 I hope that's not the whole talk, but so I want to thank the meeting organizers for this opportunity to speak to you today on what is becoming a very intriguing story in the colorectal and endometrial cancer. As you've heard from several of the other speakers during this symposium, we of us who are studying mutations kind of have a Goldilocks principle where we don't want too few mutations because we can't discern what's broken, but on the other hand if we have too many mutations the background rates go way up and it's then very difficult to discern which mutations are causing a disease. So this is going to be a story about the ultra high mutation rate where it's very difficult to tell from one patient to another which are the driver genes, but the polymerase that may be underlying that is turning out to be very interesting. So okay, so I'll pick up where we had left off with the marker paper of the colorectal cancer where we're showing mutation frequencies here. The blue line are the frequencies of non-synonymous mutations and you can see that most of the patients have a low rate between one and ten mutations per megabase. They're microsatellite stable and then there's another group of patients that are recognized as hypermutated that have also microsatellite instability as indicated down on the panel below. The microsatellite instability is associated with a very high rate of MLH1 silencing through CPG Island methylation and what we observed was that there is a small group of patients with the highest mutation rates. These are mutation rates greater than a hundred per megabase that did not have microsatellite instability and did not have MLH1 silencing apparent and in fact the MLH1 track up here shows that they weren't even mutated in MLH1 and interestingly they all had mutations in the polymerase E and so polymerase E is one of the two major replicative enzymes that replicates the genome at S phase and this came to our attention and we dubbed these ultramutated. This came to our attention when we looked at all of the mismatch repair systems across these patients and what's shown here are the different groupings of DNA repair genes. The green are mutation frequencies in patients with greater than 100 mutations per megabase the ultramutated. The red are the hyper mutated microsatellite in stable and the blue are bars represent the low mutation rate patients and generally you can see that all of the gene or many of the genes are increasing in mutation frequency as you go to these ultra high mutation rate. However interestingly not all genes show that trend in particular this blue bar here is p53 whose mutation rate actually goes down as you go to higher mutation frequency. So interestingly in the polymerases there was a single gene that was mutated in all of them and however a single gene mutated in 100% where n equals 6 is not all that much to write home about. But when we looked at the location of those mutations in the polymerase we saw that they clustered mainly in the exonuclease domain. Now all of these mutations are only from the ultra mutated patients and so there are more than six here and that's because some of these patients are mutated multiple times and what was really intriguing was not only the clustering in the exonuclease domain but the fact that S459F had been seen twice and as this was coming together a paper came out from Japan by Yoshida and colleagues where they had discovered in a single patient the F367S mutation and so which had also been seen in this study. So this recurrence at these two sites in such a small data set seemed strongly suggestive but again with n equals 6 we had a very hard cell here. So we had about 300 more patients to go in the colorectal project overall and so we went back to sequencing hoping that we would see more of these and so let me just mention why Yoshida referred to this in the title of that paper as the proofreading function. These polymerases have been studied for the last two or three decades in great detail and this shows the results of some experiments with the T4 polymerase and in the exonuclease domain of this polymerase mutations are known to cause a very high rate of mutation and this mutator phenotype as it's become called has been seen in bacteria now in yeast it's been studied extensively. Interestingly mutations in the polymerase domain over here sometimes actually improve the fidelity of the polymerase and it's thought that the polymerase has to slow down when the polymerase domain is mutated and that lets the exonuclease domain operate more efficiently. So what you have here is something like a modern day word processor where you're typing in Microsoft Word and you mistype a letter and push the space bar and the error is corrected. So we can also look at recent experiments in mice where the exonuclease domain has been mutated and knocked out in mice. These mice are viable and they die quickly. Here is a polymerase E homozygous mutant compared to the wild type. They die much faster and they're dying of cancer. The pole D1 which is the sister polymerase of pole E also has a very high rate of death. These are also dying of cancer and elsewhere in this study they showed that these mice had a mutator phenotype. So with all this together we were very excited about this and went on sequencing and this now shows the results of sequencing across 500 patients in colorectal cancer. And you can see that we have replicated mutations in sites we saw previously at P286R with a variety of different amino acids at that position, V411411L. Here is the F367S which we haven't seen again and then the S459F which has not replicated. So our colleagues at Memorial Sloan Kettering who are working on this with us also worked on the endometrial paper or the endometrial project where they also have microsatellite in stable patients with hypermutator phenotypes and so they were able to quickly confirm that this same phenomenon occurs in the endometrial cancer. So here you see replication of P286R and V411L across their patients and these patients have the same hypermutated, ultra mutated phenotype. In addition, these ultra mutated patients show a very dramatic skewing in the relative frequencies of CA mutation relative to the hyper and the low mutation rate microsatellite in stable. So we don't know for sure the origin of this yet. The mutations that we see in the patients are a combination of a mistake by the polymerase and whatever replication, sorry, whatever repair processes are going on. So we don't know for sure whether this just results in a results from a inefficient repair of this kind of mutation. However, there are some early suggestions now from further work by Nils and Nicky at Memorial Sloan Kettering that different mutation hotspots lead to slightly different frequencies of these mutations, suggesting that it might be arising from the enzyme itself. So this is then the scorecard in colorectal cancer and so what this shows that across our microsatellite stable low mutation rate patients of which there are 412 in colorectal cancer, only four of them have mutations in pole E. None of those are in the exonuclease domain and none at the recurrent sites. In the hypermutated, we actually get 19 mutations in the hypermutated, three in the exonuclease domain, but none of those in the recurrent sites and this suggests that these are just passenger mutations resulting from the hypermutated phenotype and then in the ultramutated across the whole molecule we have 23 mutations which is more than the number of patients because there are multiple mutations per patient. 