NIH CLINICAL CENTER GRAND ROUNDS
Episode 2009-020
Time: 53:18
Recorded June 10, 2009
6th Annual John Laws Decker Memorial Lecture
Transplanted Allogeneic T-cells Identify a Viral Corpse Resurrected in Renal Cell Carcinoma
Richard Childs, MD
Chief Section of Transplantation Immunotherapy Hematology Branch and Captain Select U.S. Public Health Service
ANNOUNCER: Discussing Outstanding Science of the Past, Present and Future - this is NIH Clinical Center Grand Rounds.
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ANNOUNCER: Greetings and welcome to NIH Clinical Center Grand Rounds, recorded June 10, 2009. We have a special presentation today, the sixth annual John Laws Decker Memorial Lecture, featuring the 2008 Distinguished Clinical Teacher Award winner, Dr. Richard Childs -- chief of the Transplantation Immunotherapy Hematology Branch at the NIH Clinical Center. His topic, Transplanted Allogenic T-cells Identify a Viral Corpse Resurrected in Renal Cell Carcinoma. We take you to the Lipsett Ampitheater at the NIH Clinical Center in Bethesda, Maryland, where Dr. John I. Gallin, director of the Clinical Center, will introduce today's speaker.
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GALLIN: Good afternoon and welcome to our special grand rounds session today. This is one of our contemporary clinical medicine great teachers lectures and today we are going to have our annual celebration of the John Laws Decker Memorial Lecture. This is the sixth time we have done that. This presentation honors the legacy of Dr. John Decker who serves as the Clinical Center Director from 1983 to 1990. Don was an internationally recognized clinical investigator whose reach focus on rheumatic diseases when he was the Director of the Clinical Center, lots of major things happened. So, for example, the pet program was started. The clinical use of MRI was started and the marshaling of resources to support all the work that has occurred on AIDS happened. In addition, to being a great clinician and a great investigator, John Decker had a great sensitivity to the environment that we all live: He started the art program that graces our halls and continue to grace our halls & this art program has been noted by many, including the Secretary of Health and Human Services who now has the program, down to the Office of the Secretary to work here at the Clinical Center. I’m very pleased to recognize and acknowledge Dr. Decker’s daughter, Dr. Meg Molaro, as well as John Decker’s son, David and his wife, Lisa.
We regret that Lucille, John Decker’s wife, is unable to join us today but we are certainly thinking of her. Even after retiring, John Decker continued to do important and wonderful things for the Clinical Center. Among the things he did was to write Promtomechanics, A Guide to Preparing A Clinical Research Study. Initially, that was a text and was used by everybody, and now that has been converted into a computerrer-assisted tool which many are beginning to use. So the Decker Lecture honors a remarkable legacy and each year the individual chosen to deliver the talk is an exceptional teacher and mentor. Today’s lecture is or will not disappoint us. And before we go further, I’d like to turn the microphone over to Dr. Grace Chen, a fellow in The National Institute of Allergy and Infectious Diseases, who will talk for a moment about the award.
CHEN: Thank you to our 2008 Distinguished Clinical Teacher’s Award winner, Dr. Richard Childs and many thanks to the family of Dr. Decker for your support of the series. At this time I’d like to announce the opening of nominations for the 2009 Distinguished Clinical Teacher’s Award which recognizes excellence in the clinical training and mentoring at the NIH Clinical Center. Nominations will be accepted from the following sources. All NIH clinical fellows, NIH research fellows who have direct patient care responsibilities at the NIH, and former clinical fellow whose have completed their patient care roles at the NIH within the past 6 months. E-mail submissions of the nominations may be assessed through the website at the address shown above by clicking on the DCTA subcommittee link. Submissions will be accepted through August 13 of this year and the 2009 Distinguished Clinical Teacher’s Award winner will be announced at the Grand Rounds Great Teacher Lecture Series this fall. Thank you.
GALLIN: Thank you Dr. Chen and thank you for all that you do and the clinical fellows committee does to make life better for everybody here at the NIH and including picking the awardee. So our speaker for today’s lecture is Dr. Richard Childs who is Chief of the Section of Transplantation Immunotherapy in the Hematology Branch of the National Heart, Lung and Blood Institute. He is also been recently named Captain Select in the United States Public Health Service and will become a Captain on July 1.
