Understanding Your Biomarker Report: Dr. Strickler (2025)

Manju George 0:00
Hello everyone. Welcome to Doc talks. I’m Dr Manju George, the Scientific Director at PALTOWN Development Foundation, the nonprofit that supports COLONTOWN. So all our DocTalks are recorded and the videos and the transcripts will be available in the Lecture Hall on COLONTOWN University. So today we have Dr John Strickler with us. He’s from Duke Cancer Center, and he’s going to talk to us about understanding our biomarker testing reports. And I know that Dr Strickler is a very familiar person in COLONTOWN, I’ll allow him to introduce himself. And he mentioned that he’s been leading a tumor board, so he’s been looking at a bunch of these reports, and I’m hoping that he’ll share all the wisdom with us. And Dr Strickler, welcome. And I also want to thank all the COLONTOWN members who shared their reports, so we could use those for this talk. Dr Strickler, welcome.

Dr. John Strickler 0:59
Thank you so much, Manju, it’s really a pleasure, an honor to be invited today. This is a topic that’s near and dear to my heart for many reasons. Number one, I’m an active GI medical oncologist. Number two, I run the molecular tumor board at Duke, so I literally look at 1000s of reports a year across the whole institution and help specifically our clinicians around understanding the reports. And third, I’m very involved in clinical research here at Duke, and I see that many of the people who have joined today are from the clinical research team here at Duke. And I would say this is a big pain point, not just for patients, but also for clinicians and for our clinical trial staff, these these reports are hard to understand. They can sometimes require a lot of background information that clinicians, patients and others are not given. So with that in mind, let’s go ahead and dive in. And I want to give a special shout out to the medical writer here, Ella Strickler, who’s a sophomore at Swarthmore College near Philadelphia. I asked her to help design these slides, because this is such a complex topic, and she’s so good at boiling things down in understandable ways. I thought her expertise would be perfect for this. So anyway, with that, let’s, let’s go ahead and dive in. We’re first just going to do really basic, two minute overview on some basic cancer terminology that can throw people off, and I don’t want to lose people along the way. Now, some of you watching this may know a lot of this already, so just know if it’s too basic for you, just wait. It’ll get a lot more complicated really fast. We’ll go into understanding biomarkers, interpreting biomarker report from my perspective, and then we’ll dive into some specific examples. So this will go from super basic to super complicated very quickly.

Dr. John Strickler 3:04
So let’s start first with the basic stuff. So DNA are the building blocks of life. I think of DNA as carrying the instructions for how to build a human, right? So it’s a double stranded molecule that stores genetic information in the form of these code letters that we call nucleotides. DNA gets transcribed into RNA, which is a single stranded molecule that carries instructions on how to make a protein, and proteins are what are needed to for the cells to work. Okay, so we talk a lot about genes, and we talk a lot about chromosomes. A gene is a section of a chromosome. The gene carries specific instructions for making a protein. The chromosome is where a bunch of genes are packed on, and these chromosomes are in the center of the cell, the nucleus of the cell, and cells are present in all living organisms, including us humans. And each cell is differentiated or form specific functions within our body to help us be functioning humans. When we have a tumor that’s a mass of tissue that develops in an organ due to uncontrolled cell growth. We can have benign tumors that are non cancerous and just stay in one spot, and then we can have malignant tumors that grow, spread, invade and go to other parts of the body. That’s what we’re all here to fight, are these malignant tumors. So one of the terms you’ll hear thrown around is this idea of the tumor micro environment? What is that exactly? Well, a tumor is not just living by itself in isolation. It lives in an ecosystem. It’s made up of different types of cells around it, not just the cancer cells themselves, but you have immune cells, what’s called stroma. Proteins, blood vessels, and all of that together makes up a micro environment. What I’m going to focus on though for the rest of this talk is that portion of the tumor micro environment that is comprised of cancerous cells.

Dr. John Strickler 5:14
Now, what makes cancerous cells different from normal tissue? Cells that are cancerous will divide. They grow too fast to specialize. They can trick the immune system into growing and spreading. They can invade other tissues and generally wreak havoc by spreading throughout the body. So with that, let’s dive into specific alterations that characterize these tumors. So cancers can result from alterations, sometimes mutations to a person’s genes, but not all mutations result in tumors. So an alteration is a change to a cell’s DNA. So now we’re getting kind of into the complicated stuff. A fusion is an event where maybe two genes that are normally functioning and doing their job normally, get joined together and create an aggressive, call it a super gene, right? Something that is causing a gene to cause a cell to grow uncontrollably, to invade, to spread. Amplification is where you get one gene, like right here. And instead of having one copy on the chromosome, you get multiple copies on the same chromosome. So instead of getting one protein, you get many proteins, it can create a malignant cell type. You’ll see other things like indels, insertions and deletions, and that’s where fragments of DNA are removed or inserted, thereby creating a more of a cancerous phenotype. You’ll also hear things like single nucleotide variants, where, literally, one of the nucleotides is replaced, and you can turn a functioning protein into a poorly functioning protein, which can lead to cancer. There are other confusing words that you may find in your report. I just laid it out here. You’ll hear things like copy number variations. You should have two copies of the gene normally, but if you have fewer than two copies, that’s called copy number loss. If you have more than two copies, that’s amplification, you’ll hear a term called Loss of Heterozygosity, and basically, that’s a situation where you’ve already lost maybe one copy of the gene, and you only have one left, and then you lose that, and you’ve lost your heterozygosity.

Dr. John Strickler 7:46
You’ll hear other terms, like frame shift mutations. I actually want to spend a minute on frame shift mutations. These ones I find very interesting. That’s where there’s maybe an insertion or a deletion of a sequence that causes the amino acids to shift very substantially. Amino acids make up a protein, and this can make for a very disorganized kind of appearing protein. And what’s interesting about these frame shift mutations is because they will make disorganized proteins, sometimes the immune system will recognize those proteins as foreign. So the immune system will sometimes lock in on the result of a frame shift mutation, and start to generate an anti-tumor response. So to look for frame shift mutations, look for the term Fs in the report, sometimes you’ll see that as a frame shift mutation. Stop-gain mutations isif you ever see an asterisk in the report, that’s one of these where the sequence just stopped prematurely. And you’ll also sometimes, in some reports, see the C. (dot) term at the beginning of a mutation, and that represents a variant effect at anucleotide or mRNA level, and a p. (dot) represents a variance effect at a protein level or amino acid level. So I recognize I’m moving really fast. I went from super basic to super complicated. Ultimately, the way to learn this stuff is just to spend time on it and see a few reports. But I just wanted to let you know, if you feel confused by looking at the report, you’re not alone. We as clinicians struggle with these terms. Medical students struggle with them, and our trial staff struggles. Everybody does, so don’t feel like you’re alone in that.

