 Thank you very much. I'm glad it worked out. Finally, so the title of my talk is genome and transcriptome dynamics in cancer cells. To start the most important slides, the member of my laboratories who in different capacities contributed to what I'm going to talk about, we have active collaborations. The University of Lübeck, the Karolinska Institute in Stockholm, and the University of Göttingen, which is part of a clinical research unit which is funded by the German research community. So what I will do is, at the first third or half of my talk, discuss with you what we know about chromosomal changes and patterns of aneuploidy in carcinomas, essentially as part, as kind of an introduction to the second topics. When I discuss briefly what the consequences of these cytogenetic abnormalities are on the transcriptome, on the gene expression in cancer cells, and then I will exploit what we can do in order to translate what we have learned from these more basic research topics to translational medicine, both in terms of genomic aberrations and alterations of the transcriptome. What you see here are chromosomes from a patient with chronic myelogenous leukemia. And you can see there is a single chromosomal translocation between chromosome 9 and 22. And at this point, I have to pause and acknowledge Janet Rowley, who actually discovered the balanced nature of this chromosomal translocation. In fact, this slide was performed in a collaboration with her. She unfortunately passed away just two months ago. She was a staunch supporter of the Intramural Research Program in general and the Genome Institute in particular, where she served as a scientific advisor since its inception. And, as you know, this translocation has profound effects, not only in terms of diagnosis, but for treatment, because if you target the genetic event, which is the fusion of BCR and ABL, which results in aberrant tyrosine kinase signal with a drug, you can get these patients into remission. And the paradigm of chromosomal translocation not only applies to CML, but to many other hematological malignancies as well, which some of them are listed here. But I chose to show you this slide for another reason as well, which is clearly there is one genetic event, which we now cause transformation, but you can also appreciate that the rest of the genome remains normal. So, what it is that there is a single genetic event, which is sufficient to transform these hematological cells. If you look at solid tumors, carcinomas in particular, tumors of epithelial origins, the picture changes dramatically. Here is a normal kerotype, and here are the metaphases from a breast cancer cell in SKBr3, and you can see a profound degree of chromosomal instability, not only numerically, but also structurally. Then you can see giant marker chromosomes, which are a reflection of oncogene amplification, in this case, the CMIC and the HER2NU oncogene. Then you have other features, this is probably a little dark, cancer cells, which we call anibloid, which is reflected in enormous degree of variability in the DNA content from one cell to another. You can also appreciate apolamytosis, which clearly will lead to a different chromosome count in these cells. You can see the phenomenon of anaphase, which results in lost chromosome and is caused by telomere attrition. Some pictures like this and this led to the perception that if you look at most solid tumors in adults, it looks like someone set off a bomb in the nucleus. This perception led to the interpretation that in solid tumors different from the hematological malignancies, we have a cytogenetic chaos, which is induced by catastrophic mitosis, you have seen one, keratobic complexity, heterogeneity of the tumor cell population and ongoing chromosomal instability. The perception of a cytogenetic chaos also led to the perception that chromosomal aberrations in solid tumors different from the hematological malignancies would rather be a consequence rather than the cause of the disease. What I will show you now is, I will discuss with you now whether this is indeed the case. Arguably, we had to develop methodologies to dissect the complex aberrations in solid tumors. Most important was comparative genomic hybridization. I will show more details later, but in short, it allows you to map genomic imbalances in the entire tumor cell population. We have spectral keratiming, which we developed years ago, which allows you to paint all chromosomes in different colors. And then we can use specific probes to enumerate chromosomal copy numbers directly in non-dividing cells, cytological preparations or tissue sections, and it can also be used to identify translocation because you see a fusion of different colors. And now, with the advent of global gene expression profiling or sequencing, we can interpret and query how these aberrations affect the gene expression levels in a global sense. So, having had these tools, we then try to understand the dynamics of changes in the transition from normal epithelium to invasive disease. And initially, we turned to two model systems which are cervical carcinogenesis, called rectal carcinogenesis, mainly for the reason because the morphological changes are very well described and they are accessible. So, you know the sequence. It goes from low-grade dysplasia to high-grade dysplasia and eventually to invasive disease. And while it looks a little different in the colon, it's the same. You have primalictan lesions that if they would be removed, you would cure the patients. So, we used a combination of molecular cytogenetic tissue