David R. Gandara, MD, FASCO, discusses the evolving role of liquid biopsy in therapeutic decision-making for Non-small Cell Lung Cancer (NSCLC).
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it is now my pleasure to introduce Dr David Gandara. He is director of the thoracic oncology program at the University of California, Davis. His topic is the evolving role of liquid biopsies. Doctor Gandara is well known for his work in the field of developmental therapeutics. Please welcome him again. Remember, if you have questions, use the chat function and they will be addressed during the panel discussion. Hello, my thanks to the organizers and to Dr Cost in particular, for the opportunity to participate in this wonderful meeting. I've had the opportunity to participate before, and of course, I hope by next year this is back in person instead of virtual. Ah, my topic today is liquid biopsy, and I will talk about the evolving role. This is really a fast moving field, and I'll use non small cell lung cancer as a model because, as you'll see from the presentation, non small cell lung cancer is really a poster child for the use of liquid biopsy. These are my disclosures. This slide is taken from a publication from the Swanton Group, and it talks about the multiple roles of liquid biopsy in therapeutic decision making and non small cell lung cancer. And, as you can see from the figure, this now in 2021 in particular extends all the way from early detection of non small cell lung cancer through patients with advanced stage disease and their initial molecular testing, which is now standard of care to the point where they subsequently have progressive disease and the use of liquid biopsy and identifying the resistance mechanisms. So you can see at the bottom the potential advantages of using plasma liquid biopsy, circulating tumor DNA over tumor biopsy or perhaps re biopsy, depending on the situation. And I think the first point is very important, and that is that shed. Um, our DNA into blood provides a global perspective. We know, particularly in non small cell lung cancer, that there is what we call tur Heterogeneous city, which means some mutations or abnormalities could be present in one area of a cancer. Let's say, the primary and not present in the metastases, which is growing. So this obra gates that issue, of course, blood is relatively non invasive. It can be repeated serially, so this is again very important. It means that we can monitor your response. We can look for early signs of progression and blood. And of course, patients are very amenable to having a blood specimen compared to getting a tissue biopsy, as I mentioned. And this is the most established role we can use liquid biopsy now to determine the mechanisms of acquired resistance prior to radiographic detection. And I'm here, I'm talking about patients with aqua gene driven, non small cell lung cancer, an emerging field and this is will turn out. I think to be very important is to be able to find minimal residual disease after surgical resection. And I'll give you a very good example of this data we're waiting for from the Adora study. And then, lastly, I'll just comment briefly about the potential for using liquid biopsy to identify early stage disease or even during the screening process with low dose CT scans. And I'll start here with the global perspective. This slide is from the NC CN guidelines for recommendations on genomic testing and non small cell lung cancer. As I mentioned, non small cell lung cancer is a poster child for genomic testing because we now have eight Anka genes that are recommended for testing in every patient with advanced stage disease, particularly non squamous. Although if the patient is a never smoker with squamous or a younger patient or has a family history, then we do recommend screening there as well for these mutations, and you can see for each of these g fr Alec Ross Juan be wrapped her, too met amplification or mutation Rhett rearrangements in track rearrangements that we have very effective drugs. I'll come back to this later for each of these categories, so that's what we have to test for. The second thing that is changing rapidly is how we test This is from a paper we published in the Journal of Clinical Oncology on Genomic Assessment of Non small cell lung Cancer was published in 2013, and we talked about the evolution of how we test. We said, Even in 2000 and 13, we've moved beyond the empirical approach. At that time, we were doing mainly single gene testing, although ourselves and others were also doing multiplex so you could test for multiple of these Anka genes at the same time. But we predicted in that paper in 2013 that in the near future. We would be using high throughput next generation sequencing in the clinic, and some people said, Well, we don't think this is going to happen any time soon. But in fact, within 18 months of this publication, we had commercially available next generation sequencing, we've moved beyond research tools such as whole xom sequencing to now comprehensive genomic profiling, which is done through commercial tasks. Uh, several of these now are too. I should say FDA approved within the United States, both in tissue and in blood. What we could not predict in 2013 is that when in a reasonable time after then we would do the same next generation sequencing in blood for both genomics and for, um reputational burden. This is from a paper we published just recently on strategies for how do you implement plasma base that is liquid biopsy genotyping in clinical practice for non small cell lung cancer. And on this slide, you see a variety of differences between tissue genotyping and plasma circulating tur genotyping. I've already gone over several of these, so I won't dwell on them. But the summary here is that in this paper we recommended