Dr. John Strickler 9:37
Okay. Now let’s pull out from some of the complicated stuff to a more general message, okay? It’s important to know you’re going to get a report. We call it next generation sequencing. They’re looking at 300, 400, 500 sometimes more, genes all at the same time, and you’ll see a huge laundry list of mutations. And it’s important to know that some of these mutations are passengers. They’re along for the ride. They’re not doing anything to support the tumor’s growth. Some of these mutations are what I call drivers. Okay? They are making that tumor go. They are the fuel for that cancer. And sometimes it can be very hard to know what’s a passenger and what’s a driver, but that will be something that I come back to here in in the next few minutes of this talk. Let’s go back, dive into some more technical terms that those of you who have seen these reports may have struggled with. You may see something on your report called tumor mutational burden, that is literally just a count of how many mutations there are in a section of DNA.

Dr. John Strickler 10:53
There are some excellent questions that Manju shared with me about tumor mutational burden. This topic generates a ton of questions, not just for patients, but also for clinicians. We can go into that in more detail, but just think of it as kind of a raw count, of the frequency of mutations in a sequence, in a section of DNA. Then there’s a last term that I think is super important, called micro satellite instability. If you or a loved one have a diagnosis of colon cancer, colorectal cancer, this is the one biomarker you need to know. This is probably the most important across all stages andmicro satellite instability basically refers to how many errors there are in the micro satellites. Why is this so actionable? Because we have therapies. This is what I call an immunotherapy biomarker. We have therapies designed for people with what’s called high levels of micro satellite instability, or MSI high. I’ll come back to these terms a little bit later. It also, in some cases, can be associated with what we call germ line disorders, or disorders you’re born with that predispose you to cancer. So this is my number one biomarker that I’m looking for in all patients. The challenge that you’ve got as a patient is we will use the same name, we will have many names for the same thing, and that’s where it can get very complicated. I will come back to all the different names for this in a moment. All right, so there are a lot of different ways that gene alterations can cause cancer. Not only do we get mutations, you can get what’s called hyper methylation, which can turn off a gene without a mutation that can cause cancer. Additionally, you can get a mutation in an oncogene that can basically, kind of flip that gene on perpetually, so that you’re driving a cancer. Or you can actually hypomethylate Something, thereby turning it on all the time. And then finally, you can get DNA repair defects. Normally in cells, we have these editors that go through and fix mistakes in DNA. And sometimes there are mutations that will cause errors to not be fixed. So the way I’ve explained this to some patients, the DNA in our body, in our tumors, in our chromosomes, that DNA, it divides every time the cell divides, and every time it divides, it has to make a copy of itself. But every time it makes a copy of itself, there are mistakes made, and so we need editors to come back and fix those mistakes. But if the editor is gone or missing, then you can get kind of an accumulation of mistakes witheach time the cell divides. So if you read a book and there’s a period in the wrong position or a comma in the wrong position, you can still read the book and understand it. It’s still a functioning book, but you can imagine if the book made errors every single time, eventually, you would just get kind of a garbled mess that you’re trying to read, and you wouldn’t understand it anymore. So that’s what’s going on with DNA repair, where this book is no longer readable and it can lead to cancer, okay,

Dr. John Strickler 14:28
Another really important term, germ line, there’s germ line DNA and somatic DNA. I think, is this is something that I think is important for most patients to know germline DNA is found in reproductive cells. It’s found in eggs and sperm, and these are passed on from parent to child, and some people can inherit germ line mutations that can predispose them to cancer. I know Manju that you’ve had a talk on Lynch syndrome, and DNA repair errors. Maybe you could put that in the chat, but that’s an example of a germ line DNA mutation that can lead to cancer that requires testing you not the cancer. Then there’s somatic DNA. This is only found in non reproductive cells, and these are accumulated during your lifetime. These are not passed on to family members who did not get this from a parent. Basically, this is something thathappened over the course of one’s lifetime, and these are usually the cause of cancer here. But just know there’s a way to test for these kinds of mutations, and then there’s a way to test for these. We’re going to focus on somatic DNA testing, though. All right, so let’s dive in now to biomarkers.

Dr. John Strickler 15:58
What is a biomarker? A biomarker is something that’s measurable in the body. It can help us detect and understand tumors. They’re usually biological molecules found in blood, tissue or fluids. So what are we looking for? We’re looking usually for DNA, but it could be proteins, could be DNA, could be RNA. There are a couple of different types of cancer biomarkers, there are biomarkers that will give prognosis, and then there are biomarkers that predict for outcomes. Both are very important. Prognostic biomarkers tell us whether we’ll do well or do poorly regardless of what we give. So they’re kind of not really modifiable factors by treatments. And then predictive biomarkers are biomarkers that I really like, because this tells me Aha, I found this biomarker. This patient will benefit from a special kind of drug. They’ll benefit from immune therapy, right? So these are the ones that we’re really going for when we’re looking for these reports. But prognostic biomarkers are also important.

Dr. John Strickler 17:10
How do we test for these biomarkers? We’ve got blood and we’ve got tissue now. Tissue does require collecting that. Tissue could have come from the operating room. Could be coming from a biopsy of, say, a spot in the liver, but it does require an invasive procedure. Blood is much easier. It just requires a blood draw. They do have different purposes and different strengths and weaknesses. I won’t go into too many details on that, but I generally like to use blood and tissue in a very complimentary way. It’s important for me as a clinician to know what blood can do and what tissue can do and how one can make up for the strengths and weaknesses of the other. So now let’s get into the specific tests. This first one, PCR, is called polymerase chain reaction. This has largely fallen out of favor. This requires taking a tiny piece, usually of DNA from a sample and making many copies to find biomarkers. The only time we will order PCR now in the United States is for micro satellite instability testing, typically in early stage disease, but we rarely order this anymore. When I started practice in 2011 this was our main way we tested for gene mutations, but it is not commonly used now, there’s another really important test that we use a lot, and we’re going to be using it a lot more, immunohistochemistry, IHC. This is actually not looking for changes in DNA or RNA. This is looking at the end result of the DNA or RNA. This is looking for protein expression. So this typically requires giving a special dye with protein tags to look for the presence of a protein. The classic one here would be like HER2 IHC. Then we’ve got FISH (Flurorescence In-Situ Hybridization), which is a really old test, and this basically sets up probes to bind to portions of the DNA that have a gene of interest. So classic one would be like HER2 or ERBB2, and we’re literally just counting how many probes there are on that chromosome to see if something is amplified. So you’ll typically only get FISH for colon cancer to look for HER2 amplification.