Dr. Childs received his BS in Medical Degrees from Georgetown. He then completed an internship, residency, and Chief Residency in Internal Medicine at the University of Florida in Gainesville. He also completed a fellowship in Medical Oncology at the NCI and Hematology Fellowship in the NHLBI. He was appointed to a tenured track investigator in the Institutes Hematology Branch in 1999 and received tenure in 2006. Dr. Childs research focused on Tumor Iminologiy and Allogeneic Immunotherapy to treat nonmalignant Heme Ta Logical disorders and solid tumors.
He was the first to establish the existence of a grass verses solid tumor affect mediated by transplanted donor T-cells that could cure patients with Renal Cell Carcinoma. This observation defined a new therapeutic application of Allogeneic Immunotherapy. He has gone on to characterize the immune mechanisms mediating his graft verses solid tumor affect. Novel investigations using Allogeneic T-cells from responding patients identified a solid tumor antigen derived from an endogenous retrovirus that is immunogenic in-vivo. His honors include the NIH Director of Scientific Medal Award and the NHLBI’s Award for Scientific Research. Dinnery, He received 3 NIH Bench to Bedside Awards. He is a member of The American Society for Clinical Investigations, The Society for Clinical Oncology and The American Society of Hematology. He serves on the editorial board of The Journal of Translational Medicine, Drug Discovery Today, Bone Marrow Transplantation, and Immunotherapy Insights. So it is now a real pleasure to invite Dr. Childs to the podium to discuss his topic, Transplanted Allogeneic T-cells, Identify a Viral Corpse resurrected in Renal Cell Carcinoma. Rick.
[APPLAUSE]
CHILDS: Thank, John for that very kind introduction. My wife is in the audience today. This is the first time she ever heard me give a medical lecture. So I'm hoping after that introduction she might cut me a break and I won't have to do the dishes this week. [LAUGHTER]
It’s a real honor to receive the Distinguished Clinical Teacher Award, particularly when you consider the number of outstanding clinical teachers that practice medicine in the NIH Clinical Center. And I’m very honored to receive this award and also very honored to be given the John Law Decker Memorial lecture today. First I have to make it clear that I have no relevant financial relationships with commercial interests to disclose. The goals of my presentation today will be to characterize studies investigating for graft-verses-tumor effects in patients with Metastaffic Kidney Cancer that have undergone hemat poetic stem cell transplantation. To review translational research conducted in our laboratory aimed at defining reck films to which graft-verses-tumor effects occur and to describe a novel Renal Cell Carcinoma antigen derived from a retrovirus identified using allogeneic T-cells from a responding patient that appears to be immunogenic in-vivo and potentially in-vivo - could be an excellent tumor antigen for future immunotherapy protocols.
At most know, for 30 years now, allogeneic and hematopoietic stem cell plan plantation has been the mow dalt tow treat patients with these maglignancies and it even curious those with leukemias and lymphomas. A significant component is mediated to the consequence of the graft verses tumor affect, which occurs from transplanting immunoconfident lympoid tissue from the donor. T-cells and possibly NK cells that mediate these curative affects.
To highlight the power of the graft verses leukemia affect, there is a patient that we treated in the clinical center that had stage 4 mycroseis, tumor stage cutaneous t-cell limb people that was refactory to all conventional chemotherapy this patient underwent a reduced intensity transplant on an NHLBI protocol. And shortly after the transplant, the first month hollowing conditioning, her tumor progressed thereafter, her psychospore in was tapered to try to enhance a graft verses tumor effect and 6-7 months post transplant, the tumor nodules began to flatten and she ultimately achieved a complete response and remains in remission 3 years post transplant with no evidence of disease.
In all likelihood, it's probably cured of her malignancy. So interestingly, hematologic malignancies vary in susceptibility tow graft verses leukemia affect diseases are sensitive to GBLN contrast, diseases that have rapid tumor kinetics, such as refactory all and aml, are resistant to the GBL affect and that may account for the disparity. Up until recently, there was relatively little data to inform us whether scott id tumors might be susceptible -- solid tumors -- comparable to the graft verses leukemia affect. And I think the high risk of transplant related mortality associated with myeloid ablated transplants, which 15 years ago we quoted patients up to 40% they would die from the procedure, precluded such studies from being conducted. 13 years ago when I was a fellow in John Bear on the's lab, we became interested in exploring whether select solid tumors might be susceptible to a graft verses tumor affect. And one of the tumors we became interested in was Renal Cell Carcinoma as a target for allogeneic immunotherapy. We thought this might be a good target for a number of reasons. First, it was a common tumor, about 30,000 Americans are diagnosed with Renal Cell Carcinoma annually. About half of those patients will ultimately go on to develop metastatic disease. And their prognosis, once they develop this is quite grim. Median survival is barely over a year. And although there have been advance in the treatment of Renal Cell Carcinoma including 4 New FDA approved drugs listed here, these drugs can prolong survival unfortunately that is relatively short. It's measured in months and it does not appear these agents are capable of inducing complete responses or curing patients with metastatic disease. So one of the reasons we thought Renal Cell Carcinoma would be a good target is the it's an immunoresponsive tumor, unusual among solid tumors.
