 So today I'd like to give you an overview, not just of my work, but also of much work by others in, whoops, I just broke the podium, thank you. So this is an exciting time for us in cancer research because as I'll get into, we have tools that are just jaw-dropping in their power to examine the human genome. And I've been using these for quite a few years to study lymphoma, as mentioned. I also now have two jobs, two full-time jobs, the other one being directing all the large genomic efforts that the National Cancer Institute is conducting right now. And these then hope to do what I've been doing for lymphoma for all cancers and do it on a grand scale that will lift the entire field up. So I'll begin today's talk, giving that overview based on what's going on out there, and then I will focus in on my own research and show you some more in-depth studies on how we think about the genetics of cancer. But the broad take-home message is that the current ways that we're diagnosing cancer and treating cancer are changing before our eyes and we're going from a histological diagnosis to a molecular diagnosis of cancer as fast as we can. So when I speak of genomics, what I'm talking about are the somatic changes that occur in the cancer cell, not in the germ line of the individual, although that contributes. But what I'm going to be talking about today are the alterations in the tumor cell that make it different from a normal cell. And of course this begins with mutations that sometimes alter the structure of proteins. There are structural rearrangements of the genome, either DNA copy gains or losses or sometimes translocations of the genome. There's a need to look at the active part of the genome that is expressed as messenger RNA that can be dysregulated in cancer. There are a class of small regulatory RNAs called microRNAs that are important to know about and then there are modifications of DNA such as DNA methylation that lead us into insights into the abnormalities of cancer. And in the latter part of the talk, I'll talk about these I would class as structural genomics and then there's another class of studies called functional genomics where we aim to find out which genes are important by directly manipulating those genes and seeing what happens. The tool that all of us are relying on now are these so-called next generation sequencers. One from Illumina and what we took, we being the broad genomics community took more than a decade to finish one human genome. Now two human genomes can be done in 11 days of sequencing on this one machine. So this is an outpouring of data that obviously requires a lot of computational power to make sense out of and this is courtesy of the Genome Institute is one of their more commonly shown slides. I'm sure I'm showing that here is the drop in cost of computing. I'm sure you all have heard of Moore's law and here's the drop in the cost of sequencing the genome. So it's plummeting faster than Moore's law and we're now somewhere around a $4,000 genome and we hope to be soon at a $1,000 genome, really making it very personal for all of us very soon. So as I mentioned, I'm directing these programs in genomics for the NCI now and they include something called the Cancer Genome Atlas which is looking at adult cancers very deeply. There's a program looking at pediatric cancers and then there's a functional genomics program. And so first I'll tell you a little bit about recent progress in this TCGA program from structural genomics of cancer and the goal here was to study a large number of cancers because we believe that there's this great diversity of human cancer and until we see that in all of its glory we won't know how to understand any individual patient and what's going on. So here are the tumors that we've studied and by the end of the year we will have brought into the program 10,000 tumors that will then be finished in their analysis about two years later. And the first thing we have realized is something we knew before but is something we deal with now that the mutational burden in cancer is very, very high and some of this is of course self-inflicted. We have carcinogens of various varieties, smoking, sunlight, possibly food that we eat leading to very high rates of mutation in, for example, lung cancer, melanoma and stomach cancer way on this side whereas on the other side you have the childhood cancers where very little action is going on. So out of this rather noisy picture we have to discern the biological signals. Some of this is just noise and some of it has led to changes that make the cancer cell malignant. So a recent study that was published out of the TCGA was about kidney cancer, renal cell, clear cell carcinoma and in this cancer, this is a busy slide but it shows you that cancer information is busy and rich and it focuses on what was found to be a recurrent signaling abnormalities in a signaling pathway known as the PI3 kinase pathway that promotes the survival and metabolism of cells. The first important point is to say it's not one abnormality but all large number of genes were either mutated, amplified or deleted such that when you put them in a pathway diagram you can see that in small numbers of patients there were mutations somewhere in the pathway leading to a grand total of 28% of all of this cancer type having some abnormality in this pathway and some of the point mutations in a key kinase called mTOR that in this pathway are shown here and they cluster in particular parts of the protein. Important point is that from a clinical point of view, a clinical study had already been done with a drug that targets mTOR known as Everolimus and you'll note that in fact in clear cell carcinoma there was a signal. There were responders, it's not a home run, we're not curing anyone but the drug works and it's probably because this type of cancer relies and has turned on this signaling pathway which is and of course now we have to figure out well who are these responders to this drug, did they have these mutations whereas the other ones did not. Now this is mislabeled, this is a study of recent study of endometrial