Dr. John Strickler 19:26
Another tool that we use, we’re using more lately, is RNA sequencing, and that looks at our what’s called RNA expression. So we’re literally looking for the presence of exons. This one we don’t use very much, so I will not focus on that one. This is the big one, NGS. Classically, we use this in the metastatic setting, stage IV, where we’re using next generation sequencing to take a small piece of tissue and look for, say, 300, 400, 500 biomarkers all at the same time. This is a very powerful tool. It’s also expensive, but it is probably the source of your confusion if you’re looking at a report, because it has tons of information. Then there’s one other test Manju, that you asked me to include, karyotyping. We don’t see this that often right now, but it may show up on a report. This basically looks at a picture of all the chromosomes and looks at their appearance, looking for alterations in number or shape. The classic karyotype example would be down syndrome, where see this little 21 right here, Down Syndrome would be like one extra chromosome. You’d see three copies of chromosome 21 we call that Trisomy 21

Dr. John Strickler 20:56
So those are all the different tests we have. I’m moving fast now. So let’s go into the colon cancer world, or colorectal cancer world. So what are the actionable biomarkers for colorectal cancer? There are many biomarkers linked to colon cancer. This is my map of the biomarkers I’m looking for. We’ve got microsatellite instability, MSI,-high or deficient mismatch repair, BRAF, V600 E, HER2 amplification, KRAS G12 C. There’s a bucket of fusions. You’ll see tumor mutational burden is kind of overlapped in here. This group of patients with no biomarker is shrinking every day or adding new biomarkers. But I use this term actionable. What does that actually mean to be actionable? Well, I think what it means for a patient might be different than what it means for a doctor. It’s kind of in the eye of the beholder. But when I look at something on a report and I’m defining it as actionable, it could mean something that I use to qualify that patient for an FDA approved therapy. So an example would be like an anti HER2 therapy that’s FDA approved for HER2 positive colon cancer, that would be an actionable finding. HER2 positive disease. Could be used to qualify a patient for a trial. Could be used to qualify a patient for an existing FDA approved therapy, but it’s approved for a different tumor type. So this can happen, where you find a biomarker that maybe it’s approved for breast cancer, but it’s not approved for colon cancer yet, but that could still be actionable. And then there are occasionally actionable findings that mean we need to test for something else. So an example would be a gene like BRCA2, if that’s found in a report, sometimes that means we need to test for whether that’s present in the germ line, if we find it in a somatic report. So that may be actionable for other reasons, there are other ways we can use to define actionability.

Dr. John Strickler 23:14
So when I’m confused about how actionable a gene is, I go to a website called OncoKB.org, and I will type in that gene, and it will classify that gene and that mutation into five different categories. One is level one, which is F for FDA approved drugs. Level two is it’s maybe not an FDA approved therapy, but it gives you access to a standard of care therapy. Level three means maybe there’s some weak evidence on how to do it, and then it goes down from there. Now level R1 R2 means that it is actionable, but it predicts resistance to some kind of therapy. So this is a tool that I’ve used when I get confused about actionability, but I’m focusing on somatic testing. There’s also germ line testing that many patients would have received. That’s testing of them, not of the tumor. Here, it’s a much simpler grouping. When you run that test, you could have a pathogenic finding, which means that the genetic alteration is known to predispose that patient to disease. You could have what’s called a variant of unknown significance, and that means exactly what it says. We just don’t know if that alteration increases the patient’s risk of cancer. And then finally, sometimes we see these benign variants, something that’s not normal, but it doesn’t cause cancer. It doesn’t cause cells to behave in any negative way.

Dr. John Strickler 24:43
All right, I want to go back to this question of biomarkers for metastatic colon cancer one more time. So I said, if you know one thing, go for this number one, MSI, or MMR. MSI stands for micro satellite instability. MMR stands for mismatch repair. We can test for this in three different ways. One is by immunohistochemistry, protein expression. So there, the proteins either work, meaning they’re proficient-pMMR, or they don’t work. They’re deficient, dMMR . Deficient mismatch repair predicts for immunotherapy sensitivity. But we could also run the exact same biomarker with PCR, Microsatellite instability-high is that high level of errors in those microsatellites. Microsatellite stable means that there’s not. MSI high also predicts for immunotherapy sensitivity. And then our next generation sequencing tests can also test for this, and they will list it as microsatellite instability-high or microsatellite stable. So there’s once again, it’s one biomarker, but there’s two different names for it and three different tests you can use to find it. Is that confusing enough? It is for many of us out in the clinic. The other thing where it gets really confusing, sometimes the tests will disagree with each other, and that causes all kinds of mayhem for us on the clinical side of things, the gold standard, the best one, we’ve got is PCR. The challenge is that many hospitals don’t do PCR. Most of them do IHC, all right.

Dr. John Strickler 26:33
The number two is KRAS, NRAS, exons, two through four and BRAF V600E. These are almost always tested out of NGS. In the old days, we used to do these with PCR. You can do them with tissue. That’s my preferred way to do it, but blood is a good backup and a complimentary tool. HER2 is an important biomarker, and you can test HER2 a number of ways as well. Immunohistochemistry for protein expression, FISH for gene amplification, or NGS for gene amplification. And by the way, this has many names too. HER2 is the protein, the gene is called ERBB2. So on your report, you may see ERBB2 amplification. That’s the same as HER2, okay, so it can be a little confusing. The one thing that I will say is a trick that can be a little bit confusing, is occasionally you’ll see an ERBB2 mutation on a report. That’s not the same thing as an amplification. Amplification means more copies of the gene. Mutation means that the gene is got an alteration that maybe changes its function or form. But it doesn’t mean that there’s more copies of it, and it’s truly the amplification, IHC 3+ that is actionable, not the mutation.

Dr. John Strickler 28:02
Then there are a whole host of other rare alterations that are usually found in less than one in 500 patients. So I won’t focus too much on those today. TMB causes all kinds of confusion, and we’ll come back to that one during the questionsthat’s Tumor Mutational Burden. Okay, so I went from very simple to very confusing very quickly. So let’s end with some advice and thoughts before we dive into some of these reports. I think number one, biomarker testing is essential. It is how we manage colorectal cancer today, regardless of your stage, there is some type of biomarker testing that needs tobe done, but that biomarker you need and the test for it depends on the stage. Stages one through three were typically focused, most cases, just on microsatellite instability or mismatch repair, but not always, but that’s really the key focus when you get into stage four, metastatic, that’s when you need to test for all the biomarkers. And as I’ve said many times before, be careful. There are many different ways to test for the same biomarker, and the same biomarker could have multiple names, and they all mean the same thing. So it’s okay to ask your doctor, ask Manju, you know, these things are very confusing. COLONTOWN University is an incredible resource. Many of these biomarkers have been explained very well here at this website.

Dr. John Strickler 29:35
The other thing is, and when you talk to doctors about why they are not ordering some biomarker tests, they will usually say NGS testing is expensive, and I don’t want my patient to get nailed with an unexpected charge. Some of these list prices are 5000, 10,00 25,000$. What I tell patients around cost– it is highly likely you will see on your Explanation of Benefits some extraordinarily high charge for this test, and it is not uncommon for your insurer to say denied. Almost never does that lead to an actual charge coming to you as an invoice. If you ever do see an invoice that is exorbitant. I mean, and I would call exorbitant, more than $500, talk to your doctor, and sometimes you can call the diagnostic vendor themselves, and they can give you a form to fill out. And as long as you’re not a billionaire, they will waive most of the charge, not always, but it is extremely rare when, in my role here at Duke running molecular tumor board, it’s extremely rare that a patient gets a bill that is not manageable through this process. So just know there are resources out there. I’ve also never heard of a reputable commercial vendor sending a patient to collections. Please correct me if I’m wrong on that Manju, but I’ve never heard of a single case of a patient being sent to collections for a biomarker test. But these tests are expensive and they’re very difficult to keep track of, so, I beg of you keep a copy of your biomarker report. I cannot tell you how many times I see a patient from the outside they have a $25,000 biomarker report. It lives in some other parallel universe outside of the electronic medical record, and the patient doesn’t have it, and it didn’t make it to me with the referral paperwork. You can really save yourself a lot of grief by keeping a copy for yourself, sometimes electronically, maybe even as a paper copy, and bringing it with you to an appointment with a new doctor, new oncologist.

