Adrenal Gland Cancer

tom giordano:so it's a pleasure to be here to present some highlights from the thyroid project. i'm goingto start with a very simple model of thyroid cancer. let's see. so, we start with a normalfollicular cell and we progress to a well-differentiated tumors, either papillary or follicular tumors,follicular adenoma, and follicular carcinoma. most tumors stop here, but rarely they evolveinto poorly differentiated anaplastic carcinoma. and the point i want to make here is thatthere's a progressive loss of differentiation. differentiation is very important for thyroidcancer and it's actually the foundation of our classification. we talk about differentiatedcarcinoma and undifferentiated anaplastic carcinoma with poorly differentiated in themiddle, so it's really a foundational thing

for thyroid cancer. so, when tcga started this with 85 percentof the cases being papillary, it really became clear that we needed to focus on papillary.so, our project is restricted to papillary thyroid carcinoma. so, oops. so, there's threemain types of papillary carcinoma. there is a classical type that gets its name becauseit has these well developed papillary structures. there's actually a follicular variant thatis recapitulating normal thyroid architecture. and then there's a tall cell variant thathas abundant cytoplasm and tall cells. and those are the three main types. there's othertypes but we wouldn't get enough to really power an analysis of the rare types so wekept it simple and there's a very strong genotype/phenotype

correlation as shown here. so, before the tcga, this was sort of theview of what genes were known to be mutated in papillary carcinoma. mostly in braf andras, but also rearrangements of ret and ntrk1, and with infrequent pi3k [unintelligible]mutations. so that's sort of the foundation from which we started. so this is our cohort.we had up to 496 tumors with 391 on all platforms, and we had 49 whole genomes that we targetedat tumors that didn't have obvious driver mutations. so, i really like this slide. i think it explainsa lot. it shows that amongst all these tumor types that thyroid carcinoma, papillary thyroidcarcinoma, has a relatively low mutation density

and actually the lowest, if you restrict yourview of this, to solid tumors like, especially carcinomas. so it's, you know, the next onethat starts in his prostate here, and breast cell. so we think this is partly the reasonwhy it's such an indolent carcinoma. whoops. a little jumpy. so this is our overview ofour somatic alterations. i'll go through just a point, a few points here. so, we have mutationrate, clinical information, and significantly mutated genes. the first thing to notice isthere's not much up there. right? there's a lot of white spaces here. so, it's a relativelyquiet genome. we can see that braf alterations are common in about 60 percent of cases. andthen we have a mutually exclusive ras mutations. we have a new gene mutation here, eif1ax,which i'll talk a little bit more about, which

is mutually exclusive with raf and braf. andthen we also discovered ppm1d and in check to a significantly mutated. and then we jump to the fusions and you cansee we have a diverse collection of fusions that make up about 15 percent of the cohort.some expected ret but also diverse fusions of braf. so that's one of our interestingfindings that we have -- these braf fusions. and you can see that these are mutually exclusivewith each other and also with the point mutations. so that's a very nice story. and then we jumpdown to the copy number changes and again, many, many of the braf mutated tumors don'thave a lot going on in terms of copy number change. and then you can see very interestingly,we have a concentration of arm level copy

number changes that start up when the pointmutations and the fusions go away. so we're speculating that their drivers imbedded inhere, we obviously can't pinpoint the genes, but we think this is an interesting resultand very provocative. and so if you allow us to count those as potential drivers, weend up with only about 14 cases without a driving event out of 400. and this is relevant because, you know, inusing existing genes, if you genotyped 100 cancers, you'd only find a driver in about75 percent. so, we've expanded the universe of driving events and that will have profoundinfluences on molecular diagnostics. so this is eif1ax, and here's our mutationsand they're sort of landing in the same region

as those reported in the cosmic database andin this paper here on uveal melanoma. so we haven't functionally proven this. jim fagin[spelled phonetically] is actually working on this at memorial, but we haven't proventhis driver, but we think it will turn out to be a real event. so here's our fusions.we have some new ret partners. we have -- we think they're real because they retain thekinase domain and express the kinase domain. we have diverse braf fusions. we've discovered,and a few other people have discovered, that alk fusions are present in about 1 percentof the cohort. and then etv6-ntrk3, which also came up in the screen of radiation inducedpapillary carcinomas recently. so, we think this is a big part of our story -- these newfusions. oops. this is a little jumpy.