microdissection techniques, gene expression profiling, and they asked the question, is there a non-random distribution of chromosomal changes in one of those different tumor entities? Is there a stage-specific sequence of events? In other words, some changes early, others late. And which additional genetic or epigenetic changes do occur? And it is now completely clear that the distribution of chromosomal gains and losses is non-random. In red, you see the aberrations for the cervix. Let's just look at chromosome three. On the left side are the gains. On the left side are the losses. Chromosome five has gained. But the rest of the genome is relatively unaffected. In the colon, which is displayed in green, you can see invariably gains of chromosome seven, extra copies of the long arm of chromosome eight, chromosome 20, and very specific and unique for colorectal tumor genesis gains of chromosome 13. And I just want to point out, we and others have now studied more than 500 cases of cervical carcinomas here. The blood is normalized for 10. And you can appreciate that all cervical carcinomas have extra copies of the long arm of chromosome three. In other words, this aberration is as common as is the Philadelphia chromosome in CML. We do know that we have risk factors, high risk HPV, most commonly 16 and 18, which is required for cervical cancer to occur, but it's not sufficient. And in colorectal tumors, you know that inflammation, colitis, ulcerative Crohn's disease, greatly increases the risk for colorectal tumors to occur. But the distribution in these conditions is the same as in sporadic tumors. Conclusion, we do not have a cytogenetic chaos, but we have a stability on a different plateau of copy number changes. It's like a speciation. So, but we do have the problem that we have an enormous degree of variability. So the question comes up, how can we reconcile, excuse me, anibloidy and ongoing chromosomal instability, which is a fact with a strictly conserved pattern of genomic imbalances that we observe. And in order to answer this question, we engage in a collaboration with the Navy at that time across straight, across two streets from here, and collected patient samples where we have ductal carcinoma in situ and invasive ductal carcinoma on the same slide. For that, we use tissue microdisection, and we've prepared from both of these lesions independent slides where we had these single-cell suspension here, you see, for one case, the histology. And then we chose, we looked at the distribution of genomic imbalances that is specific for breast cancer. You can again appreciate that it's specific, but it's different from both colon and cervix. And we chose probes, fish probes, fluorescence in cytohibrosis to enumerate a copy number of these regions together with control probes directly on these cells that we prepared from DCIS and IDC, and that was done, this is a technical detail in repeat hybridization, but at the end of the day, we could enumerate 10 independent loci on many of these cells that were on the slide DCIS and synchronous IDC. And we don't have to go through here in detail, but that allowed us to enumerate in the entire population of the cancer copy number gains for a certain chromosome and copy number losses for a certain chromosome. And here you can see there are many cells that have the same pattern, and this is a DCIS and this is a synchronous IDC. You can appreciate that here we have a major pattern that is recapitulated in the invasive component, but you can also appreciate that we have an enormous degree of heterogeneity. In some instances, the clone that was predominant in the DCIS actually disappeared in the IDC. And together with our colleagues from NCBI and in the collaboration, very fruitful and pleasant collaboration with Russell Schwartz, Kahn-Eggie-Millen, we then could reconstruct the dynamics that occurred from the transition from DCIS to invasive disease. And two pathways occurred. One was clonal stability. Here you can see the DCIS on the left and the IDC on the right. You can see the major clone, which is defined by these copy number orations, prevailed and was also found in this component. And here as well, and in this case, it was the same. But much to our surprise, we also noted that in some cases the entire population of clones that were present in the pre-invasive disease disappeared. Only those that acquired extra copy of the MIG oncogene made it to invasive disease. And that again told us that the transition from pre-invasive disease, in many cases, is determined by the acquisition of extra copies of the C-MIG oncogene. But you can also appreciate that in many cases the ductal carcinoma in situ are already governed by a enormous degree of chromosomal instability. Therefore, I'm not a clinician, but I just cannot see that any other intervention than surgery would remove these clones. But anyway, despite this chromosomal instability, if we then look at all these clones that we have analyzed in copy number changes, we can conclude that chromosome 1Q, which is frequently gained, is very rarely lost. And chromosome 8Q, MIG oncogene, which is frequently gained, is very rarely lost. And those chromosomes here, P53, for instance, which is frequently lost, is rarely gained, despite this enormous degree of chromosomal instability. And this makes it then consistent with the genomic imbalances that are specific for copy number changes. So despite chromosomal instability, what is the pressure for Darwinian selection is the maintenance of the genomic copy number changes that are the defining features for