 Thank you. It is my pleasure to present some of our results and discoveries from the pheochromocytoma and periganglioma analysis working group and I co-chair this with Carol Pasek and Kate Nathanson and Just to provide some background on the disease. It's a rare chromophon cell neoplasm with a thousand cases per year in the United States chromophon cells are neuroendocrine cells that see create catecholamines such as epinephrine and and see Wet and the tumors arising in chromophon cells are named based on their location They rise in the medulla portion the interior portion the adrenal gland they're called pheochromocytomas And if they're located elsewhere in the sympathetic nervous system, they're called perigangliomas But for brevity here, I'm just gonna use the word pheo in speaking refer to both pheochromocytoma and periganglioma and call This distinction out if necessary Up to 30% are malignant and if you have malignant disease Outcome is not good with 50 less than 50% progression free survival at one year and there's few markers of metastatic risk Interestingly pheo has the greatest rate of underlying inherited mutations estimated at 30 to 40 percent Which is the highest of any tumor type and this inherited Increased risk for developing pheo is conferred through 19 susceptibility genes including NF1, RET, the succinate dehydrogenases and some a variety of other less Frequently common least less frequently mutated genes. So pheo can be familial or sporadic Based on inheriting one a mutation only susceptibility genes There's also been some interesting work on RNA expression close RNA expression clustering which is to find two main clusters a pseudo hypoxia cluster In a kinase signaling cluster and this has potentially been Divided up into five clusters and more recent work that are associated with mutations either Somatically or in the germline in these susceptibility genes And so for the pheo cancer genome atlas the the aim is to identify genomic alterations Integrated as classifications than markers of benign versus metastatic disease We have a cohort of 173 patients Each case has the seven to eight platforms of the TCJ, which makes this really unique resource for pheo And in a cohort of this size So tape first result I wanted to show is just taking a step back and either by number of somatic mutations number of DNA chromosome breaks RNA proliferation or DNA methylation pheo here in PCPG is at the extreme low tail end it just Doesn't have a lot of somatic alteration on a large scale however There is a lot of diversity in mutations in pheo and so looking at the 19 susceptibility genes We detected mutations in these led by SDHB at the top ret VHL and in some this covers 25 percent of patients, which is strikingly high and close to published estimates Adding to this somatic mutations that we've detected in this list those with an asterix were significant by a mutsig analysis and Most of these are previously Have been previously associated with pheo with the exception of CSDE one I forgot to mention the rest of the un asterix genes are significant in pheo or in other cancer types But what I take from this is there's just a striking exclusivity Across the germline mutations and the somatic mutations patients are basically mutes in Exactly one or zero of these genes with few exceptions and we're looking into these double mutants. There is a ret Somatic and germline double mutant that appears to be authentic and we're following up on the rest of the cases So in some we have 65 percent of pheo patients with a mutation one of these genes Some of the genes are mutant in the germline and somatically ret VHL and NF1 and this is consistent with prior work The the ret the by mutation origin ret mutations collect in this extracellular domain of ret and Somatically clear collect in this terracing kinase domain, and this is this is previously reported, but it's interesting that the different portions of ret Depending on how the mutation happens occur in different spots. They end up being in the same expression subtype And I'll show that later an NF1 of VHL didn't show any Mutational tendency preference Turning to our novel driver gene CSDE one the mutations are shown in this portion The gene to splice site to frame shift mutations when it's mutant it's copy number lost and under expressed extremely under expressed So it seems to be a loss of function role and from the literature knocking out CSD one causes irregular neuronal migration and brain development Two of the mutations were spiced sites that sometimes have uncertain consequence on the protein product of the transcript So from UNC or analysis that makes mutation calls based on the integration of DNA and RNA evidence We observe the mutation in tumor RNA This is the acceptor site of this intron of CSD one in trans retained protein product of the transcript is disrupted So we're able to confirm that for this CSD one mutation in terms of somatic copy number we see one P lost three and And three Q and some other regions Lost at other rates and these associate with where the inherited susceptibility genes are and it's consistent with what we've seen before In terms of focal deletions There's a set of deletions including CSD one which is right near the peak of the one P loss NF1 But interestingly the folk I want to focus on the focal copy number amplifications. There's very few But when looking at the samples that had the amplification on for Q They were the same samples that had the amplification on this 17 Q and then looking at the fusion Transcripts detected from RNA sequencing by map splice I found that this these same samples had a fusion