There are cases of spontaneous regression occurring with Renal Cell Carcinoma thought to be remediated although this incident is low. And unlike most solid tumors, Metastatic renal cell carcinoma appears to be sensitive to high dose interleukin 2 therapy. So studies at NCI showed high dose IL2 could reduce emissions in up to 10% of the patients with metastatic kidney cancer and these remissions turned out to be variable. And IL2 is the only FDA trug to treat metastatic kidney cancer that has the capacity tow cure patients with metastatic disease. So this was the transplant strategy that we developed to try to investigate for graft verses tumor affect in kidney cancer. Patients that were eligible for this study were required to have metastatic disease that was progressive raid logically. They were required to have Cytokine Refactory disease and they were required to have an identical SIB I think there serve as a stem cell donor. We used -- sibling to serve -- we used immunosuppression to knock down the immune system to prevent graft rejection and we knew these drugs would not be ablative. They are considered nonablative drugs but highly immunosuppressive and since these patients would be chemotherapy naive, our hope was they would reject the graft suppression. Donors underwent mobilization and on day 5 underwent a t-cell ieplete allogravand it was collected and infused intravenously on day zero. We used psychospore in as Prophylaxis and our goal was to get patients off this agent as quickly as possible because we knew that placing patients on an immunosuppressive agent wouldn't be conducive to inducing a GBT affect but you do have to use some immunosuppression because lethal graft verses host disease would occur at an exceedingly high risk if you didn't have any post transplant immunosuppression in the context of a t-cell replete graft. So, we used t-cell chimerism tol guide us in terms of how rapidly we tapered this in patients in had mixed T-cell camers and we knew it would probably also prevent TBHG. And patients that had full donor T-cell chimerism after the transplant, these are patients we knew were at higher risk for graft verses host disease, they continued on full dose until 60 days after the proceed and you are then underwent a rapid Taper. Our goal was to have the patients off psyche low spore in 100 days after the procedure. We then assessed them with CT scans and patients that it is had evidence for disease progression were then eligible to be treated with donor Lymphocyte infusions we collected from the original stem cell donor and had preserved with the intent to use them in the context of disease progression. So this is a timeline for our transplant procedure. It was approved by the NHLBI/ IRB in 1997. We transplanted our first patient with kidney cancer in February of 1998. In ninety nine, we reported the first case of a graft verses tumor affect in the JCL in a patient that had Metastatic Kidney Cancer. This patient had Metastatic to the lungs. Biopsy confirmed had a very remarkable graft verses tumor affect and achieved a complete remission by 180 days profit transplant.
The -- post transplant. We went on to describe the first 19 patients with Metastatic Kidney cans they're underwent this procedure. -- Cancer. 10 of 19 had evidence for graft verses tumor affect. Most of these responses were partial but we did see 3 patients that had a complete response. Also remarkable was the fact that many of these patients had failed high dose interleukin 2 therapy and had bulky tumors such as this patient that had a large anterior mass and bilateral knop thee that had disease regression which was delayed after the transplant. You can see tumor regressing by 270 days post transplant. We also observed the patient that is had a history of graft verses host disease were more likely to have response in patients that did not develop GBH & this had been observed in patients who were you should going allogeneic transplantation - around going. This potentially could be mede eighth the GBT effects. Occasionally we would see patients in individual a response -complete. This had the disease in the owns and had literally hundreds of lung lesions. This was clear cell carcinoma but was biopsy confirmed. This patient had a complete Reons and remains in remission more than 11 years after the transplant. So since our initial report there have been a number of small case series describing transplant outcome in patients with Metastatic Kidney Cancer. I listed them here.