any one of those carcinomas. This now, of course, triggers the questions. We have these changes, mostly whole chromosome arms or, in other instances, whole chromosomes in the colon gain of entire chromosome 7. This now triggers, I think, a fundamental question. How does it affect the transcriptome? You could ask the question, but you could test the hypothesis. The expression of all or most genes located on a chromosome is affected by chromosomal gain or loss. Or the expression of only a few genes whose reduce or increased expression is critical for tumor genesis is the target of chromosomal aneuploidy during tumor genesis, and this was not clear. And in order to identify... So the conclusion so far, chromosomal aneuploidies are a defining feature of carcinomas. The distribution of genomic imbalances is cancer-specific to an extent that you don't have to have any other information, but the distribution of genomic imbalances is a cervical carcinoma or a colorectal carcinoma. Specific genomic imbalances occur before the transition to invasive disease, which is very important if you want to convert that to a diagnostic test, which I will show you later. And, as I said before, there is no cytogenetic chaos but a stability on a different plateau of genomic copy number changes. So, as I said, we then ask, what are the consequences of chromosomal aneuploidy of the transcriptome, which I will discuss briefly in the next few slides, and then I will turn to how we can use this knowledge to improve the diagnosis of cancer and premalignant lesions. So in order to address the question what are the consequences on the transcriptome, we use the...an old technique which is called microcell mediated chromosome transfer to generate artificial chromosomal trisomies and this is how it looks. Here we have a normal kerotype and when we do this manipulation, we can put in an extra chromosome, in that case chromosome 3, but you can see, which is now present in 3 copies, but you can see that the rest of the kerotype is unaffected. Then we collected RNA from the control and RNA from the plus 3 cell and performed global gene expression profiling. Here again the control, some genes are up, some genes are down, which is what you would expect, but if you look at the plus 3, you can appreciate that most, if not all genes, go up in expression levels. And this, by the way, is the same if you look at the constitutional chromosomal trisomies 13, 18, 21, where this increase is also not restricted to a few genes but affects most of the genes and in a resting on a site, it's tolerated for chromosome 13, 18, 21, because these are the gene poorest chromosomes despite they have a larger size, 13, of course it's larger than chromosome 20. So aneuploidy results in the significant increase of average message levels of genes on the affected chromosomes. The degree of increase closely follows genomic copy number. Therefore, chromosomal aneuploidy is not only target a few specific genes, but result in a massive and complex deregulation of the cancer transcriptome. And just contrast that to what we see as a consequence of the Philadelphia chromosome where you have one aberration. Now you have a thousand genes that go up and you also have to appreciate of course there are numerous transcription factors that affect genes and other chromosomes. So it's really a massive deregulation which we have not yet understood in its complexity. This is not only the case in our model system but also in real tumors. Here a control, a normal DNA copy number and this is the gene expression level which is normal as well. But if you have copy number decreases or copy number increases, the gene expression goes down in this region, goes up in this region. It's completely clear. Function follows form. And this is established not only from our laboratory but now accepted in the scientific community. To summarize we have a normal cell carried typically stable. Then we have chronic inflammation for instance in the colon, HPV infection. We have for whatever reasons in the breast hyperproliferation we have just a cell cycle accident. What happens then? That we have extra copies of a given chromosome and this chromosome is tissue specific that can occur with or without additional mutation. Then what I call nuclear aneuploidy is very low so that there is not much difference in the DNA content from one cell to another. The trisomies affect global gene expression levels and as you will see later these events are the basis for global expansion of these early lesions. If this is not being treated then we arrive at a cancer cell where we have additional mutations. As we know P53 occurs relatively late in colorectal tumor genesis. We have an enormous degree of variability in the DNA content from one cell to another. But the genomic aneuploidy persists that why CGH was successful. As I have shown you before what is the selective pressure is the maintenance of the specific genomic imbalances. So now after this introduction I want to show you how we convert that to translational applications. Three topics I hope I can cover them all. First I will discuss with you the role of genomic instability in the prognosis of breast cancer and then we look at cervical carcinoma and if time how we use transcriptional profiling to predict treatment response in patients with rectal cancer. So breast cancers usually present in two flavors. One have a relatively stable genome which are called diploid and others are genomically instable have high degree of aneuploidy use if you take home lessons this is the Washington Monument and