 First disclaimer, I am not a molecular biologist, and I'm not a computational biologist, and I don't have a lab like these distinguished laboratory investigators. I'm privileged and humbled to be sitting with them, giving a talk on molecular biology. But my interest started several years ago when I joined the staff at M. Genderson, when I had the opportunity and the privilege to see young patients with two rare kidney cancer tumors, Translocation Carcinoma and Renal Medallori Carcinoma. These are individuals who are afflicted with these devastating tumors at a young age, and unfortunately no effective therapy is instilled today, has been established. This slide shows you the 2004 World Health Organization classification of epithelial renal tumors. I think you all recognize on top is clear cell, but as Chris Wood showed in his talk, 24%, somewhere around a quarter of the patients with RCC have non-clear cell histologies, which are diverse group of patients, and sadly to say they have been neglected in research over the past several years, the focus has been on VHL and HIF in clear cell. I think unclassified RCC is in this list here, not because it's a biologic, distinct biologic entity, but because it is the frustration of pathologists after immunohistochemical stains and different consultants looking at the tumor, they cannot come up with a diagnosis of what subtype of RCC it is. And we all recognize oncocytoma as a good model to study because of its relationship, close relationship to chromophobes. So just to briefly review for you the psychogenetics and the genomics of several of these non-clear cell histologies, there are two genetic defects in pepillary type 1 RCCs that lead to hyper-activation of the mutate of the MET pathway, and that's either through a single nucleotide mutation in the exon that harbors the gene MET, which is on chromosome 7, or there is increased DNA copy number through either trisomy 7 or tetramus tetramus tetrasomy 7, leading to hyper-activation of the MET pathway. However, people think that this may not be enough if it is through the increased copy number, and then this is where leucine-rich repeat kinase 2 present on chromosome 12 is cooperating with CMAT 2 both being overexpressed and hyper-metallate the pathway. Now the story with papillary type 2 is different. I don't think it's as well understood as type 1 on CMAT pathway, but I think there is emerging evidence to strongly indicate that there is activation of NR2F in patients with papillary type 2. I think this is also the most plausible mechanism for this is mutation of the fumarate hydratase gene that leads to accumulation of fumarate, which is critical in the CREP cycle that leads to covalently modifying KEEP 1, and KEEP 1 is a negative regulator of NR2F. When NR2F2 is upregulated as a result of KEEP 1, then there is increased signaling of survival pathways in tumors. The story with chromophobes and oncocytoma is similar as you have mitochondrial DNA mutations and that leads to increased levels of genes associated with oxidative phosphorylation, and then cycling the one gene is just proximal of the breakpoint of 11Q13, which is translocated in 50% of the oncocytomas. I will be discussing the two tumors I mentioned, translocation and renal medullary, and show you some of our work here. Translocation RCC is a common tumor in children and in adolescents. However, in adults, it is estimated that only 1.5% of tumors have translocation Xp11.2. We looked at the TCGA set data, 460 tumors, only seven had the Xp11.2 translocation. As I said, this is a disease that afflicts young individuals, predominantly females, and these mostly present with advanced disease at diagnosis, and there have been five partners identified that are the hallmark of the TFE3 gene translocation to these partners, and they are here PRCC, SFPQ, and the ASPSCR, and nono gene, and the CLTC. So the hallmark of this is identification of the translocation of TFE3 gene and fusion with one of these partners leading to the product that we can identify histologically in this panel there, the HNE, where you see both clear cell features and pepular features in the same tumor, and TFE3 antibody is now used to diagnose these, and then you can confirm that with fish. So a few years ago, as I said, I saw this young 20-year-old nursing student, female, with metastatic disease and primary in place, and that's when we began our journey, and we partnered with a French group. They have a very good juvenile RCC network where we were able to collect tissues from these patients. So we had 16 primary frozen samples from nephrectomy specimens, and five were pediatrics, 11 were adults, and we had also other 13 additional paraffin-embedded samples, and we established three novel cell lines, and I'll discuss one of them, and then we did SNP arrays and line 1 methylation, which is a surrogate of global DNA methylation on these. So the results of the SNP arrays in these 16 cases are presented here. 31% had SNP arrays consistent with clear cell, with 3p loss, 3 cases had plus 7 consistent with papillary, five cases had novel cariotypes, and three cases had normal cariotypes. In the five cases that had 3p loss, four had also 9p loss. But the most important finding we found in this study is we identified 17q in seven patients, or 44%. And this was also importance of this is was patients who had 17q gain had a poor prognosis. When we looked at age, separating the samples results by age, under 18 and 18 and older, because our observation was children and young adolescents, so with terrestrial tissue, with TFE3 had a good prognosis, usually no metastasis, they lived longer, or if they had metastasis they had indolent disease. Unlike older patients, those that I have seen who had a poor prognosis. So when we look at separating them by age, under 18 had fewer genetic aberrations compared to patients who were older than 18. The blue bars represent the gains and the red bars represent losses of chromosome, and then the numbers of chromosomes are listed there. Again, this shows what I was talking about, it was statistically significant difference, even though we had a small sample of only 16 patients. And this is again the same data showing that patients who had 17q gain had a worse prognosis than those who didn't. And again, 17q gain was associated with a much larger number of genetic aberrations. So this is a cell line that we established from a tumor of the 20-year-old female patient I told you about, and what this spectral karyotype shows, six