have a Labrador with a sponic mass. Um what about the psychology there to get an accurate and earlier diagnosis?

Janet Grimes 3:41

Yeah, it's it's really tough. Um, so I I have a golden retriever, and of course, they're the poster child for hemanger sarcoma. So, you know, any golden retriever, you really worry about having that. Um, I think it's hard because certain breeds are more at risk, but even within those breeds, there's still a chance that those masses are benign. And so I think in some of our previous work, um, we've tried to look at other things to help that out. And so if they are bleeding into their abdomen, the chance that it's a mangiosarcoma is much higher than if it's just an incidentally identified mass. So you take your dog into the vet, they palpate the abdomen on a routine exam and feel a splenic mass, or you get an ultrasound of the abdomen for something else and they find a splenic mass. Um, so those dogs have a much better chance of having benign disease. So I think for me right now, that's one of the biggest things that we can use is whether they're bleeding into their abdomen or not. Um, but we still, you know, there's still a large percentage of dogs with large enough to investigate further of dogs with uh hemoabdomen that will have benign disease. And then even some dogs with without tumor rupture and no hemoabdomen, they're gonna have malignant disease. But at least we've caught them earlier. So I think it's just it's such a challenge, and it's really hard to help owners. And if they are familiar with their breed, like a golden retriever, labrador, German Shepherd, they're probably gonna already know about it and be worrying about that. And so it'd be really nice to be able to give them more definitive information before they spend all this money on surgery.

MicroRNAs Explained For Cancer Testing

Lisa Fortier 5:23

Yeah, very good. Uh so the uh center of your manuscript is really microRNAs. Can you explain to our listeners what microRNAs are and why they might be useful in cancer diagnostics?

Janet Grimes 5:34

Yeah, absolutely. So microRNAs are short, approximately 22 nucleotide non-coding RNAs. And so even though they don't code for anything, um, they're they do interact with messenger RNAs and affect gene expression by suppressing or promoting certain genes that drive cancer progression. Uh, and so they can bind to several different messenger RNAs and act in different ways and different diseases. And what's really great about them is they're altered not only in tissues, but also in circulation. And so because they're already so short, just being 22 nucleotides, they're highly resistant to further degradation, whereas longer messenger RNAs degrade rapidly in circulation. And so this makes microRNAs a really promising, minimally invasive biomarker for cancer. Uh, and so that's kind of why I'm investigating them further. Very cool. And within that same concept, what was the hypothesis of this study? So, our hypothesis was that we would identify a panel of microRNAs that could accurately discriminate dogs with hemangious sarcoma from those with benign disease. Um, and so our our thought was really that a panel of microRNA would have a higher sensitivity and specificity than a single microRNA. So, similar to some of the other tests that have been done to kind of determine what these masses are preoperatively, if you can use multiple ways to come at it, you're gonna get more higher sensitivity and specificity. And so our hope was to identify a panel of, you know, four to six microRNA that would that would tell us this dog has some anger sarcoma or is more likely to have benign disease.

Sarah Wright 7:23

Yeah, sounds a lot better than the rule of two-thirds, which I feel like was what I was taught in vet school, what I used when I practiced ER. Actually, what while we have you, what's your what's the current, I guess, stance on using that to guide clients as far as prognosis goes for splenic masses?

Janet Grimes 7:38

So that's a good question. So I still I still use that rule. I I do think nowadays um I would say if the so that that rule, I traced it back to kind of its origin, and it was pretty much for any dog with a splenic mass, whether they had a hemoabdomen or not. And so um I think Jabmer published a study a while back where a group looked at just benign disease, or I'm sorry, just dogs with with no hemoabdomen. So incidentally identified splenic masses, and they found that actually only 30% of those dogs had a malignancy or hemangiosarcoma. And so that's a a huge different difference from the double two-thirds rule, of course. And so um we we did a study here, kind of a systematic review looking at dogs specifically with hemoabdomen, and it was a much higher percentage of uh hemangiosarcoma in that group. And so I think what I tell owners now is I probably still use the double two-thirds rule for uh dogs with a hemoabdomen, but for dogs where it's not ruptured, not bleeding, I tell owners 50-50. I have a hard time saying there's a 70% chance because if their dog is in that unfortunate group that has semantial sarcoma, it just feels even worse. So I kind of say 50-50, but hopefully it's a better chance than that.