so, if you look now at the 14 cases that areofficially dark, they're actually not that dark. we do have some interesting mutationsatm, apc. we have some hits on some potential fusions, so, you know, if you allow us tocount maybe some of these as potential drivers, we're really down to about five cases thatdon't have any explained cancer mutation. so, there was a report that suggested brafwas subclonal. so we specifically looked at the five drivers and using absolute to makea statement about their clonality. and we're making a strong statement in the paper that,you know, these drivers are in fact, clonal, which has implications for targeted therapy. so, what were some of the challenges thatwe faced when we did this project? one, it's

restricted to papillary carcinoma, which isa very indolent, well-differentiated tumor, that's cured 95 percent of patients. and ifyou're going to study the outcome of this disease, you really need 20--year data andour cases don't have anything close to that. so, that was a challenge. and then we havethis relatively low mutation density. so that presented us with really, with two choices.we can sort of write up a bio-marker paper that, you know, had some new point mutations,talk about the fusions, do the clustering, and call it a day, or we can push. and gaddy[spelled phonetically] and i maybe synergized and we pushed. so we focused on the fact thatthese drivers were mutually exclusive, that we had a relatively quiet background genomein which we could explore the role of these

drivers, we had all this multi-dimensionaldata, and we had a very, you know, great and imaginative awg. so the first thing we did is we said, "well,let's explore this difference between braf and ras." and we developed a signature -- a71 gene expression signature that separated these tumors out and then we converted thatinto a score of braf-v600e-ras score that we call brs, and we scaled from minus one-to-oneand then displayed the tumors. so what you can see here is that it displays the tumorsalong this gradient and it's not black and white. there's a transition that goes on herein the middle, and then we can use this score to explore how the other mutations, you know,where they fall on this gradient. what's really

fascinating, you can see that there's somebraf mutations that are not v600e that are actually ras-like. and this one's actuallythe braf k601e mutation and that's consistent with the literature because those are thoughtto be, you know, the follicular variant which tend to be more ras-like tumors. and then you can see some of the other fusionslike the pax-ap per gamma are weakly ras-like and that makes sense. so, this was a niceway to explore how these other mutations might signal. and then if you bring in all the otherdata into this figure, you can really see that the biology of these ras-like tumorsis very different across all platforms from the braf-like tumors. and that's one of ouroverarching conclusions that these are fundamentally

very different tumors. then we turn to thyroid differentiation andthis was known that when you pick up a braf mutation, that you have a loss of differentiationand particularly silencing of the iodine metabolism machinery. and so we wanted to explore thisin our cohort. and so, oops. so, this is a complicated slide, but basically, these genesare those responsible for the iodine metabolism machinery. and we displayed them in contextwith genotype. here's braf v600e. here's ras. here's a fusions. and you can immediatelysee that the tumors on this side, which are the follicular variant tumors, are more differentiatedthan the braf tumors. that's not a shock. but what's really interesting is within thebraf v600e cohort, we see a range of differentiation.

we said, "well, why is that? why do we care?"we care because there's probably at least 100 papers that have looked at braf as a bio-markerin isolation. in other words, the field is treating braf v600e papillary carcinoma asa homogeneous tumor group. and our data suggests that may not be appropriate. so then we got interested in what are thedrivers of that, what are the potential drivers, what's correlated to that, and we found outsome interesting genes like trefoil factor three and two alchamiers [spelled phonetically]mere21 [spelled phonetically] and 146, and then potential tumor suppressor mere werecorrelated to the tds score that we derived from these genes. and so we have some interestingpossibilities and keep those in mind because

they'll resurface later. so, we used different kinds of data. we usedthe messenger rna and the rppa data. the group at memorial spent a lot of time in figuringout the signaling consequences of these two drivers. the take home point is that the braf-liketumors signal pretty much exclusively through map kinase and that the ras tumors are -- havea much more complicated -- little bit more pi3 kinase, but also some map kinase. so,we explore this in really show that there's fundamental differences here. and then ontothe clustering. we -- i don't really have time to go through each platform so i'll showthe super cluster. basically, all platforms, like i showed in the earlier figure, showsthat there's a striking difference between

ras-driven tumors and braf v600-- like driventumors. and there's histologic differences, et cetera. so that's not shocking; again,it confirms our big conclusion. what's interesting though, is that there's a cluster here, thatis very robust and it matches up between the different platforms -- methylation, messagerna, micro rna. and that it's maybe hard to see, but these are enriched for the tall celltumors. so we think that there's a distinct cluster of tall cell tumors that have distinctexpression profiling. and so that becomes more important in the context of this, wherewe really focused on the meres as part of the story. and i'll spend a little time goingthrough this. so, basically, mere cluster one are ras-like tumors and then we have fiveclasses of the braf-like tumors. and you can

start to see that there's some interestingmolecules that are preferentially expressed in some of these mere classes that i'll focusedon this one -- mere21, known occemere [spelled phonetically] is here. and why do we thinkthat's relevant? well, we actually took the scores that we developed in the middle partof the paper, the braf-ras score, and the thyroid differentiation score, and used themthroughout the clustering section to make it more rich and informative. and you can see here, this cluster has three-fourthsof the tall cell tumors, it's in a braf background, there's not a lot of other mutations goingon, it has a higher risk, it has -- i graded all the tumors, and it has a higher grade,it has clearly different messenger rna profiles,

and -- but, most importantly, it's the mostbraf-like, because it has the lowest braf-ras scores. and it's the least differentiated.it has the lowest thyroid differentiation scores. and, so, why do we care about this?you know, because i admitted that we -- that the tall cell is, you know, a recognized variant.well, we care about it because as come through it all pathology talks, we tend to disagree,and so what i call a tall cell another person may not call a tall cell. so if we can actuallyuncover a molecular marker of the tall cell that would be adapted widely by the thyroidcommunity. so we're very excited about this and then there's a similar story here forcluster five and mere-146, which, i remind you, were the mere's we uncovered back in,when we were looking for correlative tds.