a simultaneous testing For all the actionable oncogene targets, that is all the eight and maybe 1/9 coming. That would be G 12 c K wrasse mutation, and that next generation sequencing was preferred, whether it is in tissue or blood. These recommendations in our paper are similar to those from multiple other organizations has shown in that same paper. We talked about patients at their initial diagnosis with stage for non small cell lung cancer. And when might you use tissue versus blood? And I'll just make a couple of points here. If tissue appears adequate for genotyping, then tissue would be done first or possibly concurrent, and I'll show you the reason for doing concurrent a little later. This requires that the oncologist knows what that tissue is like to be able to say. Is it adequate or not? You don't want to wait two weeks for the tissue to come back, and then they say it was inadequate. So this requires talking to the pathologist, knowing about the tumor content. It usually needs to be at least 20% and any other issues in terms of the tumor adequacy. On the other hand, if the tumor tissue is unavailable. Maybe it's been used up. It was a small specimen, so for immune artistic chemistry PD L one testing that's been used up there. You would start even initially, with plasma circulating tumor DNA, and only if it shows nothing or does not show any of the targets. Would you then reflex to getting another specimen? Well, in order to implement liquid biopsy for initial genotyping, the test needs to be sensitive. So there are two FDA approved circulating tumor DNA platforms and the United States one from foundation medicine, one from Garden. These are data from our publication in a large number of patients multiple tumor types, 21,000 patients. You can see the various tumor types using the garden essay. And as you can see, there was an 85% detection of circulating tumor DNA across all the cancers. Here's non small cell lung cancer, non small cell, small cell, around 90%. But the main purpose of this slide is to show that the variant khalil frequency how much of the mutation is in the blood is very low. You can see the median here less than 1% 0.41%. So many oncologists say, how could this be clinically important? I think you have to realize that this percentage is based on all the cell free DNA, not just the circulating tumor, the cell free DNA in that patient. So you and I all have cell free DNA and our blood, hopefully not mutant. About one nanogram per ml. So this is an accurate reflection of the, um are even at a low level. How do we know that we have multiple publications, including this Very nice one from Dr Aggarwal, where she did, uh, in this case, it's the garden test, uh, for liquid biopsy, and she showed the chance of response in a patient with an driver. Oncogene was just as good regardless of the variant Leo frequency. So oncologists typically asked me, They say, Well, I have, you know, an al translocation that was picked up in blood, but it's only 1% as that actionable. And the answer clearly is yes. This is from our own study about the additional use of circulating tumor, DNA and blood to help genotype patients who had quantity non sufficient in the tissue. Or maybe they were only tested for each fr or out so you can see in patients here. This is 1288. About 30% of them were found to have a driver oncogene in tissue, the remainder quantity non sufficient or under genotype. And, as you can see here, employing circulating tumor DNA, we were able to increase the yield with an additional 19% of patients having actionable biomarkers. The other issue is a complementary nature of circulating tumor, DNA and tissue. This is a very nice study, the so called Nile Study, in which physicians were asked to do their standard tissue genotyping, whatever it was. And at the same time they did circulating tumor DNA next generation sequencing. In this case, it was with the garden essay. Well, the first thing is, in this study, only 18% of patients had complete genotyping For all the aid guideline recommended biomarkers and in the study, if you did genomic testing by circulating tumor DNA and blood first you were able to identify a biomarker in 87% compared to 67% with tissue alone. So they were complementary. There are, of course, issues with liquid biopsy in terms of false, negative and false positive. These issues assume that tissue is the gold standard, and as we'll see sometimes plasma might be more accurate than tissue. For example, due to heterogeneity, some of these issues have been overcome because the publications are talking about liquid biopsy, as it was three or four years ago. The techniques have gotten much better, both sensitivity and specificity. The most common reason for a false negative is that that patient either has low tumor volume. They are not shedding DNA into plasma very much, or it's been eliminated by therapy. Oncologists very commonly asked me, You know, I know this patient has an E g F R mutation from tissue testing. I did a blood assay and it wasn't there and I say, Well, how's the patient doing? And they say, Oh, they're in a complete remission from an E g f r T k I. And I say That's actually great. That's the reason that you have a false negative in blood. You've eliminated it. False positives. Well, one of the most common is that there is a time difference between the tissue and the blood, and something has evolved which is showing up in the blood. That's exactly what we want to look for. There is an issue of germline variants, so called Colonial Hamad up Oasis. These are some mutations P 53 is relatively common, one that come from white blood cells in the specimen. Again, technologically, this has been much improved. And so it's not as much of an issue now as it used to be. And then lastly, as I mentioned you are heterogeneous city, in