Dr. John Strickler 31:55
And then I would say, know your actionable biomarkers, which I am calling the big five. And this is really for metastatic disease, the big five. This is micro satellite instability, mismatch repair, BRAF V600E, KRAS, NRAS and HER2. Those are the big five. That’s what I’m honing in on, on these reports. And when you’re confused about what a report says, Ask your doctor, Ask your nurse, and use the amazing resources here at PALTOWN. All right, there were such great questions that made it to me. Manju, I thought I should answer some of them preemptively. So here’s one that I get. My doctor called me triple wild type. Is that good? And many patients are really curious about what it means to be wild type. Wild type is another medical term for not mutated. I actually consider it to be, well, let me back up. No patient is wild type. The tumor is wild type. Okay, so just know if you’re called wild type, it’s actually the tumor, you are normal. So a patient is not wild type. Triple wild type means that the three major biomarkers that we look at look at KRAS, NRAS and BRAF V600E are not mutated. That means that the patient is a candidate for anti EGFR therapies, so I would consider it to be good, but it’s not bad, if you have a mutation, it’s just what is driving that cancer.

Dr. John Strickler 33:30
Let’s go to the next question here. What do these mutations mean in terms of my prognosis? Most mutations have no major impact on prognosis. There’s one exception to that rule, and that’s BRAF V600 E, which used to be a marker of bad prognosis, but now we have lots of treatments for that. But in general, I do get lots of questions about, is this bad orgood? I would usually it just is. We want to know what’s driving that cancer. We want to know what it has. Why did that normal cell turn into a cancerous cell? Most of them have no good or bad connotation to them. It’s good to do the test so that we know what’s driving that cancer. Here’s a great question. I have a TP 53 APC and a SMAD4 mutation. How do I use this information to find a trial? We are still in an era where we get hundreds of of biomarkers being tested on a next gen sequencing report, and yet very few of them have therapies associated. I do think it’s a little bit unfair on thesereports where they will say there’s clinical trials for SMAD$, APC mutations. It’s a little bit unfair because in most cases, those are not trials that are geared, really, to attack that mutation in a specific way. So I would say, use all the resources of COLONTOWN, but just know that the vast majority of these still don’t have drugs available yet, and that’s okay. We we just want to be smart about how we direct people towards trials. I think there is a desire for when we talk about trials, really, at the end of the day, we want treatments that will help our patients, right? So we don’t want to direct people towards things that are not likely to benefit, away from things that are likely to benefit. So if we look at this list of mutations, and we say this is not a good fit for trials. It’s okay. It means that we’re going to find a better fit for things that might help more.

Dr. John Strickler 35:48
All right, my doctor says my mutations don’t matter, but I have four. How could these not matter? What a great question. Every mutation matters, right? But this goes back to that question of some mutations are drivers and some are passengers. And I suspect what the doctor was talking about is like, there’s not a treatment for any four of those mutations. But they matter because they probably together were responsible for the formation of that cancer. So they do matter, but from the standpoint of clinician may not always direct clinical decision making at that particular moment. Why does my tumor mutational burden keep changing every time it’s checked? Oh, I love this question. Tumor mutational burden is a very frustrating number. All that’s doing is counting the number of mutations on some portion of the DNA, and tumors evolve over time, so they can change. They’re allowed to change the number of mutations they have on their DNA. Typically, the number of mutations would go up over time. But the other part of it is it’s not a reproducible number. So if you send two different samples, you can get two different numbers from the same patient. Additionally, the tests don’t agree on what tumor mutational burden even means. So a blood tumor mutational burden will never match a tissue tumor mutational burden. They use different algorithms, different bioinformatics. They might use different sections of the chromosome, so they’re counting different things. So I think we put probably too much stock in these tumor mutational burden numbers, and in fact, in colorectal cancer, having a tumor mutational burden that’s elevated does not always predict for benefit from immune therapy. So I tend to downplay this number a little bit. We’ll come back to this percentage question. But variant allele frequency, I’ll save this one for the reports.

Dr. John Strickler 37:52
My tumor mutational burden is zero, but my report has two mutations. How could this be? Well, probably the portion of the DNA they were using to count the mutations didn’t have any on it, but the portion they were not using for tumor mutational burden had mutations, so that that’s probably how that came about. And then, when is the best time to get a liquid biopsy? Fantastic question. Liquid biopsy is looking at fragments of tumor DNA in the bloodstream. So when you’re on active treatment, it will tend to suppress those fragments in the bloodstream, so that will be a lower yield of mutations. So typically we will get this when the patient is off treatment, and it’s actually a much more effective test. The more disease there is, the more fragments of DNA and the more helpful information you can get from it. And then, why does my liquid biopsy show fewer mutations than my tissue NGS? I would say typically, that’s the case, well, there’s two reasons. Number one, because the liquid biopsies will typically have fewer genes that are covered on the reports than the tissue, so it’s a smaller panel, but the other portion is you need enough fragments of DNA to be detected on the liquid biopsy to report it out. So tissue NGS sometimes will give a more comprehensive panel of that portion of the tumor, but the liquid will just look at the fragments of the DNA in the bloodstream. So if the person is on active treatment, sometimes there will not be a lot of fragments floating around for that test to find.

Dr. John Strickler 39:35
All right, let’s dive into the reports. What I like to do first, I look at the first page, always the top line summary, start with the most actionable findings, and that is MSI. You’ll see TMB. These are the immunotherapy markers, MSI being the key one. Then I’m always looking for KRAS NRAS and BRAF. And then I’m rounding out my Big Five. If I’m looking for ERBB2 or HER2 amplification, I want to see the big five in every patient with a colorectal cancer diagnosis. And then I’m thinking, after I get through these big five, I’m thinking about other FDA approved therapies or trial options. Here’s an example of one, circulating tumor DNA report number one, I’m thinking about, Okay, why did I get this circulating tumor DNA report? What’s the clinical context?

Dr. John Strickler 40:29
And then I’m looking at the genes which are listed right here. TP53 APC, CDK6, ERBB2, GATA3, SRC. Here, looking at the percentage, this is the percentage, mutant allele frequency. This is what percentage of this gene is mutated in the bloodstream. This is a pretty high percentage, at 46%. The other thing I’m looking at is the names. R282W, this is the normal, arginine position, 282, has been– isn’t this tryptophan as W, all right, thank you. So this arginine has been replaced with tryptophan at this position. It’s almost half of all the DNA that they found in the bloodstream is this variant here, which is not normal. Further down, you’ll see this asterisk. That’s one of those premature stops. So here at serine 86 there’s this premature stop. And further down, you see amp that stands for amplification, and they just gave three pluses here to indicate that it’s the highest level of amplification. So you’re putting all that into context for the patient, and from that, getting a picture of what might be actionable here in this report, I would say I’m looking for the big five, right? Where’s my MSI, well, it’s not here on this report. It’s not always on a report. KRAS, NRAS. BRAF, not here, but there’s a HER2 amplification. ERBB, two stands for HER2. Okay, all right, let’s jump over to these actual reports.

