 Thank you very much. On behalf of Paul Spellman and myself and the rest of the members of the papillary renal cell carcinoma working group, analysis working group, we want to really thank the NCI and NHGRI for putting this together. As on a personal note, as a urologic surgeon who's worked on the genetic basis of kidney cancer for 31 years now, I would say this project and the previous project we worked on, the TCGA clear cell and the TCGA chromophobe kidney cancer really are sort of the dream of a lifetime. So what we're going to talk about is the comprehensive molecular characterization of papillary kidney cancer. Just for background, kidney cancer is not kidney cancer. It's a number of different types of cancer that happen to occur in this organ, different histologies, different clinical courses. We'll talk a little bit about the genetic basis of them, obviously. What we're talking about today is papillary kidney cancer which makes up 15% of kidney cancer and what we knew when we started this project, what has been a given for a long time, about 25 years now, is that papillary kidney cancer roughly is divided into two types. Type one and type two papillary kidney cancer. Type one, from a clinical point of view, is a relatively homogeneous disease. Appears clinically to be more indolent than type two kidney cancer. At this point in May of 2015, by the way, we have no effective forms of therapy for any type of papillary kidney cancer, the NCCN guidelines. If a patient comes to someone like me with advanced papillary kidney cancer is to put them on an experimental trial. We have seven drugs approved for kidney cancer, primarily targeting the DHL pathway in clear cell kidney cancer. We have no drugs for this. What we know or the field has known most about the genetic basis of papillary kidney cancer really comes from a couple hereditary types of kidney cancer. The first one being this hereditary papillary carcinoma, HBRC, which is the inherited form of type one papillary kidney cancer. The gene for this, of course, is met. In all of those families you find mutations in the activating mutations in the tyrosine kinase domain, and a few met mutations have been found in sporadic papillary kidney cancer prior to the starting of the study. Now, most of us who manage patients, you look at type two papillary kidney cancer, you say my word, that is a very heterogeneous disease. At the NCI we even refer to them sometimes as non-type one to tell ourselves we really don't understand these cancers very well. You could say clinically and phenotypically that these must be about three or four different types of cancer, and we'll talk about that. Again what's been known a little bit has been found from studying hereditary types of this of type two papillary kidney cancer, a disorder called hereditary Lyomaomatosis renal cell cancer, a very aggressive form of type two papillary kidney cancer caused by mutation of the Krebs cycle enzyme fumarate hydratase. So, in this analysis, of course, 161 samples looked at copy numbers, somatic mutation analysis, methylation, mRNA, microRNA, and protein expression analysis. We will not talk about whole genome sequencing in this talk. So initially then we had a lot of discussion about this, I must say a little controversy about it, whether we should actually have our pathologists look at these and characterize them. We decided as a group that it was the thing to do because that's what we do clinically and that's what we think about these, and we think they're very different. So we put together a group of expert pathologists, really are expert pathologists. I would say the five best kidney cancer pathologists I would say in the world, but we'll say in the U.S., and they looked at an incredible job, looked at these, had a single slide to look at, off of frozen, which is not perfect, but they did a great job, characterized 75 of them as type one papillary kidney cancer, 60 as type two papillary kidney cancer, and 26, they really couldn't call, they called them unclassified. And I think it was their sense and our sense that most likely most of the unclassifieds were actually type two. Now, in this analysis looking at copy numbers, so when you looked at copy numbers, there were basically three different clusters. Cluster one was a relatively stable, genomically, a few numbers of gains and losses. If you looked at cluster two, very different. You most notably, big time, increased copies, chromosome seven, also some 16 and 17. But the big thing here in cluster two is increased copy number seven. For cluster three, a lot of deletions, very genomically, you might say unstable type of kidney cancer, and most notably we would say would be deletions of chromosome nine, and we'll come back to that. Now, if you look at the clusters for survival, cluster one and cluster two, clustered for survival pretty close. Cluster one was more type two papillaries, cluster two was almost exclusively type two, and cluster three had the poorest survival that was mostly type two papillary and high stage, stage three and four. Now, when you looked at mutations, and this was done by looking at Mutsig 2.0, and then also we evaluated genes that were identified in Pancan 21, and a number of notable genes were identified, met, of course, fat one, set D2, which we'd seen, of course, in clear cell, NF2, KDM6A, BAP1, PBRM1, SMARC B1, NERF2, STAG2, P53, the chromosome three, chromatin remodeling gene mutations that we had seen in clear cell kidney cancer, that being set D2, BAP1, and PBRM1, chromosome three, chromatin remodelers, were mutated