cancer that the TCGA did and these are about 500 samples from different patients and then you classify them by their molecular abnormalities in a variety of ways that I won't go into but the categories are fairly self-explanatory. There's what is called an ultramutated type of endometrial cancer with lots and lots of mutations due to a defect in a repair enzyme. Then there's a hyper-mutated form that's defect in a different repair enzyme, the mismatch repair enzyme and then there's two other forms that are called copy number low and copy number high meaning that there were either extra amplification of DNA here or not as many events over here. Now if you look at this classification with respect to, that's not shown here yet, I'll get to the next slide. So this leads us to the classification, what's now shown is how does this relate to the histology as a pathologist would normally do it. The pathologist sees there's an endometrial, an endometrioid type of this cancer and a serous-like type of this cancer and that's in either light blue or dark blue. You'll note that in this copy number low and these others mostly it's the endometrioid so things are lining up okay but here we have a problem. Here there's a mixture of histological diagnoses within one of these categories and in fact it would look like there's some things that are really serous by their molecular profile but the pathologist called it endometrial. Do we care about this? Well yes we do because endometrioid has a better prognosis and is treated differently from the serous type. It is given adjuvant therapy with radiotherapy is along with surgery whereas this more difficult to treat serous type chemotherapy is added to surgery where if you look at the survival of the patients classified in this way you'll see that indeed this serous type in the red curve has the worst outcome. This copy number low has an intermediate outcome and this ultramutated type at the end. These folks do incredibly well so you could say maybe all they need is surgery and nothing else but the important point again is that if we stuck with the histological diagnosis it's possible that some patients who really have this molecular serous type would have been called endometrioid and maybe gotten the less aggressive therapy and not done as well as if they had gotten chemotherapy. So now can we find new therapies for our patients based on structural genomics and here I'll highlight just one example from the pediatric target study that we've been looking at a large number of these childhood cancers shown here and this was a 10 year old boy and he had a refractory form of B cell acute lymphoblastic leukemia of course is pretty treatable except when it's not and in this case it wasn't and the RNA sequencing was done another way to look at the RNA not the DNA but nonetheless it showed that this boy's tumor had a translocation that involved a receptor tyrosine kinase known as the PDGF receptor. So fortunately there's a drug for this and it's called a matinib you may have used it or heard about it as a treatment for chronic myelodysl leukemia but it also is a very good inhibitor of the PDGF receptor and he started this treatment got immediate clinical improvement within one week there was a morphological remission in the blood and by two weeks there was essentially no molecularly detected disease and that's shown here in this PCR assay on before treatment this is the the translocation mRNA and on treatment you just can't detect it anymore. Now this is a very new unpublished work so we don't know how this individual is doing long term but it's a dramatic response and it's this kind of N of 1 experience anecdotal experience that we've been seeing with some of these targeted agents that have gotten a lot of people excited but of course anecdotes are not how we do good clinical medicine so the Cancer Institute is trying to do this more seriously and essentially the problem is how do we find the right drug for the right patient and there's two ways to approach that the first is in this genotype to phenotype that is I discover that the tumor has this mutation I have a drug I think is good for that aberrant protein made by that mutation and let's see how it works and we're not going to just do this in one patient we're going to do this in a thousand patients and to find these thousand patients that have some targetable molecular abnormality will screen about 1500 to 3000 of them in in CLIA labs to get the these molecular genetic changes we've gotten drugs over 30 of them committed by pharmaceutical companies that may target one or the other of these mutant proteins and they are going to give them to the NCI for this trial and then we're going to assign a sort of algorithm if you have this abnormality or the other you get drug A but these other abnormalities you get drug B and so on and then we're going to see how often it is that we can as I say predict the T-leaves predict the response is this a successful approach and that will go through the clinical trial design the second the second way to look for the right drug and the right person is so called phenotype to genotype here the doctor is the investigator the doctor sees this remarkable response in one individual that the same drug in other individuals seemingly of the same cancer type did not respond and so these are the so called exceptional responders and here's one celebrated case from Memorial Sloan Kettering 73 year old gentleman I think it's actually I don't know that a 73 year old with bladder cancer and and and this person had been put on and failing a lot of other therapies had put on the same drug that I introduced to you this Everolimus the target the mTOR protein and had a remarkable complete response and it was it's been quite durable no evidence of disease 24 months after starting the therapy and this is just the abdominal scan showing large clusters of tumor masses that then resolve and are still gone at 18 months so why is it that this one patient had a response whereas the clinical trial that he was in was declared a a failed trial the statistical endpoint was