The Double Two Thirds Rule

Lisa Fortier 9:03

We do get a fair number of listeners who are uh non-veterinarians interested in the subject topic. So would you mind explaining what the double two-thirds rule is?

Janet Grimes 9:13

Yeah, absolutely. So the the double two-thirds rule is that two-thirds of dogs with splenic masses will have a malignancy, so a cancer that can spread. And then of those two-thirds that have a malignant tumor, two-thirds of those will be hemangiosarcoma.

Blood Sample Workflow And PCR

Lisa Fortier 9:31

So, Janet, can you walk us through uh how you collect it and then how you analyze the blood samples from these patients?

Janet Grimes 9:37

Sure. So um kind of since I started on faculty at UGA, I've been prospectively collecting blood samples on dogs undergoing splenectomy. So any dog that comes in that's going for splenectomy for a splenic mass, we talk to the owners and um ask if we can get a blood sample. It's been approved by our research committee and everything. So we do informed owner consent. And then I'm just banking these samples and holding on to them until I have an idea and funds to do a project. Um, and so I kind of went back through my sample bank and I identified dogs with hemandrocercoma and those with benign disease. And then I kind of look at those dogs as a whole and look through their record, and any dog that has a concurrent different cancer, so for example, mast cell tumor or something like that, I try to exclude them because that could affect the microRNA profile. Um, and so once I had that, I kind of went through the literature and we found kind of or chose eight microRNAs that have been associated with canine hemangiosarcoma and human angiosarcoma, which is the corollary in people. Um, and so eight microRNAs that have been associated with those in previous studies. And then we did a quantitative RTPCR to evaluate the levels of those microRNA in each of our patients. And then we also had two reference genes. So those are genes that are going to stay stable in our samples, and so we can then compare if our microRNA are higher or lower to those reference genes and then compare the two groups. And so that's kind of how we proceeded.

Building The Four MicroRNA Panel

Sarah Wright 11:18

And what were the key criteria you used to select the four microRNAs that ended up in the diagnostic panel?

Janet Grimes 11:24

Yeah, so this was really a statistical uh decision, I think. And so we built a multivariable model. So that means that we put all the microRNAs in at once because we, again, we wanted to get that panel rather than just one single microRNA. And then I did what's called backwards elimination. So I put all eight into the model, and then each time I removed the least significant microRNA one at a time until the whole model as a group reached significance. And so as I removed those, once we got down to those four, that's when the model became significant to be able to tell the dogs with hemandrocercoma from the dogs with benign disease.

Lisa Fortier 12:02

While we're talking about stats, what did your findings reveal about sensitivity and specificity of your panel?

Janet Grimes 12:09

So the four microRNA panel had a sensitivity of 80% and a specificity of 90% for discriminating hemangiosarcoma from a non-cancerous mass. And then kind of another way to look at that is our area under the curve for our receiver operating characteristic curve was 0.93, which is highly discriminatory. So a one is perfect for the area under the curve. And so 0.93 is very high. So we're capturing quite a few of those of those dogs correctly.

Sarah Wright 12:39

And now

How The Panel Could Change Care

Sarah Wright 12:40

on to a question that I bet our listeners are probably gonna enjoy the most. How could this diagnostic panel change how veterinarians and pet owners approach a suspected splenic tumor?

Janet Grimes 12:51

So I think my my long-term goal is to eventually have a cage side diagnostic test. Um, and so that's gonna take a little while to develop. But the first way I think this could be helpful is again for those dogs with the incidentally identified non-ruptured splenic masses, because you have a little more time to work with. So until a cage side diagnostic is developed, this could be a send-off test like you would for a COVID swab or something like that. Um, so any dog that's found to have a splenic mass, the veterinarian could draw blood, send it off to us, and we could perform PCR on the sample, and then report a likelihood that this dog has hemangiosarcoma or not. And then the owner, the veterinarian can take that information back to the owner and they can work together to kind of decide how they want to proceed. And again, I think this can really help encourage owners to proceed with surgery for dogs that have a high likelihood of having benign disease. But I think another way this could be used is even just routine screening with ultrasound on at-risk breeds at a certain age. So golden retrievers, by the time they turn seven or you know, some age, uh, we can perform abdominal ultrasound. And as soon as you see a splenic mass, you then send this test off to determine what it is. And so even if it comes back as homangiosarcoma, which is of course devastating, we'll at least have identified them prior to rupture of the mass, which does lead to longer survival with treatment.