this is the Manhattan Skyline and many years ago our collaborator Greta Auer at Kowalinska I was not on that paper at the time discovered that patients who have a genomically stable tumor have a far better prognosis than patients who have an aneuploid tumor. Profound differences. And then in 2002 and as I said many other papers followed Wanderweber published a paper in the New England Journal who showed that there is by gene expression a good prognosis signature and a poor prognosis signature. They even came to the same conclusion and if you look at these curves you cannot argue that they are very similar. So we try to understand the nature of the similarity and perform gene expression profiling of a set of diploid tumors and a set of aneuploid tumors. And we could easily separate the aneuploid ones from the diploid ones and this separation was based on a signature of 12 genes which perfectly separated almost perfectly separated the stable from the instable breast cancers. And then we asked the question can we use this aneuploidy specific gene expression signature to predict to recapitulate the published data sets and that worked in all instances and the numbers are very high with very high statistical significance. And then the next question was whether we can ask the prognostic signatures that were derived from these data sets to predict the degree of genomic instability in our cases. And that worked perfectly well. The oncotype DX which is used in the clinic as you know and the good prognosis signature of the oncotype DX predicted that the tumors were diploid and the poor prognosis signatures predicted that the tumors are aneuploid and you could see that works very well. And the same occurred for the mama print. The good prognosis signature predicted genomic stability. The poor prognosis signature predicted aneuploidy. And just on the side luminal area is another gene expression signature which is now which you know is associated with the good prognosis predicted genomic stability and the basal signature under her two new signature associated with the poor prognosis predicted genomic instability. In summary, the degree of genomic instability and gene expression signature of poor prognosis are linked. The degree of genomic instability is the major biologically determinant of poor prognosis. And it is not only the degree of biological genomic instability if you look at the tumor cell but it's the fact that you have many multiple clones which provide the tumor with the nimbleness to react to environmental challenges including therapy. The next application which we developed for over more than 10 years is the identification of individual progression risk in cervical dysplasia. And here you remember what I showed you that there is invariably a gain of chromosome 3 the long arm of chromosome 3 in cervical carcinomas. And this gain never occurs in normal cells. So we developed in collaboration with Abbott a probe set that targets these regions that just control probes which help us to just control probes which help us to in normal rate you have the same scenario if you test for her to do amplification you also use a centromere for chromosome 17 as a control. And then we applied this to routinely collected PAP cervical smears. And I want to really make the point in cervical cytology the challenge is not to diagnose cancer. Because we all actually do not want to diagnose cancer because it's too late. We want to diagnose early lesions which can be cured by surgery. The challenge in cervical cytology is that there are two challenges. I mean everybody can say well this looks different than this but you have a normal cervical cell which is defined by a small nucleus and a large cytoplasm and you can easily discern that from these cancer cells but the distinction from here to here is much harder. Even the distinction from here to here is essentially impossible. What is even more important is that only 15% of the low grade dysplastic lesions would progress. Therefore it would require treatment despite the fact that about 90% of these low grade lesions are already positive for HPV. So HPV does not really help. It only helps if it's negative because then you know there won't be a cervical carcinoma but most of them are positive so it doesn't really help. So we argued that if all these carcinomas have a gain of a chromosome 3Q but none of the normal have it. And there is only a fraction of the low grade lesions would progress and would require treatment that those that progress already have the aberration that defines the invasive disease and that those 85% that would spontaneously regress are cytogenetically normal. And we then explored that and you can see here with this probe set for three copies 2, 2, 2, 2, 2, 2. This is normal. Cervical interaberythal neoblasia grade 2 which is a moderate dysplasia. You can see here 1, 2, 3 copies of chromosome 3Q. The rest of the genome is still normal. And in some of the carcinomas you can see clouds of 3Q of the turc oncogene which we choose as a probe that is reminiscent of the amplification of her 2 in breast cancer. And then this is unfortunately very difficult to see. I come back to the what I said it is the basis for clonal expansion. Here you can see a pap smear which was stained. We then destained it. Here a normal cell 2 copies. And here we have a nest of cells which were collected. They all have 3 copies of chromosome 3Q. But they are next to each other. Meaning they originated from each other. So once a cell has acquired 3 copies that is the basis for clonal expansion. So not only do we have