not met well the whole genome sequencing was done on this patient and here's a way we depict all the molecular abnormalities where all the chromosomes are arrayed in a circle one through 22 X and Y and and then these various marks refer to whether where there are abnormalities and some of them you can't see these little dots are point mutations in the genome and I've a circle circled one green dot which indicates a frame shift mutation in a tumor suppressor pro protein called TSC one and TSC one is a negative regulator of this mTOR signaling complex so when you remove the break on this complex you get spontaneous signaling and that is presumably why this patient had such a good response now with this knowledge if you go back to the clinical trial and look at the response of patients on this trial and ask whether there were mutations in this TSC one disease gene you find here this big this is the depiction of the tumor volume so going over to the left is the loss of tumor volume so this is the patient we've been talking about with the remarkable response but here's some other partial responses or actually didn't even make it into partial response but they are the ones that have the mutations whereas the ones that don't have a mutation all those tumors grew so this is now sparked more work there's another clinical trial to evaluate whether this is indeed true and so to capitalize on this kind of clinical observation we're encouraging physicians from all over the country all over the world for that matter to send us your exceptional responders that is we would like to study these unusual responses if the patient is willing to participate have their genome sequenced and then see how often we can figure out the puzzle of why we had such a clinical response so in the last part of the talk then I'll tell you about how we've approached a particular really aggressive type of lymphoma known as diffuse large B cell lymphoma and I will introduce first the idea of functional genomics and we depict this by Achilles here being felled by the arrow to his heel and I modified this to include on his shield a histological picture of diffuse large B cell lymphoma and the metaphor is so rich I can't believe to tell you but if you remember the Iliad then the shield of Achilles which is described in several pages in that book basically if you read and understood the shield of Achilles you would know how the world works and worked at that time. There were depictions of the various cities and there were depictions of wars and there were mountains and rivers and oceans so same way we feel that if we can understand in a molecular way this cancer then we will find this vulnerability that we can target therapeutically. So the way we do this is do a generic screen of course genetic screens have been done since Mendel and famous people studying fruit flies and we just couldn't do that with the cancer cells because of course we can't breed our organism of choice the human at will so but we have new techniques that allow us to do genetics without heredity in a way and what we do is we have tools called small hairpin RNAs that are able to surgically inactivate a particular one gene at a time and in so doing we can see what that gene does for the cancer cell and that's depicted here so we have a library of these SH RNA vectors each one in different color targeting a different one of the 24,000 human genes and we introduce that large B cell lymphoma library into a cancer cell line so you can see that every cell growing in this flask in my laboratory would have a different SH RNA and therefore have inactivated a different gene so this is the beginning of a genetic experiment you have genetic diversity in a population of cells you now apply a selective pressure and the selective pressure is simply can any cell in that flask grow and proliferate and survive for three weeks in culture so you'll note that in this population of cells one is missing right and presumably that is because this SH RNA this turquoise SH RNA knocked out an essential gene in that cancer cell that it cannot live without and therefore it is depleted from the population and so then all we have to do is compare the population of SH RNA vectors at the end of the experiment to that at the beginning of the experiment and figure out what the essential genes for proliferation survival are so these are our so-called Achilles heel screens important point to make is that it's not at odds in any way with the structure genomics I just got telling you about in fact what we find over and over again is when we perform an Achilles heel screen and then look within the pathway we've identified as essential pathway somewhere in there in that pathway is a mutation that tells us why that pathway was active and so this is the goal finding these essential cancer pathways so diffuse large B cell lymphoma I'm sure you've all had some experience with it in one way or the other is the most common type of non-Achilles lymphoma it's a aggressive but obviously can be treated and the cure rate currently with CHOP chemotherapy regimen plus Rituximab is on the order of 50% so great 50% much better than other solid cancers but we're trying to figure out something to do for the other 50% and this leaves us with a large number of deaths every year 10,000 in the United States alone and so we thought that perhaps the problem was that by looking into the microscope and describing the cells as large and diffuse in spread maybe that was just not sufficiently accurate to perceive the differences that were leading to different responses to chemotherapy so we used molecular profiling to find subtypes of diffuse large B cell lymphoma and the molecular profiling the available technology to me 10 years, 13 years ago was not this fancy next gen sequencing but rather ability to profile the levels of messenger RNAs of various genes within the cell so-called gene expression profiling and the way we usually depict this is if a gene is very active and there's a lot of messenger RNA tumor is given a red color and if there's very little expression of gene you get a green