100% in the exonuclease domain and about 80% in the recurrent sites. The phenomenon looks very similar in endometrial except that in endometrial the frequency of microsatellite in stable is higher and the frequency of the ultramutated is correspondingly higher, but we come down to very close to 80% of the patients with mutation in the recurrent sites. So this shows that there is actually detectable similarity between T4 phage and human pole E. These systems are for DNA replication was pretty much solved once in evolutionary history and is now recognizably similar across all most species and not only the polymerases but the other components of the replication machinery. So this enables us to easily map the mutation locations onto a X-ray crystallography structure of T4 polymerase and the gray domain here is the polymerase domain. You can see the double helical DNA moving through here. This purple ish is the exonuclease domain and the red and yellow are amino acids that are mutated in our data set and so they're all clustering in this one area of the exonuclease domain. So earlier I mentioned that there are actually two polymerases. One is pole E and the other is pole D. These have been known for decades and over the last five or six years studies in yeast where yeast origins of replication are well known have knocked out the exonuclease domain and looking at the skewed ratios of mutation arising from pole E mutants in yeast, researchers have been able to show that the pole E is functions on replication of the leading strand and likewise experiments with mutation in pole D show that pole D functions on the lagging strand. So there's this asymmetry in function of these two polymerases. So there's been a recently published collection of origins of replication in human and so Nils Weinholt looked at the mutation skewing in our polymerase E mutants which are effectively the yeast experiment in our tumors and found a 60-40 bias in CA on the leading strand suggesting that the pole E is operating on the leading strand even in humans. This would be the first time that human have replicated this what's known in yeast although it's widely assumed to hold in yeast as well, hold in human as well. So just to remind you of this high mutation rate again and cancer in the mice, these cancers are different from pole E and pole D in the mouse. The pole E mutants lead to primarily intestinal cancers. So that kind of leads to the question well what about pole D in these cancers? Do we ever see pole D mutated? And this on-co-print from the C-bio portal shows that for all these pole E mutants we never see a mutation in the nucleus domain of pole D. So this suggests that that the pole D may be required for some essential function in these tumors and although we don't know what that function is yet this asymmetry is very intriguing. We've also looked at the rate of mutation as a function of the expression levels and you can see that as expression goes up the mutation rate goes down suggesting that transcription coupled repair is operating in these patients. When you look at the hyper mutated this line is flat across the expression levels. And finally we are just getting the first look at progression free survival and Doug Levine showed this slide this morning showing that the patients that are ultra mutated have a better prognosis than patients that are hyper mutated. Similarly in colorectal cancer the microsatellite in stable patients are known to have a better prognosis and so now it's become a very urgent question to find out whether this is a generalizable feature that high mutation rate leads to better prognosis. So in conclusion the rare exonuclease mutation in pole E leads to an ultra mutator phenotype in colorectal and endometrioid tumors. The ultra mutator phenotype defines a new subtype of these tumors that may have unique prognostic features and interesting biological properties and so at this point we're gathering with our colleagues Gordon Mills and Stan Hamilton and MD Anderson cohorts of patients that will be able to test that have outcomes that will be able to verify what the prognosis is. The ultra mutator patients exhibit a signature of transcription coupled repair and the absence of pole D1 mutators suggests that it may perform an essential function in this new subtype of colorectal and endometrioid cancers. Maybe that's transcription coupled repair but it'll be interesting to try to figure that out. The Stan specific mutation pattern associated with putative origins of replication in humans is the first suggestive evidence for confirmation of the yeast model of replication in a higher u-cariat and so we are now sequencing whole genome where we'll be able to look at more origins of replication and get out of the transcribed regions where things could be biased and get a better look at this phenomenon. So with that I'd like to thank all my collaborators especially Nils Winhold and Nikki Schultz at Memorial Sloan Kettering and the rest of the crew at the Baylor Genome Center, the WashU sequencing center who sequenced the endometrial and many of the colon cancers, my colleagues at the Broad as well. Thank you. One question. It was very interesting fact that you didn't see pole delta mutations but you saw pole epsilon mutations and I want to bring up here the analogy with yeast. Many of your samples they actually are defective in mismatch repair either by MLH1 or by combination of MSH2 or MSH3, MSH6 and it is known in yeast that pole delta proofreading deficiency in combination with mismatch repair deficiency just kills the yeast cell because of hyper extreme hyper mutability. However pole epsilon proofreading deficiency is in combination with mismatch repair deficiency is hyper mutable but still can live. So that may be another factor that can be included into all considerations here and we can talk about it later. Okay thank you very much.