mostly in type two papillary kidney cancer. We then looked at a number of pathways, pathway analysis looked at mutations and pathways, found a number of pathways that were notable, we thought, one of course being met, other pathways such as hippo pathway and a number of chromatin modifier pathways. The SWE SNF complex was mutated in genes in that pathway in about 20, 27% of type one and type two, chromatin modifiers in 35 and 38%, both in one and two, and the hippo pathway mutations were primarily in type two papillary kidney cancer. Now the MET mutations that were found, 14 MET mutations were somatic, three were germline, most of them were in the tyrosine kinase domain, had a couple outside the tyrosine kinase domain, one in the gesticular domain and one in the semidomain, and they were all except one type one papillary, well, either type one papillary or unclassified, also found a specific MET splice variant in eight samples, resulting in loss of the first two exons and the gain of the novel exon of MET, not unexpectedly, but when you look at MET mRNA significantly higher in type one versus type two, same thing for phospho MET. We find amplification of seven, mutation, splicing, an increased copy number of chromosome seven, primarily in type one papillary kidney cancer, we'll come back to that. Also, gestic analysis revealed a specific region deleted on chromosome nine containing the CDK-N2A P16 gene. CDK-N2A hypermethylation was identified in 10 tumors, each of these, both the mutation and methylation correlated with low expression of P16, and when you looked at the 21 altered cases of either silencing, loss, or mutation, they were almost all either type two papillary, 71% or unclassified. When we looked at survival of all the tumors, all the type twos, the ones with CDK-N2A mutation had a significantly decreased survival versus those that were wild type. This was all of the papillary tumors. We also found TFE3 and TFEB fusions in 12% of type two papillary renal carcinoma, which was surprising. This is a surprisingly high number of TFE3 and TFEB fusions. Traditionally we would think of it as being more like 1%, including patients in their seventh and eighth decade, which is also surprising. Usually we think this was originally TFE3, we originally described in kidney cancer in 1996, and we traditionally think of it as tumor in children and young adults, but it turns out that's not the case. The TFE3 fusions also included four known fusion partners as well as two novel fusion partners, and both of the TFEB fusion partners that we identified were also novel. Now, when we did methylation analysis, we saw a number of things. We saw three different clusters. One of them which demonstrated a CPGiolin methylated phenotype, the SMP phenotype, and eight of the nine SMP phenotype papillary renal carcinomas were type two papillary renal carcinoma, and the SMP phenotype strongly associated with somatic or germline fumarate hydratase mutation or decrease levels of FH. That's the Krebs cycle enzyme. Again, when we looked at the SMP papillary renal cell carcinoma phenotype, we found that it was, it characterized early onset papillary renal cell carcinoma and low survival. So it's a very aggressive early onset phenotype. Now, when we looked at a number of metabolic genes, what we found was we think pretty remarkable. When we looked at glycolysis, fatty acid synthesis, AMPK, and TCA cycle, what we found was in the SMP phenotype, so here we have the SMP phenotype, here we have type two, and here we have type one papillary kidney cancer. In the SMP phenotype, this was very, very characteristic. Increased glycolysis, increased pentosephosphate chunt, decreased TCA cycle. This is like your classic Warburg metabolic shift. Increased glycolysis, decreased TCA cycle. That, if you look then at the type two papillary kidney cancers, what you see is a shift toward increased glycolysis and pentosephosphate chunt, but decreased, but normal oxfoss activity, looking at gene expression patterns. So this would be consistent with what we see clinically, either patients with papillary kidney cancer, very high PET scans. So what this would suggest then would be that those tumors are doing, have shifted to, made a significant shift to aerobic glycolysis. Now when you think about that, then you could also imagine that those tumors also would be characterized by increased oxidative stress, increased reactive oxygen. So we're going to come back to that in just a minute. So think about these type two papillary kidney cancers, the poster child being the SMP tumors, but the other papillaries as being very metabolically active. So how do you put this together? Well this was the model, very similar model that we saw in clear cell kidney cancer in the, in the Kirk paper and the clear cell TCGA paper, in which we saw with high grade, high stage, low survival, aggressive clear cell kidney cancers, we saw a metabolic shift toward aerobic glycolysis and decreased oxfoss. We're seeing the same thing here, exact same thing here in the SMP phenotype, increased glycolysis, increased pentose phosphate shunt, and decreased TCA cycle activity. Now when we did look at cluster of cluster analysis, the COCA analysis, we saw four different, four different, four different clusters basically, and this was Chad Creighton and Katie Hundley, did a beautiful job putting all this together. And the first cluster, C1, you can see here, is primarily type one papillary renal carcinoma. And the cluster, cluster 2A, 2B and 2C are type two papillary. And the cluster 2C is, went completely