color and black is somewhere in between and then what you see in this diagram are lymphoma biopsies probably a couple of hundred of them each in a different column from a different patient and then we have a number of genes probably about 200 genes here and you can see that in tumors of this variety which we call the activated B cell or ABC they have this signature of genes being very active and these genes being inactive and just the opposite in the germinal center type GCB you have a different signature of gene activity and these different activities didn't just arise from from know where they're actually a signature of the type of normal B cell from whence this lymphoma derived so as I mentioned one of our goals was to figure out which of the patients were doing well with chemotherapy and which weren't and it certainly is the case that these ABC tumors are the bad actors here we're only curing at best about 40% of the patients with our chop chemotherapy whereas the germinal center type is coming in at about 75% so we're not done worrying about the germinal center type but we've spent most of our work in the lab on the the activated type so how do you know that this is reality well that is maybe there's an infinite number of ways to use all this complex data to to divide up tumors and why is this the right way to divide them up well I've shown you that there was some clinical differences so that's one thing to hang your hat on the other thing to hang your hat on is that then you look in those tumors classified as ABC or GCB ask what are the genetic abnormalities which mutations which deletions amplifications of genes which structural genomic changes are found in one type or the other and what you find is that all of the abnormalities shown on the left side here are only found in the activated B cell type whereas all these others on the right side are only found in the germinal center type so this Yin Yang appearance tells you that these are pathogenetically distinct diseases and why are they distinct as I mentioned these tumors come from a different starting point a different normal B cell and probably because of that different start point then it has to take different steps to become malignant and the way that we think about how this particular ABC malignancy arises is boiled down to a very simple molecular wiring diagram what we noticed very early on that was that there was one signaling pathway within the malignant cells known as the NF Kappa B pathway which was seemingly stuck on in the on position in all these tumors and this is a key pathway that in any cell type will prevent cell death so of course that's one of the hallmarks of cancer that the cells don't die appropriately now NF Kappa B does another important thing to a B lymphocyte it turns on a transcription factor called IRF4 which propels that B cell down the differentiation pathway to becoming a plasma cell and if it went all the way down to the end then that might be the substrate of multiple myeloma a plasmacitic neoplasm but it wouldn't be this ABC diffuse lymphoma so instead there's a second block having to do with a loss of another transcription factor known as blimp 1 and these cells pile up in a differentiation stage that's normally only transiently present so this plasmablastic stage is probably no more than about 12 hours in the life of a B cell but perhaps now this abnormal accumulation in this stage allows for further oncogenic changes to lead to the full malignancy what we wanted to understand though was why this NF Kappa B pathway that was promoting the abnormal survival of these malignant cells what was the cause of this normally this is turned on in a B lymphocyte in response to exposure to an antigen in the environment and it gets the cell activated but we didn't know that there was any antigen here so this pathway was on all the time why was that and in particular what upstream signaling pathways lead into the key regulatory kinase in this pathway known as ICAP B kinase that turns on NF Kappa B and what we discovered which I'll take you through in a step by step fashion is that it is the B cell receptor signaling pathway that is important and the B cell receptor pathway of course is arguably the B cell receptor is arguably the most important receptor to a B cell without it you can't even make a B cell from a bone marrow progenitor cell you have no B cells and then even if you had those B cells and when you were exposed to an antigen, a pathogen, a virus there would be no response because it's the receptor for those antigens this is the key receptor and it does many many things for the B cell that promote its survival and proliferation so what we discovered in our genetic screen initially was that there was a signaling complex downstream of the B cell receptor and upstream of ICAP B kinase involving three proteins card 11 malt 1 BCL 10 and if we inactivated any one of these three proteins by that SH RNA technique then the cells died our cell line models of this type of lymphoma so that was our first clue that this entire pathway which had been worked out previously by immunologists was important then we started sequencing the tumors and found that in 10% of the tumors of this variety there were mutations that affected the card 11 adapter protein and these mutations were really fascinating because they created mutant proteins that spontaneously turned on the NF Kappa B pathway if they were put into another cell so these were the oncogenes if you will within these cells but note that 90% of the cases did not have any mutations in card 11 yet we had other cell line models that with with no mutations of card 11 but still if I knocked down card 11 they die so something was was turning on wild type card 11 so we surmised there must be some signal upstream in this pathway that was tickling card 11 and turning it on and in fact in our genetic screen we had already observed a strong dependency of those cell lines on Bruton's tyrosine kinase which I'm sure you remember as important to even make a B lymphocyte the the boys with this X linked inherited disease don't have B cells and