transcript joining a portion of the gene in for Q and a portion of the gene 17 Q and this is the UBTF mastermind UBTF is on chromosome 17 and mastermind like 3 is on chromosome 4 It's setting and this is a display of the bridging and spanning read evidence from a visualization output of the map splice program And we have two we have seven positive cases Joining these two genes two different isoforms a different Exa that are specific to different samples. You don't observe both isoforms in the same sample So it suggests two different sites of a DNA Intrachromosome translocation at different exons exon 14 and 16 UBTF also have a TCF for its on chromosome 18 In a fusion transcript with mastermind like 3 and so these are this is an overexpressed transcription factor is an overexpressed Transcription factor it's an activating fusion mastermind 3 is highly overexpressed in these cases to a large degree And then in addition the exonic expression pattern also supports a promoter swap for of UBTF or transcription factor for To mastermind like 3 and so these diagrams show over relative over or under expression Normalized within the gene normalized relative to other samples and this is the fusion point at these triangles You can so you can see there's overexpression prior to the fusion point under expression under expression prior to the fusion That's not part of the transcript in this and in the tumor cells at least and overexpression of the downstream portion of mastermind like 3 and it's this is pretty uniform This is a consistent pattern across the three species Some of the tail ends appear to be under expressed, but this composite view compresses some of the alternative initiation. That's found in these genes So this supports It's a gain of the UBTF and transcription factor for a drive in the overexpression of mastermind like 3 And so what is it doing? Well mastermind Like 3 is known to be a notch co-activator, but the fusion lacks the notch binding site and similar to another fusion reported in Cytonasal sarcoma and so it's not notch and so we analyzed by our platforms To see to get a sense of what this fusion event might be conferring to the tumor What enhanced properties and so we see we saw by expression There's overexpression of genes in the wind receptor signaling pathway by micro RNA expression Mere 375 is the most under expressed mirror in fusion fusion positive samples And some of its known binding partners that it are significantly anti correlated with mere 375 expression includes one of the frizzle genes By DNA methylation mastermind fusion samples of a very divergent methylation pattern and some of the hypo methylated probes include some of the frizzles and some of the winds and so Pretty consistent story on three different platforms that mastermind fusion positive samples are associated with increased wind signaling pathway expression So to put to classify our tumors we detect we applied unsupervised expression clustering we found four clusters that were statistically significant by SIG cluster analysis is represent the major patterns in FIO and we compared to the earlier study that I mentioned who had a five class scheme and our four classes Correspond to their five classes one of their classes one of our classes joins to their classes, but What this shows is that the subtypes are reproducible across cohorts This is there's more than two clusters and our data support that there are four clusters Some of the marker genes of the expression subtypes are shown here such as ret being overexpressed and what's marked as the the black subtype And so having these expression subtypes we wanted to ask is there differential Potentially differential molecular pathogenesis that gave rise to these subtypes and so we analyzed all of our mutations and Fusions and genomic events by this subtype stratification to answer that question and cutting to our most Novel finding all of the mastermind fusion positive samples occur in exactly one subtype which we call the mastermind subtype In addition most of the CSD ones Somatic mutations occur in the mastermind subtype and so the subtype previously didn't have Anything known about its molecular pathogenesis. We've assigned two events to it and we've also shown that this appears to be a Form of sporadic pheochromocytoma, not a familial form The kind of signaling subtype has all of the or most of the NF one hrass and ret mutations germline or somatic this Property has been previously reported. We add to that three new events that are related in similar pathways B ref fusion and NGFR fusion, which are both activating appear to be activating an NF one in activating fusion The pseudo hypoxia subtype has almost all of the perigangliomas. It has all of the SDHB VHL and E-POS1 or HIF2A mutations and so in this coordination of alterations has been appreciated in this tumor type in addition There's hyper methylation of many of the samples in this subtype and that's related to the succinate dehydrogenase mutations That's a previously known phenomena The last subtype cortex admixture had very divergent expression characteristics and on secondary Histopathology review we found that there were subtle cortex cell presence and In these in these specimens that was enriched in these specimens not completely specific but enriched and This subtype also had other increased indices of reduced tumor purity Taken these exclusive mutations and putting them in the pathways in which they occur to main pathways Which are appreciated in pheo? Pop out the pseudo hypoxia pathway and the PI3 kinase a kt and map the kinase signaling pathways And which leads to the pseudo hypoxia subtype