a diagnostic test but we also can visualize the emergence of cancer by looking at the copy number changes of that particular chromosome. We confirmed that in many different studies this was one in collaboration with women who had pap smear randomly and those that had a suspicious pap smear then underwent colposcopy and biopsy. So we could correlate the findings under pap smear with the histology with a gold standard and the conclusion is that the gain of 3Q the human telomerase gene which is on there has the highest combined sensitivity and specificity for the detection of histologically confirmed high grade lesions. And we are now conducting an even larger study to extend that to low grade lesions and to ascus. We conducted another study which was aimed at validating 3Q as a molecular marker of progression which as I showed you at the beginning is the most important and this is how it was done. We collected a group of patients who are now much higher which were diagnosed with severe dysplasia and before they had a low grade dysplasia. Then we had another group of women which had the same low grade dysplasia but returned to normal. We had another group of women which were diagnosed despite being enrolled in an active surveillance program with severe dysplasia or even carcinoma but the pap smear before was normal so that should not happen. So we then hypothesized that those we know already these are positive these are all negative, these are all positive but we hypothesized that those low grade lesions that show progression are positive for 3Q and those that show regression are negative for 3Q and this worked essentially with the sensitivity of 100% and the specificity of 95% and we can discuss that later it's a particular feature of HPV but you can see that the point of no return in the progression risk for individual lesion is the acquisition of the specific cytogenetic abnormality that then defines the cancer entity. We also were expected a little bit shocked that in some cases where the pap smear was assessed as being normal and after a relatively short latency the woman was diagnosed with a carcinoma we could detect extra cubbies in a third of the cases in the cases that were cytologically assessed as being normal and I'm just showing you some examples here here there is a syn-1 lesion which regressed two copies, two copies so there would not have been any other treatment required here we have a syn-2 lesions and you can see here for instance these two cells and they have both three copies this lesion is progressed to high grade dysplasia and carcinoma and this is the case that I mentioned to you before which was assessed as being normal but you can see you have four copies of chromosome 3Q in these set of cells and again they are next to each other indicating that the acquisition of this operation is the basis for clonal expansion I shall also emphasize so I talked to cytologists at Hopkins and they come in the training program twice a year and every time at the beginning the wrong diagnosis goes up and then they learn a little and it goes down because it's obviously relatively difficult to unambiguously assess the morphology and when I go to cytology meetings there are whole days where they discuss on how to best repeat the reading of a pap smear to avoid false negatives and the fact that it's relatively often repeated is an acknowledgement of the fact that an individual pap smear is relatively ambiguous but you cannot argue that there are four copies so it's a binary you can ask your six year old to count to four and make the statement this is not true therefore they cannot even say make the statement as cervical carcinoma they can even say there is a woman whose low grade dysplastic lesion will eventually be an invasive carcinoma so it took us a long time to convince gynecologists and pathologists to embrace that because the procedure for the collection of this material would not even have to be changed because in particular with liquid based cytology there is always leftover but now finally quest diagnostic has embraced the test and is offering it in its portfolio so now I want to switch gear a little bit I don't know what the time is I'm fine and just talk a little bit how we not only use genomic operations but aberrations of the transcriptome and inject genomic information into a problem of treatment response in patients with rectal cancer and you know that better than I do there are many treatment options in solitumis it can be surgery alone surgery plus adjuvant chemo radiation or chemotherapy in colon carcinomas or near adjuvant chemo radiotherapy followed by surgery or other approaches or in some instances dependent on the mobility of the patient chemo radiation alone and the problem in rectal cancer is a real clinical problem because based on a large study which was conducted from the term on rectal cancer study group and has been adopted now in most of Europe I know it's not quite completely adopted in the United States the standard treatment of locally advanced rectal cancer is near adjuvant chemo radiotherapy mostly based on five of you in radiation followed by surgery and then chemo therapy and the reason for that is that they found out that if pre-surgery chemo radiotherapy is administered there is a higher rate of R0 there was a higher rate of sphincter preserving resections and there was a reduction of local recurrences significant from I believe 14 to 6% the profound clinical problem is that the response to near adjuvant chemo radiotherapy is very heterogeneous you can have complete pathological response