suffer from infections and and and and other things so this is important kinase that links the B cell receptor to the downstream NF Kappa B pathway but then we started sequencing oh there must be mutations in BTK so we sequenced that and there were no mutations in any of our patients we sequenced all the other kinases in the cascade no mutations and finally tumbled to the fact well maybe it's just at the the source of this river right maybe it's at the B cell receptor itself so we went and knocked down components of the B cell receptor and the cells croaked so the and turned off this NF Kappa B pathway so these cells depend on the B cell receptor but why well in 21% of the tumors we found mutations in in two of the components of the B cell receptor known as CD79A and CD79B this seems to be going in and out this microphone sorry and and I'll take you through these appear to be gain of function mutations and and and but but they're not perhaps as strong as that card 11 mutation I told you about and we'll get into that so how does B cell receptor signaling work well this is sort of the cartoon version and this is what you would find in an immunology textbook that there are the two immunoglobulin chains that interact with antigen and then there's a signaling chain CD79A and B within the cytoplasmic part of CD79A and B there's a series of amino acids a motif known as an ITAM motif and this is the signaling motif that's important and what happens is some Sark family kinases phosphorylate the tyrosines there recruit another kinase known as sick and then a lot of downstream signaling occurs so mutations in CD79A and B occurred in 21% of the patients with this ABC type of lymphoma and essentially never or very rare occurring in the other types of lymphomas that we decided so this says that this type of B cell receptor signaling is a key part of the pathogenesis of the ABC lymphoma and not these other lymphomes again highlighting the molecular differences between these sub times so what are the functional consequences of those mutations in the B cell receptor well here's the cartoon that I showed you and what we found the mutations first what were they well they were fascinating in that they affected one single amino acid this tyrosine a proximal tyrosine in CD79A and CD79B in the vast majority of cases and basically you could put any other amino acid in there one case actually deleted that tyrosine any of those appeared to be functionally the same type of mutation now at a lower frequency we found deletions in the other signaling component CD79A that surgically took out the ITAM region of CD79A so what do these do why are they selected for well the first thing to understand way to understand this is that one of the consequences of engaging the B cell receptor with this red antigen this foreign substance is like all receptors it will then be down modulated off the cell surface in an endocytic recycling and thereby terminating the response well we discovered that if you have a mutation in CD79B this endocytic recycling is blocked so the so the receptor simply stays at higher levels at all times on the cell surface and thereby promoting signaling. The second mechanism that we discovered had to do with a negative feedback for B cell receptor signaling so all receptors both turn on signaling and then have ways of turning it off because if you didn't this would be pathological and in this particular receptor there's a kind one of the Sark family kinase is known as LYN that phosphorylates another protein CD22 and recruits a phosphatase that takes off those phosphotyrosines and turns the receptor back to its native state and what we found that in tumors that have the mutant form of CD79B that kinase although active is much less active and does not engage those negative regulatory elements so two ways we get more B cell receptor at the surface and it's more active. Now life was fine at that point but the molecular biology and genetics of cancer doesn't have to be simple and so we had in our genetics screen an equally important survival pathway that seemed to emanate from a signaling adapter known as Mighty 88 and it also turns on the NFKB pathway very strongly. Well and in 39% of our tumors we found activating mutations in Mighty 88 that make it constitutionally able to turn on NFKB. So what is Mighty 88? It's a very famous receptor for immunologists because it is the adapter rather for the toll like receptors that are involved in innate immunity. They just see danger somewhere in the environment. They see a virus with a viral RNA or a viral DNA or they see a lipid from a bacteria and they alert the immune response. These are the so-called pattern recognition receptors and they utilize the signaling adapter Mighty 88 and they are powerful receptors just like the B cell receptor that turn on the NFKB pathway, P30 map kinase, engage a number of cytokines, IL-6 and IL-10 and interferon. And this is becoming this particular mutation, one of the, I call it sort of the RAS oncogene for late B cell malignancies. So here is the frequency of this mutation within various mature B cell malignancies. Here's our ABC lymphomas coming in at 29% with one particular mutation I'm showing you. But there's a primary central nervous system diffused lymphoma that has about a 40% occurrence of this mutation, a cutaneous type of diffused lymphoma with a 70% testicular diffused lymphoma, 80% and a type of indolent plasma cell neoplasm, Walden stones, macular, and anemia. 