cancer de colon diagnostico

sandra orsulic:good afternoon. i will be talking about something completely different today. this slide isfor computational biologists in the audience who usually look at raw digital data. thisis what the raw analogue data look like when you put it into computer. if you then slicethis tumor, this is what you get. you get something that is very complex. it has a numberof different cell types that you can see here. some of these tumor cells, some of these arestromal cells with lots of fat, blood vessels, lots of collagen, lots of extracellular matrix.so all this complexity goes into your analysis. and i remember when we were first decidingwhat kind of tumors should we put in, and we decided for ovarian cancer that it shouldbe at least 80 percent of tumor content in

-- of cancer -- malignant cell content inthis tumor. but it was actually difficult to find tumors with 80 percent tumor cellcontent. and that should tell us something. that tells us that that the majority of tumorsactually have other stuff in there, and this other stuff is what i will be talking about,and hopefully i will convince you that this other stuff is also very important and wehave been neglecting it to some extent in the tcga analysis. but maybe it requires thatcancer associated analysis. this is a section of -- through a tumor andyou can see the cancer cells in pink, and you can see also that it is surrounded bysome sort of fibrotic stuff. this is the stroma that is usually present. and the questionis, what is stroma really doing in tumors?

some people believe it's actually preventingfrom cancer progression; it's kind of encapsulating the tumor. others would say, well stroma actuallyinfiltrates the tumors, and in fact helps the progression and invasion of tumors. infact, tumor cells can glide on top of these pink sheaths like on highways and progressand metastasize more efficiently. one important distinction between cancer cells and stromais that cancer cells are highly proliferative and stroma is not, so what you see here iski-67 is a marker of proliferation. and you can see the cancer cells are highly proliferative,but you can also see some nuclei here in the -- in this white space, and they are not stainedwith the marker because they're not proliferative. this is the stroma.

so the cancer cells are proliferative, andthis is also what is typically targeted with chemotherapy, because most chemotherapiestarget proliferating cells. one other distinction is that, as we know, cancer cells are verygenomically, genetically unstable. and this is a bit of a problem with all the chemotherapiesthat we are applying -- because of their flexibility and genomic instability, they quickly re-adaptand start using different pathways. this is not the case with stroma. stroma is slow proliferating;it is not directly targeted by chemotherapy, although indirectly it often crumbles withthe tumors. it's genetically stable, and what is important -- one thing about being geneticallystable doesn't actually -- we may use it to our advantage when we target it, because youmay expect that genetically stable cells are

not capable of redefining themselves -- redefiningthemselves as quickly as the cancer cells. and as i will try to convince you that itis important as genetic alterations in tumor progression. one thing that we noticed is that enhancedremodeling occurs in stroma during tumor progression, and there has been in the past five to 10years, as study after study has shown, if you compare cancers and you're trying to findan expression level, what are the genes that predict poor survival, most likely you willend up with genes that are not genes that are expressed in cancer cells, but the genesthat are expressed in the stroma. and why is that? well, that tells us that there issomething in the stroma that is very important

and that predicts poor survival or maybe actuallycontributes to that poor survival. one gene that i’m specifically interestedin is collagen 11a1. this is a very rare type of collagen that we found was expressed veryhighly in tumor. here you can see tumor is blue, and then you can see these brown things;that's expression of collagen 11a1 in situ hybridization, and you can see it's kind ofdispersed throughout the tumor. but when you look at the peritumoral stroma, which is alsoa stroma, you can see that there's very little of it, and then once you go more than onemillimeter away from the tumor, you don't see any collagen 11a1, because collagen 11a1is not expressed in the normal stroma. it doesn't matter how much stroma you have inthe tumor. what matters is that this specific

-- there's specific stroma subtype that expressesthis collagen, and it's usually inside the tumor, but not all tumor cells. and we stilldon't know what is it with that stromal cell type that expresses collagen 11a1. an interesting thing about collagen 11a1 isthat if you look across different cancer types and you compare normal tissue to their correspondingcancer, you will find that in most cancers, you see much higher levels of collagen 11a1than in their corresponding normal tissues. and this is not very common, even if you look-- if you -- so this is not because there’s more stroma in cancer than in normal tissues.if you take a very common stromal markers such as -- actually even activated stromalmarkers smooth muscle acting, you don’t

see that. you see kind of comparable expressionin normal tissues because there's -- this acting is expressed in normal tissues andin the stroma. but with collagen 11a1, you see this huge disparity. and if you looking-- look at individual cancer types, such as breast cancer, and you ask which -- rank thegenes that are most differentially expressed between normal breasts and invasive ductalcarcinoma, collagen 11a1 will be ranked as the most differentially expressed genes. incolon cancer, in colorectal cancer, it's ranked as number three. but it gets even better. so it's not justnormal and cancer, but if you look at during cancer progression -- and this is an exampleof ductal carcinoma in situ and in invasive

