 All right, thank you Chuck I'd like to thank the organizers for allowing me the opportunity to speak here today I want to specifically mention to Kenna that I did just sign the release form so you can tweet and film and Facebook all you want Let's see how does this work, okay, so and of course I'm representing a large group of people here So you may think because I'm today. I'm talking about the somatic landscape of GBM, and you may think Sure, there's an easier way to do this. Oh there You may think a GBM marker study didn't we already have one of those and that's of course correct because we started in 2008 by publishing on the comprehensive genomic characterization of GBM And that was sort of the kickoff publication to and the entire TCGA and then we followed that in 2010 by two papers One describing gene expression subtypes and a second one describing a hyper methylator phenotype so The the data set that we used for those three papers is sort of represented here We had DNA sequencing of About 600 genes in about 91 cases at that time of course in an enormous amount of sequencing Whereas nowadays you can do that in a day and we had molecular profiles and copy number profiles on us on a sample cohort of about 200 cases So fast forward to 2012 now we have exome sequencing 300 cases or close to 300 cases we have copy number on almost 600 and we have some form of unlocular data on about 600 samples So this is really quite a large data set Importantly we also have whole GM sequencing of 17 samples and RNA sequencing of 164 samples First we looked for significantly mutated genes using the 300 or close to 300 exomes You'll see that the top Genes are the ones that we saw in our 2008 paper as well p53 eGFR p10 sort of the usual suspects But we also find a bunch of novel genes that have previously previously been Unassociated with cancer and Cleo Blastoma in specific So for instance, there's spta1 that is mutated in in 10% of the cases ATRX, TCHH and so on and these genes are all free present at frequencies above 3% So this slide summarizes the 26 most significantly mutated genes With the 300 samples on the x-axis and the genes on the y-axis First I'd like to point out that we see No significant gene mutation in about 10% of samples that's sort of remarkable in my mind Although we see that in most tumor types We see interesting patterns of mutual exclusivity such as There's never a co a never a mutation in both pic3r1 and pic3ca Which obviously makes sense as these are working in in complex And we also see interesting patterns of co-occurrence for instance all the cases with an ideates one mutation also harbor a mutation in p53 and the majority of ideates ones harbor a mutation in ATRX and this has been Recently reported by Vogelstein and colleagues That these also co-occur in lower grade oligodendrogliomas I'd also like to point out that we found a five B ref V600 e mutations These are of course of interest because they are very frequent in melanoma and respond to femorefinib in that disease Although in other diseases such as colon cancer these have also been described, but they were not sensitive So it doesn't automatically translate to a treatment And as John Weinstein showed this morning, we also look for Patterns of mutation in chromatin modifiers as these have recently gained great interest in different diseases We compiled a gene set of 167 genes thought to relate to chromatin modification and we then plotted the mutation Mutations in these 167 genes as you can see in this slide And we find that about 40% of GBM has a mutation in at least one of those and these occur in a strikingly mutually exclusive exclusive fashion Now we tested this for significance by doing 10,000 permutations of similar sized gene sets And we found that in 97% of these permutations We find a lower number of cases to harbor a mutation thereby suggesting that this finding is quite significant And as I mentioned we have a very large number of copy number profiles And in 2008 we reported on about 200 GBMs And we found a number of significant Amplifications as is shown in this logistic figure where you have all the chromosomes on the y-axis and each of the peaks represents a focal region of copy number gain Now fast-forward again to 2012 We find more peaks also because we have a better methodology But we also importantly find that most of these peaks harbor only a single gene Similarly, we find this for Focal copy number loss and I want to highlight QKI at 6 Q 26 Which is now the only gene in this region and this has previously been thought to target Park 2 So we looked at genomic rearrangements using a whole genome sequencing data This is a specific copy number locus chromosome 12 Q 15 This is a locus that harbors MDM 2 and each of these lines here represents a copy number rearrangements between two chromosomal segments So as you can see this in this specific sample, there's many genomic rearrangements And we could actually assemble all those genomic rearrangements into an extra chromosomal Ring structure also known as a double minute and this was confirmed by fish I see one Showed you this morning. We looked extensively for fusion transcripts And we found 84 in-frame fusion transcripts. We also found a number of out-of-frame fusions in 164 GBMs And I want to highlight FGFR3 tech 3 as this was recently published Just like the ones you want this talked about this morning TFG. This is a local small inversion And since we have and we have two cases that harbored this fusion event and in both cases This is the copy number profile. We see