90% of individuals with this type of cancer, the tumor cells have this one particular point mutation. But at a lower frequency chronic lymphocytic leukemia which is of course the most common leukemia, 3% of those cases have that same mutation. So we need to understand what this guy is doing. So on the face of it it would look like you had two parallel pathways leading to NFKB. But we had a drug, first of all, that targeted one of those two pathways, the B cell receptor pathway. And that drug is known as Ibrutinib. And so we were, we, what? Ibrutinib. Good to write this name down because we are all going to be prescribing this drug or hopefully not getting this drug, but it is, it is an unbelievable drug and it's going to be active, it is active in multiple B cell lymphomas but I predict will have activity in autoimmune and inflammatory diseases. So we hooked up with a company that makes this called Pharmacyclics and forged ahead, worrying a little bit about this pathway, the Maudi 88 pathway, whether it would somehow mitigate the effect of this drug but nonetheless decided to go ahead with its study. The drug is fantastic in how it works. It makes a covalent bond in the active site of the kinase and thereby inactivating that protein kinase for its lifetime. So this gives you wonderful pharmacodynamics, it gives you single day dosing where you get virtually complete inhibition of the kinase for 24 hours. And it gives you great selectivity. There are only 10 kinases encoded in the human genome that have this particular cysteine to which the drug attaches in that position. So as you should be aware these kinase inhibitors of which there are many, many of them are multi-kinase that hit lots of different things. This one is more specific. It does seem to cause any problems with infections but it's an excellent question. In the lab this was highly potent in this dose response curve in killing some of our lymphoma cell lines that rely on the B cell receptor but had no effect on some other cell lines that don't. So we started a clinical trial across the road in the clinical center with my colleague Wyndham Wilson to test this out in sort of an extension of the company's phase one trial. And this is a 52 year old woman whose tumor by molecular profiling was of the ABC type and had a mutation within the B cell receptor CD79B. She had had all the patients I'm going to tell you about are relapsed refractory category. They have on average had three prior therapies usually two or three. And this woman had had two chemotherapies and had had a complete response to each but in each case relapsed. And then started taking a brutinib by mouth. It's a single white pill you take once a day without any discernible side effects. By week eight she went into a complete response shown here. Here are some abdominal tumors. The others are her kidneys of course. And these abdominal tumors in this PET scan overlaid on a CT have gone away at week eight. And she is our star. She is our exceptional responder. She is now out 3.3 years living without any sign of disease taking this drug once a day by mouth. No infections. No discernible side effects. So this is a remarkable way to get your cancer treated. It's like a blood pressure pill. Here's another lady that had a 59 year old same ABC tumor. In this case though she did not have a mutation in her B cell receptor or in my D88. She had had a stormy course. She had what's called primary refractory disease where the tumor never responded once to chemotherapy. And she came to us in extremis. She had a very rapid rise in her LDH, an indication of tumor volume in her serum. Then she started getting a brutinib and you can see this dramatic fall in the LDH. Here's her tumor that was there by imaging when she arrived. She had so much tumor in her abdomen that she couldn't eat because her stomach was being compressed. And here's what her scan looked like after three weeks. So this is a tumor that never budged with chemotherapy that seemed to melt away here. It's great. I saw her at this moment. She was doing fine. And then about two weeks later the tumor came roaring back. And so she had some form of resistance. And so it's not all roses here. It's the beginning of a new type of therapy. So to take a serious look and get away from anecdotes, we did a clinical trial, phase two clinical trial, multi-center around the country led by Wyndham and myself. And this enrolled 70 patients, all relapsed refractory. Here we took all patients of all molecular subtypes and determined whether they were ABC or GCB by profiling. And then they started getting this same drug at the same dose. And we've now finished the clinical trial. And here are some of the survival curves where here's our lady that is our exceptional responder at the top. And here are a number of other individuals, some of which are still on study with these arrows. And now this is a little old slide. About two or three of them are out past the year. But you can see that many of the ABC tumors that are relapsing, but at a longer time point than the GCB tumors. And this is another way to depict the results. This is the waterfall plot looking at loss of tumor volume. And you can see we have a lot of complete responses here and partial responses and most of them are in the blue ABC type rather than the GCB type. So overall our response rate was around 40% in the ABC type with counting both complete and partial responses, whereas only 5% of the GCB type responded. So this is evidence that in response to a targeted therapy there's a need to know about this molecular distinction between these subtypes. And this translates into a statistically significant increase in progression of free survival. And in overall survival, our overall survival in the ABC type is 10.3 months and the GCB type 3.3 months. So you know, it's a benefit to these patients. Obviously we need to add something to this single agent therapy to make it really work, but it's certainly a start. So can we understand which of the ABC patients are responding and which are not by looking at the mutations that I've been telling you about in these various pathways? Sort of. So if you look at tumors that have a mutation in the B cell receptor and the numbers are small here, that 6 out of 9 of those respond, so a somewhat higher response rate than in patients that have a wild type form of the B cell receptor. While that's significant and indicating that the mutations are important, I want to emphasize that 30% response rate in patients without a genetic cause is an important signal and tells us that there may be non-genetic ways to turn on B cell receptor signaling in these cases. And to drive that home, here's one other clinical vignette, one of our patients, a 