so we think the different parts of the paperfit together nicely and support each other, and we really worked hard to tell this integratedclustering story. so, our overarching conclusions were thatras-driven ptcs and braf v600 ptcs are basically fundamentally different. and so it begs toquestion, should we reclassify thyroid cancer to sort of separate them. and there's somedata that suggests we should. there was a paper in the new england journal from jimfagin's group when you're looking at a mac-inhibitor that had differential responses for recoveringsusceptibility to radioactive iodine and it depended on what your underlying genotypewas. so i think that the days where you could lump papillary carcinoma and run a clinicaltrial without knowing what the underlying

drivers are, what the phenotype is, is just-- it's coming to an end. likewise, we identified clinically relevant subgroups of braf-drivenptcs and we have a potential role for meres, so, we think things are going to come fromthat. we're actually very excited about how this whole project played out and somebodysent me an email like,well, tom, you know, a year ago, this was kind of looking, kindof, maybe, not dull, but you know, and now, it's really turned into this kind of, interestingstory. so, we're very excited about this and if youdon't believe me then, here we are. like gordon, especially. so, we do think this will be alandmark study for the thyroid field. and so far, it's already having impact. as i mentioned,jim fagin is working on the biology of eif1ax,

in part, catalyzed by this. yuri nikiforov,for starting a working group of pathologist to explore the follicular variant and maycome up with the proper way to diagnose this. there's a lot of argument and disagreementamongst pathologist so he's actually looking for support from the nci to study this. andthen we had a collaboration -- hopkins, mayo, michigan, and cornell, looking at braf inisolation, like many other people, and then when this -- the meres came out, i convincedthe group to say, "well, let's expand this study." so now we have hundreds of cases andthe whole goal is, can we use these meres in combination with or without braf to predictcentral compartment lymph node positivity and sort of guide surgery? so i do think thatour paper will be very impactful.

of course, there's many, many people to thank,but i do need to give a special thanks to chip, who is the analysis coordinator andreally cranked through massive amounts of analyses. and of course, gaddy, who was amazingto work with. so, i want to just take a moment to thank tcga leadership for giving me theopportunity to work on this project. what's nice about this is that gary hammer [spelledphonetically], who's here from michigan, and i, you know, convinced kenna [spelled phonetically]to start a project on adrenal cancer, so, we used the opportunity of being involvedin thyroid to span adrenal cortical project and then the feo [spelled phonetically] projectwill also play a role. so, this has really been a very exciting time and a fun time forme, and i would just like to thank everyone

and acknowledge the awg. i'll be glad to takesome questions. hopefully i did it in time. [applause] han liang:tom, very nice talk. so, the question i have is for those genomically very silent ones.where do they fall? do they fall to the ras-like? or do they fall to the bref? tom giordano:the remaining dark matter tumors? han liang:yeah. tom giordano:they're enriched in the follicular variant. and so the truth is, those are the cases thatpathologist argue about. right?

tom giordano:it's really complicated but -- han liang:-- it's not, so, basically, those are different disease, basically. right? so, like, different.it happens to many, kind of, like kidney, that we see, that -- tom giordano:-- it could be, it could be. i mean, so, we can talk afterwards. i -- you know, it's along, long answer. yeah, matthew. male speaker:hey tom. so a lot of incredible stuff in there. but i was really struck by the mutations inthe thyroid globulin gene. right? and i was curious if those are loss of function mutations?

tom giordano:yeah, so we've put them in the paper, matthew, but we're not exactly sure, you know, howmeaningful they are. they're in there. we went back and forth on that, maybe chip'shere. not sure. that's why we put it in there but we didn't make a big deal about it. theguys at md anderson, steve sherman was very excited about that. but the story didn't panout as strongly as we thought. so, not sure how meaningful they are. male speaker:so it's just a striking feature of different cancer types that i don't understand. butyou have loss of function mutations of algumen [spelled phonetically] and [unintelligible].there's loss function mutations of collagen,

and convosarcoma [spelled phonetically], andthis might be part of that same pattern. tom giordano:it might be. i mean, mutic did not pick up on them because it is a very large gene. so,i mean, i think there's still more work to do on those individual mutations. so whatmatthew's saying is that we looked specifically at thyroid receptors and thyroid globulin,and it's sort of a controversial point that a lot of mouse models on receptors, but thatdidn't pan out in our data set. we had a low incidence, maybe a couple percent of thyroidglobulin mutations, but i'm not really sure how significant they are. male speaker:[inaudible]

tom giordano:it did pick up on mutic. so, you know, mutic has different versions. i think some of theearlier versions picked up thyroid globulin but not the later ones. okay. great. thankyou very much. peter laird:thank you, tom. that was a wonderful story. so now the next presentation will be by aliamin-mansour on somatic alterations in clinically relevant cancer genes among 12 tcga tumortypes. ali. [end of transcript]