in that case where after surgery there is not a single tumor cell left or you have essentially complete resistance where the tumor just doesn't bother being treated with 5FU and radiations so we generated together with a former postdoc of mine Michael Godimi who is now the chair of surgery at the university in Goettingen Germany a clinical research unit you can look it up in order to address that problem and we aimed at identifying predictors of response and identify targets of that and understand mechanisms that could explain this profound difference in response versus resistance and in order to do so we initially designed a pilot study with 30 patients they all had pre-treatment clinical stage based on rectal ultrasound we then collected tumor biopsies and performed gene expression profiling they obviously I can barely see from here but then they went to this neo adjuvant scheme which is chemotherapy followed by surgery and four rounds of 5FU and then we had a long term follow-up the pathological staging is shown here and as I said we performed gene expression profiling and then evaluated local and distant recurrences after medium follow-up times of 44 months and the gene expression profiling worked actually fairly well here we had I forgot how many, there were 30 cases approximately half of them had non-responding tumors a little fewer had tumors that show complete response and the gene expression profiling could actually fairly well discern these two groups but then we looked at the genes that were up-regulated in the tumors that were resistant and among those what a transcription factor TCF4 and this obviously rings a bell because transcription factor TCF4 is the major effect of wind signaling of the major effect of wind signaling of course not only in colorectal tumor genesis and wind signaling is intricately involved in colorectal tumor genesis affect mutations in the adenomatosis which actually poses coli gene resulted in stability of catenin which then actually increases the expression of the transcription factor TCF4 which in turn turns on cyclin D1, CMIC and other notorious oncogenes and therefore transform these cells so that was a very interesting lead so we hypothesized that silencing of a gene that is over expressed in resistant tumors would increase sensitivity to chemo-radio therapy and we did this silencing with the approach of using RNA interference and I don't go into details here but if you inject small interfering RNAs into cells you can reduce either protein synthesis you can cleave the transcript which results in a loss of function and you can do that essentially for any gene of interest so we used small interfering RNA against the transcription factor TCF4 and asked the question whether silencing of TCF4 would increase sensitivity of colorectal cancer cells to chemo-radio therapy here you can see the results it was a very effective silencing in the reduction of protein we repeated that with many different constructs and when the cells were then subjected to radiation not shown here but also to chemo-radiation this is a large scale you can see here the control and you can see here a profound sensitization after silencing the transcription factor TCF4 which in primary tumor samples was over-expressed in those that were resistant to treatment conclusion reduced expression of TCF4 leads to a sensitization to radiation we are now exploring whether we can recapitulate this effect using small molecule inhibitors of wind signaling and together with this clinical research unit in Göttingen we are about to design clinical trials to test that in patients so in summary we have performed gene expression profiling which I have shown you in rectal carcinomas that are resistant or sensitive to near adjuvant chemo-radiotherapy we have also varied many different levels of the genome using CGH to see whether we have specific genomic imbalances that could explain the sensitivity we have performed microRNA expression profiling we explored KRS and BVF mutation which by the way did not explain the different sensitivity to radiation we looked at the methylation status and in collaboration with Steven Chanock the Illumina platform to look for single nucleotide polymorphisms in the group of patients that respond differentially to explore whether certain haplotypes for instance in genes that are involved in drug metabolism could explain the profound differences in the clinical course we do all this in order to address the problem that all drives us which is that patients come into the clinic with different clinical features they have a different genetic makeup and a different tumor biology and in the case of rectal carcinomas they also have a different treatment toxicity and this is not only true for rectal carcinomas for instance primalignant lesions in the cervix could be summarized here however despite these acknowledged differences which we do not understand to the extent we should all these patients receive identical therapy the goal of all the stuff of course is to learn enough from all these different techniques I have not listed next generation sequencing here to learn enough about the genetic makeup so that we can assign patients to therapies from which they would benefit the most with that I'm at the end and I thank you for your attention I'm happy to take this I have one a question that all biology is only understandable by natural selection and evolution is it possible that by using chemotherapy in a way we encourage resistant tumors to grow out? I think it is I think it is I mean tumor heterogeneity was already I don't