48-year-old gentleman with the ABC lymphomas. By our sequencing, had a wild type B cell receptor in Mighty 88, had relapsed from several agents, went on therapy, went into a complete remission. Here's a thoracic tumor that went away at week 10. I believe he had a four-and-a-half-month response to this drug and then relapsed. Important point is that we sent the tumor off for some very deep sequencing by a company called Foundation Medicine and they found using their next generation sequencing something we hadn't found by conventional sequencing. They found at a 5% level in that biopsy, in that patient, there was a mutation in the B cell receptor that we had missed because of our techniques. And that when we looked at that tumor, here's before treatment, this looked like a wild type sequence. But when we started looking at the tumor on therapy in the pressure of a brutinum, now you see this sort of noisy line. This is another subclone that is that rare subclone that didn't show up very well. Here there's a second band. So there is a rare subclone that says that this tumor has something to do with the B cell receptor. Siggling, there's a mutation in the B cell receptor. But remember, the whole tumor went away. It's a complete response. But only 5% of the tumor had this mutation. So you don't need the mutation to be responding. But this is obviously telling us something important. Now what about the other mutation? This, well, yeah, right, good question. This is, the first one is on study, but before a brutinum treatment. The second one is three days of a brutinum, only three days of a brutinum. So within three days we start selecting out this subclone that has a higher level of B cell receptor signaling. All right, what about some of these other mutations? Card 11, if you remember, the wiring diagram is way downstream of BTK. So you might say this might be a resistance mutation. Numbers are small. Only had two patients. They didn't respond. Not going to make too much out of that. What about these MiD88 mutations? Would they somehow circumvent the ability of a brutinum to work? Well, not really. You had responses if you were mutant or wild type. But the picture got much more interesting when we asked whether they had concurrent B cell receptor mutations. And in four out of five patients that had both the MiD88 and the B cell receptor mutated, they responded, whereas zero out of seven patients that had only the MiD88 mutation responded. And why is this? Well, we think it has something to do with the fact that genetically there was an overlap amongst 155 tumors, and about 10% of the ABC tumors had both mutations. And that was more than we would expect by chance. So we think that there might be a synergy between these pathways in some patients, whereas the zero out of seven response over here may say, well, maybe there's another way to become this ABC type of tumor that has nothing to do with the B cell receptor will not respond to a brutinum. And we need a new therapy over here. Okay. And since the hour is late, I'm going to sum up saying that a brutinum, the latter part of the talk, is showing you this irreversible BTK inhibitors inducing a high rate of response in previously refractory tumors that the mutant B cell receptors have a more frequent response to this drug, but you don't absolutely require that. And here I will highlight some recently published work on this drug, a brutinum in chronic lymphocytic leukemia, and in mantel cell lymphoma to a New England journal papers you may have seen where there is a 70% and 68% response to this brutinum drug. And by genomic sequencing, there are no mutations in the B cell receptor. But those malignant tendencies are responding to this drug. Same sort of thing, this sort of non-genetic dependence on the B cell receptor. I didn't show you this for a sake of time, but a brutinum combines very favorably with a lot of other drugs, including conventional chemotherapy, leading to a lot of combination trials that we're now setting up at the NIH and around the country. And to sum up the whole talk, I would like to emphasize from the research point of view that we can combine this next gen sequencing, find the structural genomics of cancer with a functional genomic study of the essential pathways, getting some good computer scientists involved to deal with this avalanche, this big data problem, and arrive at these essential cancer pathways. But going forward in something I'm going to be focusing on in my new job is that the patient will be at the center, and we will be wanting to do all of these genomic studies in the context of clinical trials where we have the outcome and we can relate the response or not to the prolactinum molecular abnormalities. And very soon, coming to a hospital near you will be the following paradigm that the patient will show up. They'll then undergo some sort of genomic profiling of sequencing the DNA or looking at expression levels and profiling. In that way, there will be some phase of data interpretation. Then you'll make a decision to employ a particular drug based on what you found. You'll image to see whether there was a response. If and when drug resistance occurs, you'll have to get a fresh biopsy, figure out whether there were any new mutations that occurred in that tumor, and then if you find an actual mutation, employ yet a different drug. I don't think this is science fiction. I think this will be reality in the very near future. So just to sum up, I just really want to give most credit to Wyndham Wilson, who helped lead all of these clinical trials with a brute nib with me. Thanks very much. So how will this incredibly arrogant science find its way into the clinic? How will oncologists learn from this and use it? Well, it's already happening, and for better or for worse. So I'll take this opportunity. So for, I think, about $4,000, you can order a test from Foundation Medicine, and there may well be other companies, so I'm not endorsing just that one company, but they will sequence over 200 genes for you and find all sorts of abnormalities. Then you can see, oh, well, there's