ductal carcinoma, the difference is that ductalcarcinoma in situ has a member -- has a basement membrane around and it’s still envelopingand it has much better prognosis. invasive ductal carcinoma, the tumor cells have alreadypenetrated through the basement membrane, and this has worse prognosis. if you comparethese two entities and look for genes that are differentially expressed as i have donehere with four different databases that i could find that compare these two types, again,collagen 11a1 is one of the most differentially expressed genes during this type of progressionof breast cancer. this is an example in ovarian cancer. you see that levels of collagen 11a1are higher as you go from stages. these are different staging systems as system in ovariancancer have stage one is the lowest stage

combined to the ovary, to stage four, whichis a widely metastatic disease outside the peritoneal cavity, and collagen 11a1 levelsrise through metastatic progression, or stage and even better, it actually is the highestlevels of collagen 11a1 are actually during recurrence. so you see very little -- thisis in situ hybridization when we compare in matched patients -- in this case, 10 samplesnow. we have done it with 48 samples and the result is the same, that you have very littlecollagen 11a1 in the stroma of the primary tumor. you see a little bit of it in metastasesin the same patient, but then in the recurrences you see a lot of collagen 11a1. so, as i said, it is expressed in differentcancer types. so this is now where i utilize

tcga data and i asked, “what genes are coexpressedwith collagen 11a1?” and i did that for bladder cancer, so i ranked the co-expressedgenes, for their sperm score correlation, then i did the same for breast, then for colorectal,and i did it for 12 different cancer types, all of which are adenocarcinoma, so i wasinterested only in epithelial cancers. and you can see that some of these genes in differentcancer types are actually the same genes. and i noticed that it was similar genes overand over in different cancer types. so then, in all these 12 different cancers,we ranked the genes across 12 cancers by their priority score, basically, and you see thatthe closest across cancers is collagen 11a1, and then collagen 5a1, fibroblast activatingprotein, and so on. the list is about -- we

selected a cutoff -- the list is about 190genes or so. then we asked, what are the top 10 percent genes that in bladder cancer, forexample, are -- have the top 10 priority score? and we put them in pink. then we asked thesame for breast cancer, then we asked the same for cervical cancer, and so on. and whenyou do that sort of analysis, you find that -- i don't know if you can see, but the pink,you can see most of the pinkies up in the top, and that means that what is -- 10 percentmost highly associated genes with collagen 11a1 in bladder cancer, it’s similar toovarian cancer; it’s similar to breast cancer. so that means that there’s -- these genesare very homogeneous. it's a very tight correlated set of genes, and this goes across differentcancer types. you're talking about 12 different

cancers with very different genetic durations.when we did this with anti-expressed genes, which were not very highly correlated anyway,we didn't see this sort of association. so what is the signature now? it's a veryinteresting tightly correlated signature. what does it represent? it they are in tumors.is it a specific cell type that it represents? so as i said, tumors have many different celltypes. we looked for the signature in different cell types, and we actually cannot find itin the normal cell types, as we can say, except cartilage, maybe. but really, so we don'tthink it's a specific cell type. we actually think it is more a process that exists intumors, and that is exacerbated during cancer progression. and that process we think ismyofibroblast activation, and i will not go

into how we got to that. so what is myofibroblast activation? well,fibroblasts -- resident fibroblasts in tumors -- are activated by tgf-î² growth factor,and they become myofibroblasts, which are more and more tired [spelled phonetically],more stiff, and if that is true, we actually put this signature and asked what are theupstream -- most likely upstream regulators of the signature, and tgf-î²1 actually comesup as the most likely, so that makes sense. tgf-î²1 activates these myofibroblasts. andone thing that is interesting about the fibroblasts is that they produce collagen here in blue,which is kind of relaxed collagen fibers. but myofibroblasts tend to produce this straight,stiff collagen fibers, and that's actually

important for activation and kind of feedforwardloop in making them even more stiff and more stiff, and i won't go into the biology ofthat. but here, just to see if other we can reallysee it in the real tumors, you can see here, collagen 11a1 is in brown, and you can seeone of these boxes, number one, you can see that trichrome staining, which stains basicallycollagen and muscle, and then you can see number two, where collagen 11a’s not -- isnot expressed. you can see now it’s blue and wavy. but this is where collagen 11a1is expressed, it’s nice and straight, as we would expect. we also showed if we overexpressedcollagen 11a1, we make the cells stiffer. now, it's a very good gene to target, becauseit's expressed specifically intratumorally,