Dr. John Strickler 42:25
So I’m going to start sharing these examples. All right, great, now it’s working. All right, let’s go through some examples. This is a patient A, Guardant 360, so this is a liquid biopsy. This is a blood draw. October 2023, you have very kindly zoomed in on the report. So what am I looking at? The front page carries the major summary. What’s my big five? I’m looking for MSI. I’m looking for KRAS, NRAS, BRAF. I’m looking for HER2. So out of this, we see MSI down here, not detected up here, BRAF V600E. Actually, it’s right there. So I know this tumor is BRAF V600E, mutated. Look at this associated FDA approved therapies, yes, encorafenib and Cetuximab. Is FDA approved for this? Of course, we have a new, updated approval now, it’s first line with FOLFOX. Are there trials available? They always say yes, but it’s not always true. So just I will always be suspicious of this. And then this is this variant allele frequency. This tells me what percentage of the BRAF here is mutated. It’s 25% this is a pretty high number. Honestly, anything over 1% I consider to be meaningful. Any other follow up questions on this one Manju, before I advance,

Manju George 43:55
I think this is good.

Dr. John Strickler 43:56
Yeah, all right, here is patient B. Yeah, there’s tumor mutational burden. As I said, I don’t know tumor mutational burden. This one is low at three. There’s technically an FDA approval for TMB greater than 10. But for colorectal cancer, I don’t consider anything under 40 to be truly actionable, certainly not in a blood report, because these numbers tend to be inflated in the blood tests and will tell you to take immune therapy, but actually it’s not likely to be beneficial. And this is the second page. Let’s actually dive into another report.

Manju George 44:37
So this is the same patient. This is the Guardant Infinity.

Dr. John Strickler 44:40
Yeah, ah, Guardent Infinity. So they changed the test, and Infinity means it’s got a lot more targets, and it’s looking for other things. Same patient still. Wow, very different. So that the prior one was two years ago, and now two years has transpired, and we can see that this tumor evolved. So let’s start with MSI, not detected. BRAF still here. But interestingly, the mutational frequency has gone down. So something’s happened that’s different, right? Like that’s gonedown. Maybe the patient had effective treatment that knocked down those mutated fragments. But there’s new things, KRAS and NRAS. These have been acquired. They weren’t there before. One of the things we know about KRAS and N RAS is that when you treat, this right here with targeted therapies, that tumor is trying to evolve to survive that targeted therapy. So it will grow out these resistant variants, and you’ll see it right here. So these will typically be grow out to drive resistance. And look at the mutant allele frequency coming in very low at 0. 4% and 0.2% that’s a classic sign. It’s much lower than the dominant driver mutation. So this is what you call subclonal. It’s like trying to grow out to to evade the targeted therapy. The other thing we see now on this report is CHEK2 coming in at 45% Now usually when you see 45% it’s no big deal, but when I see 45% I’m worried about a germ line mutation. When things hover right around 50% you do have to put in your back of mind, maybe this person was born with this mutation. Maybe it didn’t even come from the cancer itself. And Guardant has helped us with this, and they’ve actually called it out on the report, suspected germ line variant. Let me just bring back my pen right here. Suspected germ line variant. They’ve also in this infinity panel added tumor fraction, and that’s basically telling you how, like an absolute volume of circling tumor DNA. Anything more than 1% I consider to be meaningful. And this one’s pretty high, at 13%. You see the tumor mutational burden has gone up. But I would say that that is likely to be an artifact of all the treatment the patient has received over time. It does not mean that the patient should get immune therapy. MSI high is still not detected. Now, this patient may say, Wait a second, I’ve got all these other mutations. Don’t they matter? I would call these ones passengers, not drivers. This one is the driver for sure, and then you’ve got emerging drivers right here. So, so that’s, that’s my takeaway on this. Any other comments, Manju before I advance to the next one?

Manju George 48:01
I think it’s good we can go to the next one.

Dr. John Strickler 48:05
Yeah. Oh, okay. Let’s talk about this. This one, you will see sometimes variants of potential clonal hematopoiesis with all these different mutations here. What this is telling you is these mutations do not come from the tumor. They come from some other compartment of the body, typically the myeloid compartment, the bone marrow. So these are not actionable directly. In rare cases, these can become precursors for things like leukemia, but that is extremely unusual. But just know that they will sometimes call these out. It means they’re not tumor derived, all right.

Manju George 48:54
This is the next patient.

Dr. John Strickler 48:56
Next patient. TissueNext Okay, so this is a tissue NGS report and so what am I doing when I get this? I love the first page. Where’s my favorite marker, MSI status. And here they’re calling out microsatellite stable. So this is not and we’ve got a tumor mutational burden that, for me, is low at 11. So I’m saying, okay, my immunotherapy markers are telling me no immunotherapy right now, at least not outside of a trial. Now, let’s go into KRAS, NRAS, BRAF. There’s no BRAF here, but right there. KRAS, G13D, associated FDA approved therapies. Well, this is one of these markers that tells you what not to give. So that X right here says, Don’t give Cetuximab or panitumumab because it’s known to drive resistance. And then I find this section to be quite unhelpful and confusing to patients. Clinical Trial availability. There are real trials out there for KRAS. But they will tend to, I think, paint a picture that suggests that it’s piece of cake to get on a trial for APCwhen, in fact, it’s not. And I think that can be very confusing for patients, but there are trials emerging for things like KRAS G13D, but they are extremely hard to get access to right now. Maybe next year we’ll be talking about how to attack KRAS G13D. But I’d say we’re 12 to 18 months away. Any other questions that would come up on this one? Manju?

Manju George 50:33
I think this is okay. And then we have some other tests for the same patient. I want you to compare those.

Dr. John Strickler 50:39
Yeah, all right. So this is patient B, had a TissueNext, so tissue NGS, and this is the same patient. And wait a second, where did everything go? Right? So all we see now is this one mutation, GNAS, R201H, coming in at 0.5%. Where did the KRAS mutation go? Well, this is an example of a low shedding tumor, that tumor volume is too low to make lots of DNA fragments to go into the bloodstream

Manju George 51:20
Go to the next one,

Dr. John Strickler 51:21
Yeah, or the patient is on very active treatment that’s suppressing that cancer in a way that it can’t be picked up. All right. So this one was August of 24 and now all of a sudden, we advanced to February of 2025 for the same patient. And now all of a sudden, everything’s changed. That KRAS mutation, did it come back, or was it always there? It was always there. It just wasn’t picked up. So the the liquid biopsies, these blood tests, they do not tell you you have a non mutated tumor. They will only tell you if they detect it. Okay, so there’s a difference between wild type and not detected. Liquid biopsies only tell you if you detect it and it’s coming back, but it’s coming back at a very tiny microscopic fraction, and you can follow out the individual variants on this tumor response map as well. So the KRAS went away and it came back, but actually it was never gone. It was just not detectable,

Manju George 52:22
Yeah, and if you look at the CHIP, the GNAS is in that list, yeah,

Dr. John Strickler 52:28
I love that example. So there’s the GNAS and what she’s saying here is all we saw back in August of 24 was GNAS. Why was the KRAS mutation gone and the GNASs was there? Well, because this one didn’t come from the cancer. It came from the bone marrow. Yes, so it was always there. And if something’s coming from the bone marrow, or for germ line, you can pick it up regardless of the tumor volume. So great case.