Most of these transplant centers have used varying degrees of conditioning to achieve engraftment 6 donor immune cells. Response rates have been variable, as high as 57% in one study and as low as 0% in another study. The largest series published to date was from Europe summarizing the results of 124 patients with Metastatic Kidney that were transplanted through the EBMT Group. Thrall% of the these patients achieved disease regression -- 32% -- consistent with the graft verses tumor affect. So this is an update from our original series, the results of the first 74 patients Metastatic Kidney cancer that were transplanted from 1998 through June of 2006. Most of these patients received a transplant from an HLA identical sibling. 5% received a transplant from a sibling that was of single HLA mismatch. The median age was 50 years. About half of these patients developed acute and chronic graft verses host disease. Unfortunately, 8 patients died from transplant related mortalitity and half of those deaths were caused from steroid refactory acute graft verses host disease. Over all, about 40% of patients had a disease response including 10% of patients that the had a complete response. This shows the time to response. You can see these responses were typically delayed. The median time to disease response was 4 months after the procedure & that’s probably because early on, patients have mixed T-cell camers and they are also on psyche low spore in which is in tollergenic. It takes them a while to convert the T-cell chimerism to full donor. Important lyrics the patients that had disease regression—importantly—the Y were more likely to have long term survival. Median survival in responding patients was about 2 ½ years compared to 7 ½ months in nonresponding patients this is important because this was some evidence that tumor regression occurring after the transplant was clinically meaningful. So one of our goals was to move from proof of principle to establishing more effective allogeneic transplant trials and in order to do that, we thought it would be fortunate identify the immune affects with the hope we could use T-cells to identify the target antigens. So indirect evidence suggested that these affects were mediated through transplant donor immune cells. Clinical observations to this affect included the fact that in the majority of patients that the had a disease response, tumors would grow the first couple of months after the procedure. Followed by tumor regression. So there was no evidence that the chemotherapy we gave in the conditioning regimen had any activity against the tumor. Secondly, we usually saw disease responses occurring only after T-cell chimerism had converted to full donor or was in evolution towards converting towards full donor and third, we did see disease regression that was associated with graft verses host disease and occasionally we would see patients that would have tumor regression that would occur following a donor lymphocyte infusion.
So there is also some direct evidence that donor T-cells mediate these antitumor affects and this includes the identification of minor histocompatibility T-cell clones that could be isolate Friday responding patients—isolated from—to be shown in capable of killing patient tumor cells. Is the Fred Hutchinson Cancer Center and our group was able to expand T-cell clones to target minor histocompatibility antigens from Renal Carcinoma responders. They are a population that in-vitro, are able to induce cytotoxicity against both the tumor, which could be consistent with the GBT effect, as well as against normal recipient cells such as fibroblasts hematopoietic cells. These are population es we expect would simultaneously mediate graft verses host disease as well as a graft verses tumor effect. This is a patient that we think that the observed disease regression was probably mediated by minor antigen reactive T-cell populations. This patient had disease regression post transplant and developed both acute and chronic graft verses host disease of the skin and when he would have flares of this chronic disease, we would see radio graphic evidence for his tumor regressing. So we collected mononuke laywer cells from this patient and stimulated them in-vitro with eradiated tumor cells we had and we were able to clone out by limiting deletion, CDA positive T-cells that appeared to have a cytotoxicity pattern consistent with the minor antigen. You can see this clone, a CD8 clone, kills both patient kidney cancer cells as well as tracks formed B-cells of the recipient.