have it on this slide unfortunately but what we are now doing we look at these rectal carcinomas that are complete responders and those that are resistant and ask the question whether in those that are then resistant there are minor clones that expand during chemotherapy completely passable but I can also say it's now being more and more acknowledged that we cannot think if we look at the bulk of the tumor we have understood the tumor so this is a signal to noise question really what are the comments and questions? in breast and colon cancer is there a normal tissue in the same patient like those described in the series? the question was let's just look at cervical carcinomas or colorectal carcinomas whether those cytogenetic abnormalities that are the early events in tumor genesis can already be detected in the adjacent normal epithelium not on the chromosomal level but we are now exploring the possibility that on those chromosomes that are early gained in any one of those diseases that there are genes which are already higher more highly transcribed so that the initial impetus to gain these chromosomes would be a physiological one but on the chromosomal normal tissue has no chromosomal approvals is there a normal person without cancer? no so the question was what is the role of non-coding RNAs whether they are short or long in the maintenance of genomic instability and the answer is I don't know the answer is nobody knows obviously through the genome institute and other initiatives we will learn much more about it hopefully through other initiatives as well but the role of non-coding RNAs in the maintenance of the genome is not yet established is that an indicator that you are getting increased risk genetic defects or do you look at that or it has something totally different? so the question is to which extent established serum markers would reflect the risk of developing specific aberrations I cannot, there are no studies I cannot answer that and the serum markers are very likely but there are obviously problems and many people have looked for colon and other tissue pancreas but there is no direct correlation it could be also factors that are unrelated to the presence of certain genomic imbalances which is whether you have a blood vessel through the tumor and you can shed cells so what I believe will become a possibility in the future for earlier detection is the detection of circulating tumor cells the reason I ask that is I have a lady with metastatic ovarian cancer who is in total remission and her scans are normal and she just has an increase in her CA-125 so as a non-oncologist how am I supposed to deal with that or just let the oncologist handle it which comes to me and asks me the question I'm not going to make it such a subject so can you comment on the tempo of translation of this exciting science to the clinic just a question I would have in my head is this something I'm going to have to face tomorrow a year from now or 10 years from now it seems like it's pretty soon for the cervical casinomas it's being implemented now there are several laboratories around the world who actually use it for samples samples that are ambiguous the goal would be to all the cases that are HPV positive should get this genetic test because then you know what's going to happen and I usually restrain from such statements in biology or genetics but this is black and white it's you have it, you progress you don't, you do not, period so this is probably the most advanced we are now, as I mentioned to you before we work with the Collins Institute where we have 9000 archived breast cancer cases from all of which we have the clinical follow up the suites are very good with that there is no population not so much mobility and we have the clinical follow up and we have the DNA content so we are now in the process of correlating in a large definitive data set the degree of an employee with prognostication so that's going to be next we have just received funding and getting out of trials where we can try where we can evaluate in larger numbers the value of gene expression profiling for prediction of response I recall reading a note from Ben Stavida who was a former director of the Cancer Institute that we were entering a phase where in arbitrary cancer you need a very skillful doctor and this stuff is really beginning to bring that home we need more other comments or questions? it's only done as a reflex when you start seeing abnormal cells is that likely going to change? I know with it there were several studies I believe they suffered from budget cuts and sequestration exactly addressed the question whether HPV should be the first test because it essentially would make sense because those that are negative don't get cervical cancer I completely agree but I have not seen the result of any there was one out of Seattle, there was one that was initiated by Mark Schiffman at the NCI and others but there is no I don't want to step on anyone's toes but maybe there is also a priori resistance by the cytopathologists to change or by the gynecology to change something which has been successful and I mean one should be very careful to change something that is successful into something where you first have to validate it on a large basis but from a biological point and a genetic point of view there would be no reason not to change the practice to what you just suggested It is a fact that we clinicians stumble along behind investigators and it takes a while to translate I think Other comments or questions? If you have them please come down Thank you very much