Dr. John Strickler 52:32
So we can go to the next one, sorry.

Dr. John Strickler 52:40
Oh yeah, we’re going to run out of time. Patient C, tumor tissue. Oh man. Okay. So this is a report I’ve never seen before in my life. All right, so now if, if you’re a patient out there looking at it looks like Greek to you, just know this report looks like Greek to me. I don’t recognize this. So first page tells me nothing about, well, okay, so markers,–wild type BRAF, KRAS, MSI, stable. And I’m like, well, where’s NRAS, like, did they test it? I don’t know. So now we’ve got to go to the second page. So here they’ve grouped the variables. And like I said, I want to go for the big five, first micro satellite instability, no evidence for MSI, which means it’s MSS Okay. That’s a very confusing way to word things. What about K RAS, NRAS and BRAF? Well, I’ve got to hunt around for this one. Tumor drivers. There it is. No KRAS, NRAS or BRAF. HER2, did they look for that? Well, that would be a copy number variant, right? So I’m trying to find the section on that, and I don’t, and basically, I think it’s, it would probably be in the tumor driver section, right? What a confusing report. And then you’re looking for other things. They found a germ line variant right here, this ERCC2 and MUTYH, I would recommend following that up with a genetic counselor, just so you can understand what that means better. And then this one will even go into tumor, micro environment kind of stuff, right? ,

Manju George 55:01
HRD. HRD, which is coming up here. This is a European test.

Dr. John Strickler 55:06
So, yeah, so HRD is not pertinent to colorectal cancer. We do not have any therapeutic implications for HRD. It is highly relevant to things like ovarian cancer. What happens sometimes is a vendor will create a test. And instead of just reporting HRD for ovarian cancer, they’ll report it out for everybody, even if it’s not pertinent to colorectal cancer. So my apologies if that causes confusion for anyone. Great example as well. And now you can see me struggle with these reports.

Manju George 55:40
Yeah, and here, this is just another page where they’re giving potential therapeutic relevance. So it’s the other report. Yeah, yeah. So we can go to the next

Dr. John Strickler 55:53
Okay, Altera, I love this one. Sorry. Did you want to say something?

Manju George 55:59
In the previous one, that report has different sections where they talk about copy number alterations and germ line testing, so, and then the HLA typing too. So it seems more extensive than what we see here. I

Dr. John Strickler 56:14
I agree, and they’re really in the weeds on their terminology here. Here’s that C dot terminology, boy, this is like Greek to most clinicians. So this is, I can see why this is European, because, you know, we’re very consumer oriented and clinician oriented. This one is built for pathologists. I feel like, right, all right. Altera. So many of you have had Signatera done at some point. You can check a box that gives you what’s called a next generation NGS profile from that, and this is the socalled Altera test. And so once again, this is a same report. It’s showing the same things, but in a different way. Let’s start with the immunotherapy markers, MSI and TMB micro satellite stable over here, buried.. Oh, here we go. Genomic snapshot, right there, stable. However, I said the 10 is kind of the number for an FDA approval. I like to see more than 40 to for somebody to be truly eligible for immune therapy. This one’s coming in 129 which is off the charts high. So that makes me suspicious. Something is weird with this tumor, and we’ve got a list of mutations a mile long, right? So something is wrong with this. So there, there’s clearly a DNA repair defect, and we have an editor called polymerase E, and that is mutated. I suspect what happened to this cancer is that could be germ line, could be born with this problem, or could be acquired. But this is something that is this protein, when it functions correctly, is designed to fix mistakes, but this has been broken. Either the patient was born with this or acquired it, and now we’ve got a laundry list of mutations. This predicts sensitivity to immune therapy, and I would be thinking very strongly about immune therapy for this patient.

Dr. John Strickler 56:24
So yeah, yeah, in the next few pages, they have gone into each of the mutations. So I put some a couple of samples.

Dr. John Strickler 57:39
All right,so this is for the same patient. Yes, PDL one scores are not currently validated for colorectal cancer, but they do it for everybody. So I would not focus too much on that. PMS2 is once once again, that’s an editor function. So that’s one of these proteins that’s designed to fix mistakes in DNA. Same with MSH2 and there’s MSI stable. They will give a long description of the function of the gene. I wish I could tell you that clinicians read these, but they rarely do.

Manju George 59:07
Okay, so the next page, again, it’s more of talking about the particular mutation.

Dr. John Strickler 59:13
So this is what I consider to be the actionable driver out of this. This so called POLE it’s not part of the Big Five, and what I mean by that is it’s extremely rare and but it’s when you find it. This one is actionable, because it’s created this hyper mutated phenotype that could lead to could have germline implications, so the patient needs germ line testing, and then also could put this patient in line for immune therapy. So really important,

Manju George 59:43
Yeah, one more page. I think it’s about the same patient.

Dr. John Strickler 59:48
Sorry, let me close that out. Yep, and there’s a drug, pembrolizumab, with the POLE predicted beneficial, and they give a –I think this was published Jun Gong from, he’s at Cedar Sinai, I think, right? And, and this was a 81 year old male who had this type of mutation with a high TMB that had a great response to pembrolizumab. So that’s a really nice write up there. And then there’s another example. Oh, boy, CARIS, we’re almost done today, so we’re about to wrap up, but I find CARIS is one of these mega tests that test for every biomarker under the sun, and it can be a little bit difficult to digest if you’re a patient. To understand this report, the reason why I call this a mega test is because it’s doing everything– it’s doing next generation sequencing, NGS, it’s doing immunohistochemistry. It’s doing all, it’s doing RNA, everything it’s throwing at it. So let’s go through this report. When I see a big, jumbled report, I’m going for the biomarkers that matter to me. The Big Five– start with so mismatch repair, otherwise known as MSI high, and this is stable. Okay, tumor mutational burden is out here at nine, so not an immunotherapy candidate. Then I’m looking for KRAS, NRAS and BRAF,and they very nicely put these right up front. Mutation not detected. Okay, so then I’m going from there into some of the other rare biomarkers. And I don’t see any here, unless APC, P53 etc, so this is what I would call a triple wild type microsatellite, stable, anti EGFR therapy candidate. Any questions out of this one, Manju,

Manju George 1:01:43
I think this looks good.

Dr. John Strickler 1:01:45
And, in fact, they have said this patient is a candidate for anti EGFR therapies based on you know, this profile.

Manju George 1:01:53
Yeah. The only question I have is that they’ve used some algorithm to calculate benefit from– they have this therapies of uncertain benefit, and they put capecitabine, fluorouracil, irinotecan, those kind of things. And then in another report, they have, like a some sensitivity to FOLFOX. So do you actually look at those?