with the SMP phenotype. Cluster 2B, which I'll show you is the very aggressive type of type two papillary, was also characterized by a significant increase in the number of set V2 mutations. So then if you look at survival, if you just look at type one versus type two, type two papillary kidney cancer has decreased survival over type one, which is what our clinical experience would fit with. Then when you look at the cluster of cluster analysis and look at survivals, the C1, which was type one papillary predominantly, and C2A, which was one of the clusters in the type two papillary kidney cancer cluster, they did very relatively well to each other relative to survival. The cluster 2B, T2B, is significantly worse survival than C1 and C2. And the SMP, of course, is the 2C, is the worst survival. Now finally, we looked at a number of pathways, including this one, which were our most prominent pathways when you did pathway analysis. And we talked a minute ago about the type two papillary kidney cancers being characterized by potentially by increased reactive oxygen, increased metabolic stress, and a more metabolic shift aerobic glycolysis. Now a very pretty study by Ben Tay had shown looking at array patterns in type two papillary kidney cancers that this fit with this pathway, which is the NIRF 2 pathway. And NIRF 2 is the turns on NQ01 and the antioxidant response pathway, which is essentially a defense against reactive oxygen, oxidative stress. This is also something that many of us are working very, very hard to target. And we are very hopeful that this will be a targetable pathway. So because we're so desperate to find therapy for this, we're hoping this may lead us down the right path. But when you look at the NIRF 2 pathway and the surrogate here is NQ01, which is kind of a perfect readout for NIRF 2, as you see with the C1, which is predominantly papillary renal carcinoma, and then the 2A2B and SMP, you see that the NIRF pathway is increasing in those versus the type 1 papillaries. And you can see that also correlates with mutations in this pathway, which is NIRF 2, C3, and keep 1. And when you look at survival, we see significant decrease in survival in the tumors that have increased expression of NQ01. So what we show in this study is that type 1 and type 2 papillary renal carcinoma are genetically distinctly different tumors with different clinical outcomes. The type 1 papillary renal carcinoma is associated with MET mutations, MET splice variants, and gain of chromosome 7. The type 2 papillary renal carcinoma is made up of at least three distinct subtypes with differing survival. The CDK and 2A alterations are associated with type 2 papillary renal carcinoma and with poor survival. The TFE3 and TFEB gene fusions are found in 12% of type 2 papillary renal cell carcinoma and are found also in older patients versus just in younger patients. That the SMP type 2 PRCC phenotype tumors are early onset, poor survival tumors characterized by metabolic shift to aerobic glycolysis and decreased oxidative phosphorylation. And finally, that the NIRF 2 pathway is upregulated in type 2 PRCC and is associated with high grade, low survival papillary renal carcinoma. Thank you very much. For one or two questions, if you want to come to the microphone. For your presentation, could you give more details on what you saw about the unknown subset with respect to the genomic alterations you saw? You started your talk by talking about the fact that those are unknown subsets. Do you understand that? You have a non-type 1, non-type 2, a third other category. Where did they fit in? That's the question. I'll classify. You're unclassified. Oh, I'm sorry. Oh, so sorry. The unclassifieds, I'm sorry. Sorry. Great question. The unclassifieds really, okay, so the unclassifieds, that was by pathology. And that was by looking, all we could do was take the frozen, make an H&E, PCJ did a great job on this, getting into those slides, but really hard for pathologists to make a call on that. Anyway, so the 26 unclassifieds we had, most of those fit with type 2. Okay, so our presumption is those were really type 2 papillaries. Yeah, but are related to one of the subsets of type 2 or not? You know, that's a very good question. I don't know the answer. That's a very good question. It was where would they fit in with the TFE3 or the others? That's a good question and I don't have that in my head right now. We'll have to look that up and I'll get back with you on that. That's a good question. We didn't analyze that specifically, other than we should have. So I wanted to ask about the non-MET mutated type 1 tumors. Any ideas as to what the drivers are in those that sort of make up for MET? That is a very good question and that is something we are desperate to know and I hope you recognize that we said chromosome 7 increased copy number. We didn't say that MET was the target there because there's a lot of stuff. It is incredibly characteristic of type 1 papillary. But what the driver genes are, we don't know. That's a very good question. So we would assume, and we've got the whole chromosome 7 to think about including EGF, HGF, all sorts of things. We have increased copy number. We have increased, we have increased phosphoment. We have increased MET. But is that the target? That remains to be determined. That's a very good question. We also have 16, 17, but we don't know. Thank you so much. Sure, thank you. Our next talk is Dr. Angela Books, who will be telling us about the high throughput somatic variant impact phenotyping using gene expression signatures.