 Thank you for the opportunity to present our results. The Harrod GCC team generates and performs analysis of low-pass whole genome sequence and data for different TCJ tumors, and in our case low-pass means 7x coverage on average. One aspect of our analysis is the search for viruses and bacteria in tumors and looking for possible integration events and mechanisms of such integrations. And today I'm going to present our results for head and neck carcinomas and bladder cancers. So why we search for viruses? Actually, it's a well-known fact that viral infection is one of our important risk factors for different cancer types. A significant proportion of head and neck carcinomas are caused by human papilloma virus. As for our bladder cancer, results are somehow controversial, but several previous studies reported a moderate association between the bladder cancer and HPV and some polyoma viruses. So what did we find in our TCJ data sets? So we performed analysis of whole genome sequence and data for 113 head and neck tumors and control pairs and 105 bladder cancer and control pairs. Generally, we could divide detected viruses into three groups. Different types of HPV viruses, human-herb viruses, and one case of polyoma virus-positive bladder tumor. HPV infected 8% of head and neck tumors and 4% of bladder cancers. And most cases are represented by so-called high-risk types of HPV, such as type 16, 33, and 56. And we do not see these viruses in the control pairs except for one case, but this positive control case has also a positive tumor pair. And probably this tumor contamination event, like it was discussed on yesterday's talks. So let's look more closely to HPV-positive samples. As you can see, the entire, in most cases, the entire viral genome presents in the infected cells. And the estimate number of HPV copies per cell varies from 1 to 30. However, sometimes we see only 80 or even less percent of viral genome in the cell. Such situations observed in only two positive control samples and a couple of positive tumors. And we think that it happens not only because of a low number of copies per cell in these cases, but in some cases, it happens due to large deletions in the viral genome. So here is a visualization of our sequence and data for some of HPV-positive tumors. And for those who are not familiar with IGV browser, each gray rectangle represents a sequence and read map to a reference viral genome. And I want to mention that it's a linear representation of HPV genome, which actually is a circular genome. So you can see here that genome coverage varies not only across the different samples, but also within the same sample. Some regions are amplified, whereas some other regions are completely lost, like I mentioned before. So, okay, we detected virus-positive tumors. But the critical issue, at least in the case of HPV, is the physical status of viral DNA in the infected cells. Normally, it presents there as an episode. However, when it integrates into a human genome, bad things begin to happen. Typically, integration leads to disruption of the viral E2 gene, which normally represents viral oncogenes E6 and E7. And as a result, E6 and E7 start to overexpress, and their products down to regulate human tumor suppressors P53 and PRB. So that's why, and this leads to malignant progression. So that's why it's interesting to detect integration sites. So in order to detect integration events, we look for clusters of discordant treat pairs. We take advantage of parent sequencing. And what's the discordant treat pairs clusters? They're one end of a pair mapped to a viral genome, and the other end mapped to a human genome. So here are some detailed examples of detected integration events. Besides HPV, we found two integration events for polyomovirus-positive bladder tumor. And in one case, polyomovirus integrates in the fidgetin gene, which involved in mitosis regulation. In the case of Herb's virus 6, integration happens in the telomiric regions. As for HPV integrations, I want to bring to your attention the fact that many targeted human genes are actually members of different cancer pathways. You can easily recognize some very familiar genes, like Notch1, TP63, Red51B, involved in DNA reparation, or BCL2L1, which could act as either pro or anti-apoptotic regulator. So such results support the idea that integration events probably might contribute to cancer genesis not only for a viral oncogen expression, but also for a modification of host tumor suppressors and oncogenes. So to sum up, we detected integration events in 70 HPV or polyomovirus-positive tumors, and two-thirds of them have at least one integration event involved cancer-related genes. However, we also noticed in our interest in fact that almost half of all integration events are accompanied by somatic copy number changes. For example, in one bladder tumor, HPV insertion replaces a large part of Notch1 gene, leading to a heterozygous loss of this region. However, I think that the most fascinating cases are those where we see amplification of both viral and human regions. And in at least four tumors, we think that they see that such amplification happens after the formation of sacral or chimeric viral human episomes. And in the next couple of slides, I will