Lisa Fortier 14:20

What are the issues you're facing and what are the next steps with getting this to clinical practice? IP, RD.

Janet Grimes 14:27

So there's a lot of that. Um, so I think a lot of uh RD to develop kind of this cage side test. So how do we take that PCR and translate it? You know, a lot of the um cage side tests, like the heartworm snap tests and things, are ELISA-based. And so could we do something like that with microRNA? Um, and then I think the other thing before really using this routinely clinically is we also need to look at dogs that have non-homangiosarcoma malignancies. Um, so with this study, we've left out, you know, a group of dogs. And so we just compared benign to hemangiosarcoma, but there are other malignancies. Um, some of them are not that terrible. Once you take out the spleen, you know, they can have a good long quality of life. And some of them are again gonna be pretty bad. And so we, I'd like to be able to figure out kind of do you fall into one of these three groups? And then I think the other malignancy group is still gonna be a little bit difficult because again, within that group, you could have things like marginal zone lymphoma, which is not too bad, or you could have like a stromal sarcoma, which is not good, um, and probably almost as bad as hemangiosarcoma. And so I think we just need to figure out, we need to drill down a little bit more into the different diagnoses that you can have. Um, but I think probably where we'll end up is, you know, benign other malignancy and hemangiosarcoma to start.

Limits Next Steps And Clinical Advice

Sarah Wright 15:59

So this is all really exciting. Like I love hearing about this. I feel like this could be a game changer. So, how does this fit into the broader future of veterinary oncology diagnostics?

Janet Grimes 16:08

Yeah, I agree with you. I also find it very exciting, which is why I keep keep working in this area, of course. But um I think this is just a huge area of research right now. So there's a big focus on developing minimally invasive diagnostic tests that are accurate and can identify disease before clinical signs even develop. And so this using microRNA is one method. There's many other molecules that can be detected in blood, and people have looked at nucleosomes, circulating DNA, circulating tumor cells, proteins, the list goes on and on and on. And so there's a huge increase in the availability and importantly a decrease in the cost of things like next generation sequencing, which allows us to get so much more information and data than we would otherwise. And so I think it's just a really promising area for cancer research. And microRNAs are not the only one, but they're kind of my focus. Not really, which is good when you're doing research because sometimes that's an unfortunate finding. Um, but we've, you know, I've done enough previous studies to kind of confirm that I know the expression of these microRNA are altered in dogs with hemandrousarcoma. And so we've done some work with tissue expression. We've also done some work with serum expression, and we we did expect to find a difference, and I was glad we did. Um, I don't know that I was expecting a particular number of microRNA to come out of the panel, um, but the ones that did um were kind of consistent with what I'd expect. So uh ones that are involved in angiogenesis and things like that, the hemandu sarcomas really, you know, needs those to drive it forward.

Sarah Wright 17:56

What advice would you give to veterinarians about integrating new diagnostic tools into practice?

Janet Grimes 18:01

Probably the biggest thing is you need to understand what the test is evaluating and what the results truly mean. Um, and so not all cancers release molecules into circulation that we can detect. Um, and for some of these molecules, there's a crossover between tumors. So two different tumor types may kind of release the same molecule, or like in the case of microRNA, um, you know, the same microRNA may be involved in the pathogenesis of two different tumors. And so that's why I think a panel is generally going to provide more specificity than a single marker. And then I think importantly, other illnesses can also lead to alterations in levels of the various molecules we test for. So some of these might be better as a screening tool in otherwise healthy dogs rather than a diagnostic tool for sick dogs. So that's something we need to investigate as well. Dogs that are more ill, do they have alterations in these same microRNA? And so I still think for right now, these new tools that are coming out, um, they're adjunctive to current practice, not a replacement. Um, and so it's important that we keep researching these and investigating how to improve the overall accuracy of these types of tests, but you still need to use your basics, physical exam, imaging, blood work, and things like that, and kind of use everything as a group to determine.

Sarah Wright 19:25

Sounds like another tool in the toolbox. Yes.

Where To Read And Closing

Sarah Wright 19:28

Well, Janet, thank you much so much for joining us and for sharing your important work too with our journals. Thanks, Janet. Yeah, I appreciate it. And for our listeners and viewers, you can read Janet's article on AJVR. I'm Sarah Wright here with Lisa Fortier. Be sure to tune in next week for another episode of Veterinary Vertex. And don't forget to leave us a rating and review on Apple Podcasts or wherever you listen.