This is consistent with an antigen that is broadly expressed normal tissues as the tumor. We weren’t interested in identifying these antigens because if we were to develop immune therapy against them the concern would be we would also enhance graft verses host disease in the patient. Now the Hutch Group went to characterize on a molecular level some of these minor antigens that are expressed in kidney cancer. One important observation was a significant number of patients had disease regression that occurred without any evidence at all for graft verses host disease. 8 of the 29 patients had disease regression without GBHD and this the raised possibly that these donor immune responses being mediate the in some patients might be targeting antigens restricted to the tumor F that were the case, our goal was to identify these antigens because these would be targets for immunotherapy within the context of a transplant and outside of a transplant. So this is a patient where we identified a tumor-specific antigen and Renal Cell Carcinoma a 52-year-old male with clear cell carcinoma who had disease regression after the transplant noted 150 days post transplant. Here is pulmonary lesions regressing. We collected mononuclear cells post transplant in several occasions. And a postdoc in the lab has now gone back to Japan who did this so we were able to identify in this patient’s blood CD8 positive T-cell that appear to recognize the patient’s tumor. We were fortunate enough to have tumor cells on this patient and we were able to establish tumor cell lines. And when we collected T- cells from this patient, following disease regression, we were able to find CD8 positive T-cells secreting gamma interferon but did not secrete gamma interferon against normal tissues from the patient. So this was consistent with a T-cell population recognizing an antigen that was restricted to the tumor. Rodger did our analysis and the frequency of these T-cells ranged anywhere from about .5% to 1% depending on the time period the blood cells were collected. So we took these PVMCs and stimulated them in-vitro with erate radiated tumor cells from the patient and these cells were collected at multiple different time pints and we were able to easily generate lines that were highly cytotoxic against the patient’s tumor cells. One of thieves lines contained predominantly CD8 positive T-cells B-95%. And had a cytotoxicity pattern that was consistent with recognition of an ABT JEN that was restricted to the tumor. So here you can see the CTL line and this is high degrees against the patient’s tumor but does not recognize cells that are obtained from the patient’s fibroblasts ear from patient mononuclear cells third party Renal Cell Carcinoma cells or K562 cells this. Say pattern that would be consistent with recognition of an antigen restricted to the tumor. The dominant T-cell population in the CTL line would be Beta 7 positive. We sorted that using immunomagnettic beads. And then we cloned them out and we were able to identify a number of CD8 positive clones that were consistent with tumor-specific recognition. Unfortunately, the vast majority of these clones did not expand to be useful for identifying antigens but we were lucky and had one clone that continued to expand that we called BZ4T turned out this clone recognized an antigen in the context of an HLA11 molecule. This patient was homozygous. And what was a bit surprising was when we took this clone, and we screened it against a number of renal cell carcinoma lines, we found half were recognized by this clone. That provided us with the first evidence that this antigen was expressed commonly on kidney cancer cells. So in order to identify the exact target of this immune response, we made a CDNA expression library from the patient's tumor. We then cloned the HLAA11 molecule and transfected it into cross 7 cells and then transfected these into these cells and screened them using our CTL lines. And we were basically able to work our way down until ultimately we found two transcripts that were recognized by our t-cell clone that we called CTRCC8 and 9. 8 was the larger transcript, 2155 base pairs. And the eight 9 was the smaller transcript, 578 base parse and importantly after sequencing, we found they shared a common sequence of 377 base pairs.
When we did blast sequencing on these transcripts, we found they originated from the chromosome 6 and were derived from a human endogenous retrovirus type E that had previously been identified during the Human Genome Project to be expressed in human cells but or to be present in human cells but never found to be expressed in normal human tissues. Now these two transcripts were spliced explain each other, the common sequence was derived from the endogenous retrovirus and then supplies into unique regions of the endogenous rett vo virus. What are human endogenous retroviruses? The ancestral remnants that integrated into the human genome. These insertions occurred millions of years ago. 8% of the human genome is made up of endogenous retroviral elements. So all of us are about 1/10 walking retrovirus. Although some full-length persist over time like our endogenous retrovirus, most are in activated by random mutations and there is no evidence that any retroviruses have the 80 to replicate. Ability to replicate. How is this made? Millions of years ago, retroviruses infected germ cells from either monkeys or primitive humans. Yes, that is the geiko man. Following reverse transcription, these genes are integrated into the chromosomes of jem cells and then transferred to the offspring so all cells now possess theige urine dodgeinous retrovirus. -- the endogenous. -- under the negative selection, they accumulate mutations in most genes that result in silencing of the majority of the open reading frames or truncation of the open reading frames so that only small peptides or small protein fragments would be coded. Furthermore, a significant percentage of human endogenous Rett vo viruses you should go Methylation of the promo or which nuts silencing of the endogenous retrovirus. So you can think of it as being retroviral fossils or corpses. Some people refer to them as the domesticated or tamed retrovirus. The function of these viruses in human disease is highly debated. Recently there has been a number of reports over the last 5 years that described a number of encoded proteins detected in tumor cell and in most cases we don't know the function of these proteins with a few extensions. There was one that was recently described in germ cell tumors that include the GAG and envelope protein that has been shown to be oncogenic. These proteins called RECK and MP9 when transfected into fibroblasts appear capable of transforming them. Another interesting report was from Natalie who showed that following EBV infection of B-cells, a HERV18 was transactivatedded and functions as a super antigen. One of the hypotheses is one of the reasons that patients who develop infection monnukeoseis have rapidly expanded lymphnodes and perhaps the superintendent antigen is recruiting T-cells. So this may be a function to enhance immune clearance of EBV infection. We look by ETPCR to identify the restriction pattern of the herv endogenous retrovirus this. Is pooled CDNAs obtained from 23 different normal tissue and we performed RTPCR and no evidence these normality tissues expressed the CTRC8 or 9 endogenous retrovirus. Here is our positive control obtained from the patient's tumor and importantly, pooled CDNAs from normal kidney tissue had no expression of this endogenous virus. We looked at a number of different solid tumor and leukemia lines for expression of this and we found none of these tumor express this endogenous retrovirus with restriction -- expression occurring in our patient's tumor. But importantly, we found that the majority of tumor cell lines that we had in the lab had high expression of both endogenous retroviral transcripts.