maybe a drug targeting this particular abnormality. That goes under the rubric of this N of 1 type. I think that may work sometimes. I showed you some examples where it does work. It may fail more times. We just don't know. So I think one of the virtues is that we have the data available if we want to do that, but we still need good clinical science to see how informative that data really is. Thank you so much for this talk. I've been waiting for this a long time. But I want to give you another concept to reverse things. And that is that Stage 3 and Stage 4 of rheumatoid arthritis is a disabling disease with an increased incidence of lymphoma. Yes, it is. And so for the methotrexate failures, the treatment options are an anti-TNF drug, which may increase lymphoma. And with the problems of big form, we don't know if it increases lymphoma or if it is there. Or we can use adipatocep, which is a told receptor, or we can use rituxin. And so how about thinking about using genomics to determine aggressive therapy in rheumatoid arthritis based on your genomics? Who are recording? Could you bear a phrase to the question? The question is, can this way of turning the crank on molecular view of disease be applied in this case to rheumatoid arthritis? And it's certainly what I've been trying to promote for years. The problem, the cancer is special. We know where the disease is. It's where the tumor is. Rheumatoid, where is the disease? What should we profile? Can we look at the blood? Well, maybe not. Maybe the relevant cells are not floating around the blood. Maybe they're in the joints. You've got to get a joint biopsy. Did you hit the lesion that is informative in the joint because it's all spotty? Not so simple, but not impossible. So I think there, and of course there's an underlying genetics, you can always look at the germline component. And I will emphasize what you said or restated is that there is indeed a genetic increased risk of non-Hodgkin's lymphoma in patients that have rheumatoid arthritis. There is a genetic component that we have yet. It's about a two-fold relative risk, but it's there. So there could be, and as you've seen, because the B-cell receptors can be, B-cells can be involved in both these autoimmune disease and these malignant diseases, the same receptor pathways could be used. And we don't know yet, and it'll be fascinating to see how a brutinib works in this patient population. So some oncologists won't let us use an anti-TNF in a patient that has pre-existing lymphoma and rheumatoid arthritis, and some will, you know? And do you think it's so hard? Well, I don't, because TNF is hooked up to, in so many different ways, and depending on other things that are going on, can either promote the survival or the death of cells. It's way complicated, and you have to be talking about which immune cells, which subset are you talking about. So I don't have any deep insights there. I think that this is, I mean, it's not every patient that gets these lymphomas. I mean, as I've understood, they're pretty darn rare. It's hard to even collect a series. So they're reported. They probably happen. But as you alluded to, we don't have a real study of what anti-TNF induced lymphoma looks like. I'd love to study it. I just don't have the tumors. Yeah. So that's exactly what we want to do. So the Cancer Institute has, I'm sorry, the question was we've had decades of, let's say, empirical trials. I mean, they were a new drug tried in cancer that didn't work. And can we salvage anything by the molecular analysis of tumors? Well, fortunately, we kept a lot of those tumors in tumor banks. And now one of the things I would like to do is turn this heavy-duty technology on those older tumors and see if those two patients out of 45, which is what their experience was in that bladder cancer trial, which was a failed NCI-supported trial, now we can see, well, those were the two. And now if we, you know, because when you get into common cancers, if there are one or two percent of patients that have a target of a lesion, you want to know about that. And so we have to care about cancer in all of its heterogeneity so that when we've done our clinical trials, we haven't, we've lumped everybody together. So we couldn't possibly have seen it. The example there is KRASM and colon cancer, I guess. Absolutely. One more question, and then we'll break. Yeah. One more question just to ask, and think about it again, some more clinical. In the future, is a clinical trial at the age of electronic medical residents? There's been some recent articles saying that if you use a registry-based real-time study, is that something that you're looking to have? Yes. That I, in a less fancy way, I call it a non-clinical trial, clinical trial. That's what I've been doing for all of the work that I described in lymphoma. These were not patients on a trial. These are patients that receive standard of care at good academic institutions around the country, and we just got their tumors, figured out what their response was, and made sense out of it. I would say that there's still a need to conduct things properly, but there might be a proper way to know with electronic records that a patient had actually received a particular regimen, you know, hadn't stopped early. You know, there's problems with doing what you suggest, but it's not at all insurmountable. And it could well be, and a vision also that we have is that genomic profiling will become so inexpensive that patients will just get it as a course of their care. And then what we would like to do as researchers is if the patients are willing to donate their cancer information, we would then like to just take the data, we need their tumors at that point, and take the data and then look at not just 10,000, how about 100,000 patients, how about a million patients, and then really understand more that way. Are you able to do expression studies in preserved tissue? Yes, that's very exciting new development. We can do perfectly fine RNA sequencing from formal and fixed tissue. One of the best lectures we've had here in years. Thank you very much. Thanks.