but not outside of tumors. but there’s onelittle problem, and that’s that it is also expressed in the cartilage and a little bitin your eye. we also show that you can target it with shrna, and you -- the tumors are smallerand not as metastatic. so it would be a good target. but how do you distinguish it fromcartilage? one way to distinguish it -- and this is why it’s such a great target -- it’sactually not the same one as in collagen -- as in cartilage. so cartilage collagen type xiconsists of three chains, three polypeptides: collagen 11a1, our friend; collagen 11a2;and collagen 2a1. now, when we look at tumors, only collagen11a1 is highly expressed, but the other two chains are not. that means that in tumors,collagen 11a1 must be homodimerizing [spelled

phonetically] with itself or heterodimorizing[spelled phonetically] with something else, but not with this because clearly these arenot upregulated, and actually what we find in this signature is that other collagens,5a1, 5a2, 3a1, and several others, are highly alpregulated in this signature. and what iscommon to those collagens? all of them have very slow processing of their intermini [spelledphonetically] and this is a part of collagen processing that collagen needs to go through. and because of this slow processing, theseintermini kind of hang out of the fibers, and that prevents attrition of additionalfibers, so you end up with these nice thin fibers, as you saw. so we don't know why thesefibers have to be thin, and what it -- mechanically,

why is this important and how this affectstumor progression at the moment. but, what i've shown you is that collagen 11a1 is upregulatedin normal tissues and levels increase during progression. the signature that we have identifiedis present in multiple solid cancers, regardless of cancer origin, so that means that the residentstroma is the same. it's -- or it goes through the same process of fibroblastoma or fibroblastactivation. and it’s -- is enriched during invasion, we think it's activated by tgf betasignaling. and fibers are stiff, stretched out, and -- but what is most important isthat we think that this is also targetable because it does have a unique compositionof collagen fibers in myofibroblasts, so we think that this may be a good opportunityfor therapeutic targeting. and i would like

to thank my colleagues and my lab membersand my sources of funding. thank you. [applause] katherine hoadley:any questions? male speaker:wonderful presentation. a couple of quick questions. is this expressed in scar tissue?or is it a part of the regenerative process or damage repair process? and if so, wouldthat interfere with regeneration when you think of the patients? sandra orsulic:[affirmative] so, very good question. i don't know. how are -- you suspected, yes. we havemyofibroblasts in scar tissues, so we did

look in scar tissue wound healing in fibrosis,so it is slightly expressed in keloids, not so much in scar tissue, and not during woundhealing, not during fibrosis. to some extent you see a slight expression, but this is nowherenear what you see in cancer. so we think that unlike smooth muscle actin, which you willsee, or fat, which you will see alpregulated in all of these benign nonmalignant processes,collagen 11a1 is not. it’s tumor-specific. male speaker:very good talk. i just wanted to make a comment that we studied the protein expression datain pan-cancer analyses. i presented the results last year at the symposium, and we have foundwhat is -- would be called a new subtype, a reactive subtype, where we have seen stromalmarkers are elevated in the reactive subtype.

this was first discovered in breast cancer,and we saw that the prognosis was very good for those patients with this particular subtype.we expanded it to pan-cancer, and we found that for other cancers, the prognosis waspoor. so the prognosis is cancer dependent; it's context-dependent. one of the markersof the reactive subtypes was collagen vi. we don’t have collagen 11 in the proteomicsdatabase, but i saw collagen vi was highly correlated with collagen 11a. so i think oneof the things that we should keep an open mind about is that this may have a good prognosisand better outcome, or it may have worse prognosis, depending on the disease type. sandra orsulic:that is correct. i will just comment that,

for the signature that we have been studying,we found that in all of these cancers, it actually had the worst prognosis. it wasn’ta great predictor of worse prognosis, but still -- predicted worst prognosis, exceptfor lymphoma, b-cell diffused lymphoma, which has been published several years ago, wherethis exact signature actually shows better prognosis. but we think it's because lymphoma-- it's actually the cells that are present there are not replaced by cancer, so we thinkthat because these may be resident cells in the lymphoma. so i don't know for your signature,but surely we will be finding stromal signatures that are associated with better survival,and not all stroma is the same. i think this is very important to realize that what weare looking here is a small subset of stroma.

male speaker:very intriguing data. i have a question with respect to your trimeric hypothesis. i thinkto be more convincing, have you looked at getx data to make sure that the differencesthat you see across those three strands -- different across and for gtex data because i think,you know, that would be sort of more -- sandra orsulic:right getx data doesn’t have cartilage as far as i remember, so that’s one problem,that these genes express in normal tissues, only in cartilage. so i couldn't really findthem. male speaker:thank you. katherine hoadley:thank you. so our next speaker is dr. wenyan

zhong from pfizer, who’s going to tell usabout integrated analysis of tcga and how it identified targets and patient populationsfor antibody drug conjugates. [end of transcript]