Over all 10-14 of these lines expressed endogenous retrovirus. So in order to identify what the exact peptide sequence was that was being recognized, we cloned the common sequence region of the CTRCC9 transcript and main minigenes and transfected these into 11 cells and screened with our T-cell clone and were able to work our way down until we got to the point where we were able to identify 4 candidate peptides that could be encoded by the minigenes recognized. We synthesized the peptides and pulsed them on our cross A11 cells and were able to identify 10 amino acid peptide that elicited high levels of interferon gamma secretion consistent with this being the antigen that was recognized by our t-cell clone. So in order to confirm that this was the target antigen, we generated an HLA-ALCTRCC1 TET MERTHAT was PE conjugated and we went back to our CTL line that was highly cytotoxic and we stained this tetri merand we found more than 50% of the CD8 positive T-cells in that line were reactive to this peptide. So we knew we had the antigen that was targeted for the cytotoxicity we observed in-vitro. Now we are fortunate that we had some blood mononuke lawyer cells that were left over from the patient transplant samples and when we looked for ANT JEN recognition using the tetri mer, we found no recognition by the donor pretransplant or the patient pretransplant. But important beely, we found post transplant samples that there was an in-vivo expansion of peptide reactive examples that occurred 180 days post transplant and peaked at 900 days post transplant with slightly more than 1% of the CD8 positive cells being antigen specific for this endogenous retroviral antigen.
So with most discoveries you're left with many more questions than answers. Things that we needed to look at quickly were first, was this endogenous retrovirus expressed in fresh tissues? One of the concerns was that we may I have been identifying an antigen that was expressed in-vitro as an artifact. So it was important to establish that this antigen was expressed in tumors taken directly out of a patient. Secondly, we wanted to know, do all histologies of Renal Cell Carcinoma express this represent row virus? It comes in a variety of histologies including clear cell Carcinoma representing 80% of the patients in have kidney cancer. But others occur more rarely. And we wanted to try to do more to figure out what exactly regulated the expressionful this endogenous retrovirus that would restrict the expression to Renal Cell Carcinoma. So this is work that was done by Liz Burnie who was a CRTP student in the lab. And Liz looked for expression of this endogenous Resrow Virus using real time CRP and she found ferential tumor samples that were acquired from another lab, these were metastatic lesions, indeed expressed this human endogenous retrovirus. Levels that were comparable to what we had observed from our tumor cell lines. LIZUENTED on to show that this endogenous retrovirus had expression high degrees of the expression, not only neferential tumors and clear cell lines but this expression was restricted to tumors of the clear cell histology. None of the Pap Larry or collecting duct tumors expressed this endogenous retrovirus. Now another interesting observation was when we went back and we looked at our patients that were having disease response we observed the tumor regression occurred in patients in had tumors of the clear cell histology. About half of our patients that had clear cell histology had a graft verses tumor affect in contrast to none of the 14 patients that had nonclear cell tumors. So what is special about clear cell carcinoma that makes it a target for graft verses tumor affect after austrians plant and makes it the one sub type that expresses this endogenous retrovirus? This is a slide that I borrowed from another lab. A lot is known about the molecular pathogen SIS of clear cell carcinoma. In the late 1980's it was shown that majority of clear cell carcinoma have a mutation in the suppress or jean here. The VHL protein forms a complex that is involved in ubicinating transcription factors. And it was identified that in the majority of the tumors, 80%, either a mutation of one of the 3 axons or Hypermethylation of the VHL promotor that resulted in absence of the protein and over accumulation of HIPS. It's a transcription factor for at least 50 different genes and it's known that in clear cell carcinoma, it serves to type high levels of the EGF and glute1 and platelet drive growth factor to contribute to the tumor jenisity of renal cell carcinoma, so we have recently shown that VHL regulates expression of the ctrccherve in clear cell carcinoma. VHL is absent in all of the tumors that she has identified that express the CPRCC-HERVE.