Dr. John Strickler 1:01:54
Yeah, so that is great question that is proprietary to CARIS. It is not considered accepted practice to use this data to change management. So this is an example of something that’s thrown into a report that can be kind of disconcerting for patient. Wait a second, I’m getting capecitabine and I’m getting fluorouracil you know, like, why is my doctor giving me something that has uncertain benefit? Just know that not all of the things in the report can or should lead to a change in practice. And this is an example that I think that that the vendor is hoping that one day they could use these markers to help be smarter about what chemotherapy to give. But they’re not quite there yet where it’s accepted into routine practice. Okay, I’m delicately answering because they might be watching this.

Manju George 1:03:15
Okay, I think we can go to the last one, which is the Foundation One report.

Dr. John Strickler 1:03:22
There’s another CARIS. All right, so let’s This is the one that we order a lot of here at Duke, Foundation One. This is a very clean report, very easy to read, because they put it all right there in the top on the first page, in a very simple way. So this is a patient with a peritoneal metastasis. Was it a colon primary, Manju? And so microsatellite stable TMB is six, which is on the low side. We’re not reading HRD signatures for colon, so I’ll X that out. So this is not an immunotherapy sensitive cancer. And then they’ve listed some other variants. So I’m looking KRAS, NRAS, BRAF and they are KRAS, G 12 B, is actionable, or at least tells us what the tumor is resistant to, which is anti EGFR therapy. So there’s KRASG12 D, and then I’m looking for all the all the other variants together. So we see APC and FBXW7, these are what I would call passengers. There’s PIK3CA, which could be a driver, could be a passenger. NBN is highly unusual for colon. So I’m kind of curious about that. That’s the type of one I would look up. Anything I’m missing on this one Manju for discussion.

Manju George 1:04:53
I think this is good. So basically, then we can look up one question. This is a Canadian I want to see. Include one of the Canadian almost in house tests. So one of the question is that when you have a number of mutations, how do you decide which ones are drivers versus passengers, or is it already set for colorectal cancer?

Dr. John Strickler 1:05:16
Yeah, wonderful question! A driver actually is a difficult term to define, versus passenger. I think it starts to overlap with this idea of actionability. So a driver means that that mutation is causing that tumor to grow, to spread, it might portend sensitivity to certain treatments and resistance to others. It overlaps with that clinical actionability term. I’ll switch over to passenger. Passenger mutations mean that they’re present throughout the cancer, but perform no function. Like, if you were to eradicate that mutation, there would still be a cancer there, right? So, like, I’m going to make my APC mutation go away, yes, but that cancer would still grow because it’s got some other mutation driving it, right. Sometimes mutations just happen because the cell is dividing so fast that it just accumulates that mutation and it serves no functional purpose for the cancer. A classic sign of a driver mutation is one that’s mutually exclusive from others. So frequently, at a new diagnosis, you’ll see like a KRAS G12V mutation, but not a second KRAS mutation. So that’s a sign of a driver, because it doesn’t have a second one around, right? So, you know that would be how I would define it. Sorry, that’s not a very good explanation.

Manju George 1:07:03
Okay, that’s fine. It’s past four o’clock. Do you have a little bit of time?

Dr. John Strickler 1:07:07
I can wait a few minutes?

Manju George 1:07:08
Yeah, okay, okay, so that’s great. So I’m looking at the questions in chat. I think one of the main–this has been asked a few times, is, what’s the frequency that you want to look at biomarkers? Like, if you had one at diagnosis, then when should you have the next one? Or, would you repeat them over a specific period of time?

Dr. John Strickler 1:07:30
Excellent question. So there’s two aspects of that. Number one, does a biomarker change, right? Some biomarkers change, some don’t. So you would certainly test at another time point, if you thought perhaps there was a biomarker change that happened over time, maybe under selective pressure of treatment. The second aspect of it, of a biomarker test is how resource intensive, how hard is it to even run the test? And how expensive is that? Right? So in a perfect world, we would run biomarker tests all the time on the odd chance that something might change, and, maybe that micro satellite stable tumor turns to MSI high, but the odds of that happening is extremely low. But if the cost of the test is a penny every time you ask, you might ask it every time. But if it’s $5,000 every time you ask, then you might not ask it again, right? Because it’s so unlikely. So the bottom line is, everybody should get biomarker testing at the time of diagnosis. The biomarker testing is defined by the stage and what you’re looking for. I do repeat biomarker testing every few years, because I have found evolution over many years and many patients, particularly in patients who have wild type KRAS and NRAS wild type disease. The other scenario where I’ll check biomarkers frequently is if I give a targeted therapy like an anti BRAF therapy, you’ll see emergence of resistance, and sometimes you can identify the source of the resistance out of, say, a liquid biopsy test. So all that is to say it’s okay to just get biomarker testing once in many cases, but it is not uncommon to recheck it after a targeted therapy or after a few years has transpired, so long as the cost is not great, or the effort to go run that test, meaning a new biopsy is not too difficult for the patient.

Manju George 1:09:33
Okay, thank you. The other question is, nowadays, many of the early stage patients are also getting their biomarkers tested. What advice do you have? Because, you know, patients get really anxious when they see they are stage two or three, and they see they have a KRAS, G12D mutation or something like that. What’s your advice in such situations?

Dr. John Strickler 1:09:56
Yeah, so until very recently, all we were checking in stage two three would be microsatellite instability and mismatch repair, which are very low cost tests. That is now changing, because we now have variants that we’re looking for that could potentially direct, say, for example, the use of aspirin. Have you done a Colontown DocTalk on the aspirin data. Oh, I can give this talk,

Manju George 1:10:25
Not yet, not yet. We’ll call you back!

Dr. John Strickler 1:10:29
So now all of a sudden, we as clinicians are struggling with this question, because we know that insurance will fight us tooth and nail to run more biomarkers than, say, MSI mismatch repair, but you’d have to test for everything, 300 genes together. And if you see a KRAs, G12D, no, it has absolutely no impact on what you would do, as in that for that person with stage two, three disease, meaning if they’re going to get adjuvant chemotherapy, they’re going to get it regardless of whether there’s a K RAS mutation present. It does confer slightly worse prognosis, but not much. It is just a slight increase in risk that the cancer will come back, but it is information that could be used one day, but is not relevant to a stage two, three patient in terms of what we do.

Manju George 1:11:24
Okay, thank you. And then the next question is, many patients have reports with many mutations, right? And they come in, for example, we can see it in COLONTOWN. They’ll ask, okay, how do I look for clinical trials? So what advice do you have

Dr. John Strickler 1:11:42
it’s really hard to look for trials. And I know I’ve got my trial team on this today who are watching this talk, and they know how hard it is to qualify patients for trials. We need to make it easier, is the big thing. And I know that’s a personal mission of yours. Manju, to make it easier, to enroll people to trials. I think the first thing is talk to your doctor. Say, Hey, are there any trials? For me, if you are in particularly a community setting that doesn’t do a lot of trials, and you really want to be part of a trial, my recommendation is to get a second opinion from a large trial center within a reachable distance of your home to talk about it with. You know, if you live in Denver and maybe you’re in a community practice, you could go drive to University of Colorado and say, hey, just want to make sure there’s not a trial around. It’s okay to do that, and and we support that. There are other resources out there. Do you want to talk about what trials resources you have?