which case positive plasma is almost never a false positive. Let's move briefly into immunotherapy. Here we could have a variety of biomarkers. The two best known and best applied today are PD L one and T M B. TMB, of course, is used to quantitative the neo antigens, which are produced in relationship to a tumor. TMB is an emerging biomarker. It correlates well with in the commercial essays with whole Exxon sequencing or even neo energon load as shown here. And these are data using the foundation, uh, essay in tissue and here you can see that this correlates quite well with the response rate using the foundation one test in tissue for TMB with response to a test. Eliza Mab as good as new energy and load and then lastly, PD L one and T m B are largely non overlapping, so you can use them in combination. Multiple studies have shown when you give a checkpoint inhibitor as monotherapy, that high TMB is associated with increased efficacy. This is a trial, which was negative for the clinical aspect novel Ahmad versus Platinum chemotherapy and non small cell lung cancer By whole Xom sequencing, you can see high TMB correlates very nicely with outcome and in the bottom left, using the combination of PD L one positivity and be positive, the high high patients did the best. These are our own data showing on the right using the foundation. One essay in tissue showing correlation with a tassel is Ahmad for high TMB not for chemotherapy, so agnostic to chemotherapy, the same thing being shown with the Memorial Sloan Kettering Impact and tissue again at the top. Patients treated with immunotherapy and the bottom with chemotherapy again agnostic to chemotherapy. So various essays, all positive and tissue, and this has led most recently to an approval for Pemberley is a mob in patients with high TMB and tissue greater than 10 mutations per mega base. As per this keynote 1 58 study across multiple tuna types. Well, what about blood? This is our own study. Published a couple of years ago. It is the first analytical and clinical validation of tumor mutation, a burden in blood. The computational algorithm has shown we had a test set with a large randomized phase two Study popular validation in the Phase three oak trial, which was a textualism add versus does attack cell. We analyze TMB as a continuous variable and in this algorithm we picked 16 mutations per mega bass are higher as the most appropriate cut point. This was associated with efficacy TMB greater than equal to 16 or less. And as you can see on the right, if you combine high TMB and blood with high PD L one and tissue, it is those patients that are high high that did the best for both progression free survival and overall survival. And again, as in the Venn diagram, little overlap between high TMB and height PD. L one. So this was retrospective. This essay in blood is undergoing prospective validation. The B first study, which is a proof of principle. Study has already been completed. And, as you can see, high TMB correlates very well with response to a T cell is Ahmad, and this is undergoing Phase three validation in the B fast trial, and we're waiting these data. Let's move now to mechanisms of acquired resistance. Well, these are the Anka genes that we are testing for today, and all patients. Some of these are rare, some not so rare. What are the drugs that are being used? As you can see, the response rates are quite high, but with TK eyes. Essentially, none of these patients are cured. They all eventually relapse, even if it's years later. So this is due to either secondary resistance mutation or what we call bypass mechanisms. These would be new abnormalities that come out that would allow the US to progress even though it doesn't have a secondary resistance mutation. So this leaves the oncologist. Then, in the situation where you know the patient had an egg fr mutated cancer or an out trans located cancer, they now have progressive disease. In the past, we used an empirical approach, which we do in most of oncology where the choice of the next line of therapy would be determined empirically based on the oncologist. Experience the literature, whether it's a t k or chemotherapy or immunotherapy. But we're moving now in non small cell lung cancer because we have so many drugs and we can test, especially in blood, for required resistance to a precision medicine approach where either we do a repeat biopsy at the time of progressive disease or we use circulating tumor DNA in this study, for example, in patients who had HFR mutated lung cancer treated by first or second generation HFR TKS and then had progressive disease, both tissue and plasma were looked at to determine t 79 a. M. The common resistance mutation. And as you can see, the response rate was identical whether the T 79 AM was identified in tissue or in blood on the right. So this has become quite sophisticated our ability to determine the mechanisms of resistance and act on them in the e g fr mutated space. This is a study many of you are familiar with. It's the study that got a Samaritan IB FDA approved and first line therapy. It's a comparison of that drug versus the investigator choice of Jeff it nib or lot nib. And as you can see, it was positive for both progression free survival and overall survival in favor of us murdered. Um, but what I want to show today is what happened at the time of acquired resistance. These are the data on acquired resistance in the Comparator control arm, Jeff. It never a lot in it. And this is in blood using the garden 3. 