See also found that herve could be silent by transfecting VHL into VHL deficient RCC cells. So this is a tumor cell line from our patient that we identified the endogenous retrovirus and you can see by realtime PCR, there is high expression of the endogenous retrovirus.
When you transfect in wild type VHL and here we are detecting VHL expression by RTPCR.The endogenous rett vo virus is silent. This evidence that VHL regulates expression of this retrovirus. If we go further downstream, and we knock out alpha expression using sirna, you can see the endogenous retrovirus expression is knocked down significantly. So putting this all together, regulation of the CTRCC-HERVE appears to be consequence of a lack of VHL protein which leads no increase in HIP expression. Alpha doesn't play a role in controlling this retrovirus. I didn't show you this. You'll have to take my word on it but there was no correlation to the alfy expression and expression of this. And we think that HIV 2 Alpha serves to bind a responsive element on the lTR that leads to expression of transcripts from this endogenous retrovirus and these transcripts encode antigens that is may be immunogenic in-vivo and could be targets for the graft verses tumor affect. Now if this were the only mechanism to explain expression of our endogenous retrovirus, you would expect that all VHL deficient tumors would have high HIV 2 levels and they should all express this endogenous retrovirus. Unfortunately things are not that simple. So, while we found that all clear cell carcinomas that were VHL deficient had high levels of HIV 2 alpha expression, and we found that the majority of them expressed the herve, we found a couple of cell lines that had high HIV 2 levels but had no endogenous retrovirus expression. One potential explanation would be if the 5-Time LTR of the endogenous retrovirus would be to be methylated, then it wouldn't be able to bind and that would prevent expression of the endogenous retrovirus. We performed sequencing of the LTR on all of these clear cell carcinoma lines and then what we found was that the tumor lines that did not express any endogenous retrovirus were highly methylated at the 5-LTR and the lines that had high expression of the endogenous retrovirus had low degrees of Methylation. So to confirm that methylation played a role in regulating the expression of this endogenous retrovirus, we looked at the affects of demethylating agents on these tumor cell lines and found that a number of these lines that did not express endogenous retrovirus when exposed to the demethylating agent, DAC, combined with subtle transferase inhibitors, significantly increased expression of the endogenous retrovirus that. Shows that methylation plays a role in the retrovirus. So in conclusion, using allogeneic T-cells, a tumor antigen derived from a human endogenous retrovirus found to be expressed in the majoritiy of clear cell carcinoma kidney cells. The herve appears to be antigenic in jefeo. Properties would make it a target for immunotherapy. Appears to regulate human expression in Renal Cell Carcinoma and this accounts for why the expression is restricted to clear cell carcinoma, studies are ongoing trying to characterized the function of this eat retrovirus, whether of any of these genes are important in the tumor genisity, and we are looking for peptides that are presented in the context of more common HLA molecules, that may be derived from this endogenous retrovirus that would be better targets that would be more applicable to humans as targets for immunotherapy trials.
I like to stop there and I want to acknowledge a number of key investigators to the data that I presented to you today: both have gone back to Japan who cloned the endogenous retrovirus and Burnie was a CRTP student in the lab and shown this virus had expression restricted to clear cell carcinoma, Sheila did work showing this peptide was immunogenic in-vitro and showed that Methylation played a role in regulating expression of the endogenous retrovirus. Elaina has done the work showing VHL directly regulates expression of this and I want to acknowledge John Barrett, whose lab I came from and who continues to be a colleague and a good friend. Marston who have referred patients us and given us tumor samples to help identify factors that regulate factors of this retrovirus. David who helped us with the experiments looking at the role of methylation regulating this endogenous retrovirus and last but not least, I want to thank Michael who was one of the investigators that cloned the VHL gene in the late 1980’s. Who recently retired from the NCI and who is joined our lab group as a special volunteer. He say great friend and colleague and has provided lots of insight into mechanisms that potentially explain how this retrovirus is expressed in Renal Cell Carcinoma.
And I want to quote him because it’s true. Michael typically tells me that everything with this endogenous retrovirus is exactly as I have predicted. And even though that sounds funny, it’s about 95% true. With the expection of Methylation. So I’ll stop there and I’m happy to take questions.
[Applause]
GALLIN: Thank you for a really wonderful lecture. We have time for questions. Please use the microphone.