that this Attacks along with a focal amplification We found a number of EGFR targeting Fusions they don't do not necessarily involve EGFR as a fusion partner, but all of these occur In the area of an EGFR amplification, so these are 11 samples that have an EGFR associated fusion So this is EGFR here in the middle And the red indicates the area of focal copy number gain So this suggests that there's a more complex event going on at this locus We looked for intergenic rearrangements Starting with the v3 deletion that has commonly been reported in GBM and the v3 deletion targets exon 2 to 7 and falls in the extracellular Domain of EGFR and this is also the domain where the majority of point mutations occur We then searched for C terminal deletions and we found three different C terminal deletion variants And but we are likely undercalling the true number of C terminal deletions as those would not result in a fusion Transcript with reads on both sides of delete the deletion so the 6.4 percent that we report is likely undercalling and Then we Also showed this morning. We also find two novel variants or at least relatively unknown. There's a few incidental reports on this That target exon 12 and 13 or exon 14 15 If you now combine all the data on EGFR And using all using all our samples we find that 45% of GBM harbor an EGFR associated point mutation or genomic alteration Aside from the focal amplifications that we see so EGFR is clearly one of the most critical genes for gliomagenesis and Efforts to devise a EGFR therapy which still be very worthwhile As I mentioned in the introduction we have previously looked for molecular subtypes We found a G simp or hypermethylator phenotype That fell entirely within the expression pro neural group and we also described a neural a classical and a mesenchymal expression group This slide shows you the data of about 330 cases sorted by molecular subtype and in each of the rows indicates a genomic change and It highlights associations between genomic abnormalities and molecular subtypes for instance There's the association between ideates one mutation and G simp which we of course have previously found We now also see that most of these have a mick amplification Similarly, we find EGFR amplifications in classical, but we also see cycling E1 in the classical group and so on and so forth Importantly we confirm that the G simps do a whole lot better In terms of outcome than the for non G simp expression groups And we challenge a bit of paradigm here because if you look specifically at the non G simp samples You'll see that the pro-nerals without G simp do worse than other groups Whereas this was previously thought to be one of the better survivor groups Lastly we have our PPA based protein expression profiles So I have to disappoint Doug Levine because we also looked at this in the context of the gene expression groups For instance, of course PG phospho EGFR is highly Expressed in the classical group which also had all the EGFR amplifications and we also see although It's not as visual, but we also see a small decrease in the apoptosis modules So a number of proteins that combine form an apoptosis module We see decrease in expression in that module in the classical group So in summary I described comprehensive genomic profiling of about 600 samples We detected novel significantly mutated genes such as spta1, LZTR and so on We used whole genome and RNA sequencing to detect genomic rearrangements and most notably involving EGFR And lastly I want to again point out that the pro-neral class may actually perform worse than other subtypes I want to thank Linda Chin, Cameron Brennan and Aaron McKenna with whom I have co-led this project And I want to thank the people in my lab most notably Siobhan and Rahul who performed a lot of the analysis that you saw today And lastly the TCGA G-Dec and MD Anderson Cancer Center. Thank you. Any questions for Raul? I have one which is So is EGFR amplification is that occurring in double-minutes? Or is it in the chromosomes or what? So the double-minutes that we have seen we have seen two different double-minutes. So two individual whole genome samples had one They both are contained MDM2 But one of the two additionally had portions of chromosome 7 So it was a chromosome 7, chromosome 12 double-minute and indeed it also contained EGFR So I was going to ask the significantly mutated genes that are new. Do you think they are biologically significant? Well, of course, we don't have any functional data and in terms of the statistics I would argue that they are The methods we have to identify significantly mutated genes I think have improved and Based on those methods we would argue that these are biologically significant But we don't have the functional data to support that notion Following Chuck's question, I noticed and David there's some significant mutation of PDGF RA and I think Eric Holland's group at MSKCC had reported Introgenic rearrangements and PDGF RA similar to those in EGFR Did you observe a significant number of those rearrangements as well? Thanks, that's a great question. So PDGF RA indeed has small Exxon 8, 9 deletions and we see those but at much lower frequency Very actually, I maybe one or two percent I would say How does the MGMT promote a methylation plane to your survival analysis? It's also a good question. We do have MGMT methylation status I Don't think if I remember correctly. It doesn't specifically track with one of the molecular subtypes But we haven't corrected for it in the survival figures that I showed you. Thank you, Will