60 s A. So you can see this mimics exactly what we would expect in tissue. About half the cases where secondary resistance mutation that is t 7 90 m. The other half were a variety of abnormalities which are shown here. Well, this is the comparator. What happened in the ash martini bar? Well, a smart nev was developed as a drug specifically for T 7 90 m. So, at the time of acquired resistance from a smart neve, there were no cases have acquired e g f r t 7 90 m. On the other hand, there were a variety of secondary mutations. They're called E g f R. C 797 x. There are several of these, as well as some more rare ones. But a lot of patients had bypass mechanisms like met amplification. We now know in that patient who progresses on a smart nib. If you find Met amplification, you can apply a Met inhibitor like Chris attentive and very often get a nice response and the same for the other bypass mechanisms. The other thing we learned from Florida is that you could use blood that is plasma IGF our mutation as a predictor of response and duration of response and even survival so shown here in those patients in the flora study who had plasma HFR in their blood at baseline. These patients did worse because this reflects more aggressive cancer. Higher volume shown here if the treatment eliminated the E g f R mutation from blood at six weeks after starting therapy, those patients did better in both arms of the study and then, lastly, if the fr mutation was cleared from blood at six weeks, patients who had a smart inhibits the treatment did even better than the comparator TKs, and we found these same results in our recent swag study as 14 03 with a fat nib, whether without Cytoxan mob, as you can see again, identical to the floor. A study in those patients who eliminated the HFR mutations from blood. They did better for both progression free survival and overall survival and shown as the bottom, we could take individual cases and look at the ice a grams and determine what was happening in that particular patient. And this is just an example of a patient of mine. Followed over five years. This is a never smoking patient, diagnosed in May 2016 with an egg FRX on 19 deleted cancer. The patient had initial response to a lot nib and then progressive disease developed t 79 a. M, which is shown in the green, then treated with us. Martin if and you can see it is eliminated, the T 79 a. M and all the other mutations go away in blood for a considerable period of time. And then the patient came back. We weren't sure if they had progressive disease or not, but you can see the emergence in the pink of the C 797 s mutation resistance mutation to a relative. She is then treated on a study, uh, to eliminate that mutation and you can see she goes back into remission. So all of this observed in blood Laura Latinate, very active agent in our positive non small cell lung cancer. This is an area where, even today, most oncologists will pick the next alk inhibitor empirically. But as we can see from this paper, Laura lot nib and patients who have previously been treated with alk inhibitors is much more active. If you do tissue or blood and you find a secondary resistance mutation, you can see if you find one of those mutations or lot nib has a response rate of 69%. However, if you find a bypass mechanism in these patients, then the response rate to learn Latin only 27% and the same sort of thing for progression free survival. So it is worthwhile to re biopsy or use circulating tumor DNA rather than to do an empirical approach. Detecting minimal residual disease after surgery. This again an incredibly important area that is making headway not only in lung cancer but in other cancer types as well. Shown here are four different cancer types. Non small cell bladder, colorectal breast cancer and each one of these. Those patients who have residual circulating tumor DNA after surgical resection have a very poor prognosis. That means there is still microscopic disease somewhere compared to those patients who have eliminated the circulating tumor DNA after surgery in the various different types of cancer. Well, how does this reply to each far mutated lung cancer after surgery? These are the data from the recent Adora trial of adjuvant Smertin IB in early stage, non small cell lung cancer patients. After surgical resection, you can see randomized against placebo. Patients could get chemotherapy if it was appropriate for them. Positive study for disease Free survival. Overall survival not positive at present, but this has led to FDA approval of this regiment. The question is, in this study, blood was obtained. Post surgery. Will this mimic the results I showed you on the previous slide? And might it be in the future? We'll find out who really might need as Martin Evan, who might not based on residual circulating tumor DNA and then lastly, improving detection of early stage disease. Of course, low dose CT screening is now standard of care and high risk individuals either defined by smoking plus or minus age. So there are a lot of studies ongoing now to see Can blood liquid biopsy biomarkers assist in determining which modules on CT screening or malignant which are not, and how this affects subsequent outcome and staging. And here's just one example of this. This is the so called lung clip essay in Blood, and this is circulating tumor DNA. Very sensitive, highly specific and in this study was assessed in patients with early stage, non small cell lung cancer as well as control groups without cancer. So in conclusion, plasma first is already established as a paradigm in patients with oncogene driven one cancer at the time of acquired resistance. If they have an oncogene. Initial genotyping with blood is now FDA approved, and it can simultaneously assessed for the eight oncogene drivers with typical turnaround time of 7 to 10 days, mechanisms of resistance already established and this is equal chance of subsequent response compared to tissue detection of minimal residual disease after surgery, trials ongoing and trials ongoing to see about the pre diagnostic plasma if it will complement CT screening, Thank you very much