CHILDS: Can we repeat the question for the webcast is that. We have looked at that and with some collaborators in Russia, we looked at expression in tumors that were early stage verses advanced stage. And we do not see any difference between stage 1 tumors and tumors that are isolate Friday stage three tumors. -- It isolated from stage 3 tumors. The amount of expression looks very, very similar. And that's important because if you were interested in targeting this as an antigen in patients that it is had nonmetastatic disease, did is important there be expression before the tumor were to metaft size. If you wanted to use it as some type ofage vent.
QUESTION: As I mentioned before, a fantastic story and congratulations again. Do you think the reason why the Herv is often in nonrenal tissues is because of Methylation?
CHILDS: That's a good question.We are now in the process of sequencing the 5 Prime LTRs of all of these nonclear cell carcinomas to see what degree they are methylated. One piece of information I dependent show was we have a kidney catcher line that has a mutation in VHL that does not express the endogenous retrovirus. When we exposed to to doe meth 1 lating agents we can increase the expression of the endogenous retrovirus. When we transfect VHL back into that cell line, we can no longer induce expression with the Demethylating agents. So somehow VHL is important in regulating expression. So even if we demethylate the 5 Prime LTR, if VHL is present, we don't have expression.
QUESTION: If you demethylate a non-kidney cell line?
CHILDS: Haven't done that yet. Good question. My assumption would be from the data with the 786 line that if we demethylated a nonclear cell line it probably would not induce expression. It needs to be done.
QUESTION: Hi. Can the target -- as a potential vaccine?
CHILDS: So, this is something that Sheila looked at in the throb see if it was immunogenic. And she could induce CTL from healthy donors that recognized Renal Cell Carcinoma Cells that expressed the peptide. One of the problems with the peptide is that it's HLAA11 restricted. It’s relatively rare in Caucasians that occurs in 10-12% of Caucasians. So if we wanted to use this more globally, as a vaccine therapy or as an advent, our hope would be that we could find a peptide that would be presented in the context of more common HLA molecules. That's an area that we are actively exploring to try to look for potential A2 restricted peptides that would be more globally applicable to treating patients as immunotherapy.
QUESTION: Does herve have any efficacy Ar apricative marker for the ablative bone marrow transplantation?
CHILDS: Unfortunately, that's a very, very good question. Unfortunately, we have not been able to go back and do that type of correlation because the majority of the patients we don't have fresh tumor samples to most patients, the only thing we have is pair fin samples. And it's been very, very difficult toasmlify from RNA this endogenous retrovirus. We only get pieces. I think that would be a very, very important experiment to do because if we could find that there was a correlation with expression and response and that would help stratify which patients that we would put on the protocol. One of the things that we hope to do in the future is to be able to potentially detect Humoral responses to antigen that is are derived from this human endogenous retrovirus and if we can do that, then perhaps we could correlate antibody responses with response to either transplant or maybe even response to Interleukin 2. It's still not understood why some patients with clear cell carcinoma respond to IL2 and others don't. It's also not understood why patients have spontaneous remission with metastatic kidney cancer. It might be pie in the sky but it would be nice to think that this herve derived element potentially could be an explanation for why IL2 works in some patients with kidney cancer.
GALLIN: Thank you very much. It was a great example for why you were selected for the great teacher.
[applause]
ANNOUNCER: You've been listening to NIH Clinical Center Grand Rounds, recorded June 10, 2009. Our special presentation today, the sixth annual John Laws Decker Memorial Lecture, featuring the 2008 Distinguished Clinical Teacher Award winner, Dr. Richard Childs -- chief of the Transplantation Immunotherapy Hematology Branch at the NIH Clinical Center. His topic, Transplanted Allogenic T-cells Identify a Viral Corpse Resurrected in Renal Cell Carcinoma.
You can see a closed-captioned videocast of this lecture by logging onto http://videocast.nih.gov -- click the "Past Events" link -- or by clicking the "View Videocast" link on the podcast homepage at www.cc.nih.gov/podcast. The NIH CLINICAL CENTER GRAND ROUNDS podcast is a presentation of the NIH Clinical Center, Office of Communications, Patient Recruitment and Public Liaison. For more information about clinical research going on every day at the NIH Clinical Center, log on to http://clinicalcenter.nih.gov. From America's Clinical Research Hospital, this has been NIH CLINICAL CENTER GRAND ROUNDS. In Bethesda, Maryland, I'm Bill Schmalfeldt at the National Institutes of Health, an agency of the United States Department of Health and Human Services.