show you one example of such episome. So in this tumor, HPV integrates in the gene TRPC4AP, involved in the cell cycle control. Here is a visualization of HPV sequencing. And I want to remind you that these ends are joined to each other. And here are integration breakpoints. And these regions of viral genome are involved into integration. And you see that they are amplified, 60X comparing with 30X. So let's look on the human side. This 9KB region of TRPC involved in integration. And again, we see amplification of this region, 40X comparing with 10X. Chimeric reads, detected chimeric reads, suggests that HPV sequence encompasses this human region from both sides. One possible explanation that we see some very, very complex standard duplication, something like seven duplications in a row. But the simplest and the most probable explanation is that we see the actual chimeric fragment, something like episome. So we suggest the following model of integration event. This region of viral DNA integrates into TRPC4 gene. And this chimeric fragment undergoes excision and circularization. But in such case, one might expect to see traces of the deletion on a place of excised chimeric fragment. And actually, we do not see such deletion. Is it possible? And the answer is yes. We think that in the case of chimeric viral episome, we see the same mechanism, which is responsible for formation of double-minute extra chromosomes observed in different cancer types. So there are some probable models of these mechanisms. One model is a segregation-based model. And here excision and circularization happen after the replication. And after my traces, one daughter cell gets a deleted copy of gene, and another daughter cell gets two intact copies plus episome. And maybe such combination confers selective advantage to cells. So at the end of the day, we see only descendants of these cells with amplified episomes, because episome has a viral origin of replication. And now a possible model is a re-replication-based model. And here re-reparation of a deleted region and re-replication. And after my traces, both daughter cells get two intact copies of gene, but only one gets episome. And again, due to possible selective advantage, at the end, we see only the descendants of these daughter cell with amplified episomes. And that's why we see many copies of episomes, chimeric episomes, and we do not see any trace of the chromosome scar. So as conclusions, even low-pass whole genome sequence and data together with our pipeline allow, effectively, detect not only the viral presence in the cells, but in samples, but also integration events and mechanisms of such integrations. We detected integrations in 70% of virus-positive tumors. And we think that integration events influence of malignant progression through both human genes and viral genes. And finally, almost in the quarter of all HPV integration events, we think that we see formation of chimeric human viral episome. And that might have some connections with some clinical outcomes. For example, it could be a cause of different treatment responses, but that requires further investigation. So before that, I'd like to thank everybody in the Hara GCC team, and especially Rajo, Angela, and John Seidman. And thank you for your attention. So I'll ask the first question, which is, do these events, first of all, is the virus doing this in 100% of the malignant cells? Do we know in some clonal way that this is in all the cells in the population? And then another question would be, if this can pop in and out of the genome, does it do that just on once in a given cancer, or do you see multiple events of sort of going in, picking up genes, and coming out as an episome? For a second question, I think that we see two such events in one tumor sample. Yes. As for the first question, in some cases, all clones have this event. But in some cases, I think not. So in those cases, would that be against the hypothesis that this virus was picking up a driver oncogene? Probably yes. Probably yes. Hi. I was just wondering why you think you have episomes as opposed to tandem duplications of these integrations? Well, because it's relatively hard to imagine that a cell undergoes just, for example, exact seven the same tandem duplications. Why? I mean, what's the purpose of these for a cell? The formation of episome, like the formation of double-minute extra chromosomes is more simple explanation. And it's more likely explanation. It's more likely event in terms of probability. So part of the reason I ask is I have some data that's not published but shows that we can, like in one of the projects I work on, that we have tandem integrations of bacterial genomes. So six tandem integrations in a fly. And we're pretty sure they're not episomes. And so I think there's this notion that these tandem duplications, large tandem duplications, don't occur and aren't stable. And so I just would put forward that as maybe another explanation. Because the episome argument looks is very compelling until you start having to invoke all these different mechanisms to get rid of the chromosomal scar. Well, yeah. Actually, it's just a probable model. I mean, the ultimate answer on this question would be fish analysis. Will we see it or not? But thanks.