has a variety of mutations that lead to activated HIF It's very specific for the pseudo hypoxia subtype that is showed in the subtype figure And similarly in the PI3 kinase map cut PI3 a kt and map kinase signaling pathway these alterations Cooperate or in their own independent way cooperate to alter altering this pathway and so these two pathways are very important in Theochromositoma and you get almost the same assignment from unsupervised expression clustering and so the last results which was important for us was to try to find molecular correlates of metastatic disease and in order to assign Metastatic status to our samples Lauren fishbine led a comprehensive effort to review the clinical record of every case and Assigned WHO metastatic status to every specimen and then we did we decided to add to that Samples that had local regional disease at diagnosis or experienced a positive lymph node in recurrence or for whom the patients were deceased and the union of these features Defined a clinically aggressive status. So these are benign samples and clinically aggressive samples And that's the variable we used for outcome and some of our mutations and other genomic events that we Discovered in this study SDHB is significantly associated with clinically aggressive disease and that recapitulates. What's known. That's a known marker germline mutation Mastermind fusion our new event is also associated with clinically aggressive disease. So it's a really striking finding for us As is the hyper methylated subtype The mastermind expression subtype and then one of the micro RNA subtypes on the other hand On the other side ret and all of the ret NF1 Hs and VHL are mutant by germline or somatic And it's clearly enriched in the benign group. It doesn't reach statistical significance in this version of our data This is one behind our data freeze and we've had some more germline mutations detected since then But you can see visually that they're Enriched here, although that means statistical significance So I take from this that for pio tumors you experience one set of alterations It sets you on a path towards clinically aggressive disease and a different set sets you on a path towards benign disease And so in summary of the the new discoveries that we made in the TCGA pio study 65 percent at least 65 percent of cases have a driving germline or somatic mutation and that's not including our fusion So it's we're able to explain part of the molecular pathogenesis for a large proportion of patients in this cohort We have the first recurrent fusion in piochromocytoma the mastermind fusion associates with the clinically aggressive disease It's found in exactly one expression subtype of sporadic pio, and that was a sporadic piochromocytoma had fewer markers for malignancy familiar piochromocytoma Also overexpresses the wind signaling pathway And we have the first reports of some other alterations in this disease including the CSD1 somatic mutations Fusion genes and NGFR, BRAF and activating NF1 fusion And so with that I just want to acknowledge all the people who've played a major role in this study And all the people listed here have played a major role And I just want to additionally acknowledge Vaughn Walter for his efforts and RNA sequencing analysis Lauren Fishbine for her efforts in the clinical record review my two co-chairs Kate Nathanson and Carol Pasek Ignat Leschner from the Brodeford's efforts and comprehensive somatic and germline mutation calling that's been a very large effort Brad Murray for copy number analysis Lou Miller-Tanilova for methylation analysis Gordon Robertson for microRNA expression analysis Tara Lichtenberg and Karen Lienos from the BCR we have I think a unique set of clinical features and we've had Great access to that and being able to understand the day that we have Ena Flau for coordination Keneshofer their early days this project and getting it off the ground and then most importantly all the disease experts for coming together and building a large cohort for this rare tumor type Including Art Tischler Who has reviewed the pathology Reviewed the histology of all of our cases for various metrics. And so with that I Just close and happy to take any questions Matt, that's great What is the significance of the observation that these tumors and the kidney tumors share? Both germline and somatic mutations like SDH and VHL What is the significant you mean the cross tumor type significance? I mean these are Different cell types and yet they seem to share You know the same set of both germline and somatic mutations and I wonder what the significance of that is That's a great question. I don't have a good answer for that that this time I mean is that a possibility that Some of the kidney tumors Are classified as pheochromosatomas. I really don't think I know they say adrenal glands on top of the kidney But I don't think that is an issue in this data set But that's a good that's a good suggestion. We can look into that So I've got one question for the mastermind group You have about half of them either have that fusion or the CSD one mutation. Do you know what the others? Have that's keeping them in that they have high expression or They they don't have high expression of masterminds. They're they don't appear to be false negatives for the mastermind fusion event and I don't know the relationship of those two genes But that's a great point to try to explain this remaining cases Any other questions? Thank you Okay, our next speaker is dr. Sandra Orcelik from Cedar Sinai Medical Center medical center who's gonna tell us about a multi gene signature associated with stromal activation