Manju George 1:12:54
Yeah, yeah, like in COLONTOWN, like we have, we have lots of resources. We have the clinical trials group. So that’ll be one place. So what I wanted to really say is that, like many a times, people see the mutations on their reports, and then they are really stuck on looking for a trial for a particular mutation. Instead of that, what might be more relevant is to look at, you know, if, for example, there are combination immunotherapy trials, and if they don’t have, say, mets in the liver, instead of looking for a KRAS G12V targeted trial, it might be easier to look at immunotherapy trials.

Dr. John Strickler 1:13:32
I couldn’t agree more, there are three classes of trials right there, right now that we offer. The first is targeted therapy trials. Those are designed for specific mutations. The big ones right now are K RAS, G12D in particular, but occasionally, we will get spots for some of the other KRAS mutations. Those are very much phase one. They’re early phase trials with long wait lists. The second class are the immunotherapy trials, which do not require, usually, a specific mutation. So there, your biomarker report is not helpful at all for those, it’s more where your cancer is, right. So absence of liver metastasis will put you in line for very exciting immunotherapy trials. Sometimes we will have to have another talk, another day about that data. Maybe Nick DeVito, my colleague, can give that talk, because he’s looking for patients for his trial, which is bot bal for first line metastatic colon cancer without liver metastases. That’s an intriguing trial. And then the third are what I call new ways to give chemo– antibody drug conjugates. These frequently will not require mutational testing, but they might require what’s called expression where they’re looking for protein on the cell surface to qualify a patient for treatment. So yes, I agree. It’s probably a good idea to think about what class of trial you’re thinking about, and just know that that Foundation Medicine report is going to miss a big group of trials out there that might even be more promising for the patient.

Manju George 1:15:15
Okay, okay, thank you. Then another question is like, say, for example, when you’re looking at HER2 over expression, or things like that, does say, for example, the mode of testing, like a tissue testing, is that better when compared to say, for example, a liquid biopsy?

Dr. John Strickler 1:15:31
Yes, great question. The gold standard, the one that we really lean on the most, is tissue. There’s a reason for this. You know tissue is sampling that tumor. You can identify in tissue based on IHC, FISH or NGS and qualify for FDA approved therapies. IHC looks for protein expression, FISH and NGS look for Gene amplification. Blood is effective. You can believe it if it’s positive. However, if you don’t see ERBB2 amplification in blood, it does not mean that you’re HER2 negative or wild type. It means that you’re not detecting it. So about 30% of patients can be HER2 positive, but it’ll show up as normal on the blood test. So just know you can rule in, but you can’t rule out with blood, only tissue can be used to rule out.

Manju George 1:16:33
Okay, thank you. I think there’s one question about HER2 amplification. So this person says, I have HER2 amplification, but KRAS, BRAF, NRAS negative with no micro satellite instability. Can you tell me why traditional chemo wouldn’t work for me? Why is my tumor refractory to EGFR targeted treatments?

Dr. John Strickler 1:16:54
Oh, that’s a very that’s a great question. So first of all, one, with HER2 amplification or HER2 over expressing disease, you can still benefit from standard chemotherapy. It is still effective. It is still standard. We’ve given it for years. It is still the first line standard of care. There is a lot of evidence out there that HER2 will rescue a tumor cell when you give an anti EGFR therapy. So think of it like the light switch is in the on position. And when you give an anti EGFR therapy, you’re not turning the light switch off like that, HER2 is keeping the light on. So you need a strategy really geared more towards HER2 so we will tend to favor anti HER2 therapies for a HER2 positive cancer. Am I answering all the questions from that?

Manju George 1:17:55
I think, yeah. I think so. Then one question is like, do you consider KRAS a driver or a passenger mutation?

Dr. John Strickler 1:18:03
Oh, I consider it a driver. It is a classic driver. I consider all those actionable variants in the Big Five to be drivers.

Manju George 1:18:12
Okay, and then now I’m going to dig a little deeper to that, like, are all KRAS mutations the same? Or, would you look at them differently.

Dr. John Strickler 1:18:25
Well, KRAS, KRAS exons, two, three and four, if it’s in those exons, yes, I look at them the same. They drive resistance to anti EGFR therapy. Occasionally we’ll see KRAS variants outside of those exons, and we still approach them as if they drive resistance to anti EGFR therapy. So and there may be some individual differences between the specific variants around aggressiveness, how quickly they grow, that kind of thing, but for our purposes of managing colorectal cancer, we would group them all into one category which is resistant to EGFR therapies and conferring, in general, a slightly worse prognosis, but not tremendously worse prognosis. However, we now have certain variants that we have drugs for KRAS G12C, we have FDA approved therapies for KRS G12C, mutated colon cancer, and I would be willing to bet in the next three years, we’ll have FDA approved therapies for KRAS G12D, and several others. So we are making progress. It’s really exciting.

Manju George 1:19:37
Okay, okay, this is another question about Early Stage patients. So would you recommend when compared to a stage two or a Stage 3 patient? Because many a time when they have progression, then it’s only at that time that they get tested for biomarkers. So there has been some recent push, especially for BRAF, because there are some trials. So. What advice do you have for someone with a stage three cancer?

Dr. John Strickler 1:20:04
I think we have entered it in 2025 particularly with the aspirin data. You can justify order next generation sequencing for stage two, three patients. Where there will be pushback from the clinicians is that we’re almost certain that insurance will not pay for it. Which means that you’ll see $5,000 on your explanation of benefits, denied. I think that we can work it through with some of the patient assistance programs, but just know it’s really a financial risk. I do think there’s a good rationale to order it earlier now, but this is something that I would be willing to bet that most oncologists are uncomfortable ordering all of those biomarkers early. Now, if you were to break the test up and order PCR for BRAF that’s fine. That’s reimbursed, that’s done already, in some cases, to get BRAF together with MSI. That’s okay. Just know that PCR is not the best way to pick up a BRAF mutation. And it doesn’t, yeah, I I’m increasingly ordering it earlier, though, and I suspect over time we will all start doing that.

Manju George 1:21:28
Okay, okay. And then nowadays, a lot of people in the early stages are getting Signatera testing, and Altera is a choice. So do you think that people could not choose Altera, but if they are, say, for example, ctDNA positive after finishing all their adjuvant therapy, then they could ask for an Altera?

Dr. John Strickler 1:21:48
Just procedurally a lot easier just to say, if you’re a patient newly diagnosed, and you know you’re getting Signatera, just tell your doctor to check the box for Altera and it’ll come back. And the billing experience is usually very favorable for Altera, so that would be a great way to get all your biomarkers right up front at the very beginning. Please, as a patient, make sure that they print the copy of the report out for you and keep it with you, or scan it like don’t lose that report, because that is very difficult to come by.

Manju George 1:22:22
Okay, okay, I think we have gone way past the time. Thank you so much. This was very helpful. I might come back to you for the aspirin and PIK3CA,

Dr. John Strickler 1:22:33
Yeah, that’s a big deal. Well happy to talk to you about that further later on.

Manju George 1:22:39
Okay, okay. Thank you so much. Thanks to everyone for attending. And as I said, an edited version of the video, and then the transcript will be available on COLONTOWN University. Thank you, Dr Stickler, thanks to everyone for attending. Bye.