different translocations, some are interchromosomes and some are intracromosomes, three of these six translocations involved chromosome 17. And then the same cell line, we studied it and we asked the question, why do these patients with 17q, as this patient had, gain, had a worse prognosis? And we did RNA-Seq on two specimens where we had good RNA. We did also two specimens without the 17q. So we analyzed the RNA-Seq in those four cases, two without the 17q gain, and we did gene expression profiling, and this is the ingenuity pathway analysis showing that for when we saw 17q gains, often this was associated with 17p loss. In four of those seven cases, there was an isochromosome, so balanced loss and gain on some on chromosome 17. So however, the gene expression profile here showed that TP53 was down-regulated in those two cases with 17q, and we see the genetic, the gene expression profile different between these two tumor types. And this is the RNA-Seq showing a novel in the same tumor from that same patient, the cell line I showed you, showing the, we identified a novel translocation, the Luke 7L3, which is present on chromosome 17 and TFE3. So that's a new finding from this work, and you see down at the bottom of the slide, the two proteins, diffusion product of the TFE3 and the Luke 7L3. We validated this novel translocation by Sanger sequencing, and then when we did gene expression profiling, comparing translocation with clear cell RCC and papillary RCC, as you see here in this heat map, there is clear separation of the gene profile. The red are normal kidney, and the blue is the clear cell, the clear cell cases, and in yellow are the translocation, the XP11, and in turquoise are the papillaries. So clear separation of transcrits from these different tumors. When we did DNA methylation by DREAM, this was done in collaboration with Jean-Pierre Haissa. There was clear separation also between the DNA, the methylation between pediatric cases shown in green and adult cases shown in red, whether you look at the genes in the CPG Island or outside the CPG Island. So in conclusion for the project on translocation, we identified about half of the patients, of the cases of XP11, to have a genetic profile similar to that of clear cell and papillary. 17 Q-gain is the most frequent genetic aberration in XP11 translocation and is associated with poor outcome, perhaps due to haploinsufficiency of P53, although we did not detect any somatic P53 mutation in these samples, the same way we did not detect any VHL mutation in these samples. Tumors from patients who are older than 18 display more genetic abnormalities and lower line 1 methylation than tumors from younger patients under 18. We identified three novel partners. I showed you one already, the Luke 7. And the gene expression profile is different between these three different tumors and methylation studies also can discriminate between adult and pediatric. What about in the next minute and a half, some of the budding work on renal medullary carcinoma. This is a devastating tumor, as I said, that afflicts young adults with sickle cell trait. Majority in the US will be African-American. Transient responses to TCC regimens and in my experience, the target therapies do not really work well in this disease. For the last 12 years, I've had the opportunity to see many of these patients treat them along with my colleagues. And we treated 26 patients at MD Anderson. We were able to collect 15 tissues from nephrectomy specimens and we established three xenograft and three cell lines. These are six cases. We did exome sequencing on 11 of these 15 specimens. The bottom row is the normal kidney part from those nephrectomy specimens from these patients. The top five are different tumors. What we wanted to show here is that the gene that's responsible for the sickle cell trait, which is the HBBB gene, is mutated. And the mutation results in a change from glutamic acid to valine on chromosome 11 due to a simple nucleotide change from GAG to GTG in the six codon. And then when we looked at the mutations by genes, this is, we had one case where we had almost a thousand genes that were mutated. I mean, obviously this is, we're still trying to grapple with this information, but many of these, this is a busy table, but we decided to look at genes that were mutated in at least three different samples, three different cases. And so it's shown from the bottom up, three all the way up to six. The first column is the gene that's mutated and you could recognize some of these that have some contribution in RCC. I think Ian spoke about, I think, ML4 in chromatin, and then there is the TSC1, but I read it honestly. I do not know what the function of most of the genes are. We're still studying this, but the top row is a case, is a case with six mutations. We did RPPA, reverse phase protein assay in these tumors, and we compare, this is five cases where we compared the tumor versus normal part of the kidney in those five cases, and we were able to show there is segregation between tumor from RMC and normal tissue. And then when we looked, we saw that fibronectin and some others were, especially fibronectin, significantly different than in RMC tissue than normal tissue. We are validating this in Western blots. Future studies, we're going to be doing microarrays to try to, because the RPPA platform allows you only to investigate query 160 or so antibodies, but here we will be looking at 40, 50,000 genes to see, and we will be trying to do in vitro studies and in vivo studies in xenografts models. Thank you. Here is the acknowledgement list. This work is the labor of several people who worked with me. I would like to specifically mention Gabriel Malouf from the French, who is now on staff at Hôpital Petrier, chef of the clinic there. We continue our collaboration on translocation. We have now 16 new fresh cases we received. We're going to be studying these to validate these findings we had in our cohort. Chris Wood has been tremendous help for us. He operates on most of these patients, and we generated some of these xenografts in his lab, and the methylation studies were done in collaboration with Jean-Pierre Asa, who is at Temple University. The RMC project is led by a former fellow who just joined our department as an assistant professor, JJ Gao. He is leading this effort, and I couldn't do all of this analysis without the bioinformaticians shopping soon, and we, Yao, who did a tremendous job trying to analyze these cases for us. Thank you for your attention.