cc >> Welcome to Wednesday Nite at the Lab, everyone. My name is Tom Zinnen. I work here at the University of Wisconsin Madison Biotechnology Center. I also work for the University Wisconsin Extension Cooperative Extension. And on behalf of the Wisconsin Alumni Association, welcome to Wednesday Nite at the Lab. We do this every Wednesday night, 50 times a year. Tonight I'm very pleased to be able to introduce Dr. Paul Sondel. He obtained his bachelor's and PhD degree here at the University of Wisconsin Madison and his MD at Harvard. He joined the UW faculty in 1980 as a member of the Departments of Pediatrics, Human Oncology, and Genetics. And he became the leader of the UW Comprehensive Cancer Centers program in immunology and immunotherapy in 1990 when he also became head of the Division of Pediatric Oncology. 

His research has emphasized the translation of laboratory immunology into clinical progress. Specifically his research team is pursuing ways that a patient's own immune system can be effectively directed against their own cancer. This has involved studies in vitro, studies in mice, and then first in human studies of new experimental agents and larger national studies of clinical efficacy. A recent national study of this approach in neuroblastoma, an aggressive childhood cancer, has recently shown that this approach is improving the cure rate for this disease. On this splendid night here in September, please join me in welcoming Dr. Paul Sondel to Wednesday Nite at the Lab. (applause)  >> Well, thank you very much for being here, and thank you, Tom, for that introduction. And thanks for that incredible demonstration of how you're able to work that technological device. Not only can he make it work but he can read it at arm's length. I wish I could see those small letters. So I'm going to speak to you tonight about the interaction of the immune system and cancer, and as I can tell from the schedule of upcoming Wednesday Nite at the Lab talks, this will get you ready for some other fascinating talks coming up in a month or so regarding other aspects of the immune system. 

So tonight we discuss immunology, we discuss cancer, and certainly the word cancer brings to mind, for all of us, not only the concept of cancer as a problem for us to study in the laboratory, but clearly a problem that we all face in real life with one in two men in the United States and one in three women in the United States, roughly, being diagnosed with cancer at some time in their lifetime. And with cancer over this past decade exceeding heart disease as the leading cause of death in the United States. We've got to do better. And even though we've made quite a bit of progress, there is so much room for improvement. So tonight we're going to investigate approaches being taken to try and get the immune system better directed against cancer. So I'm a believer in cancer research, and I'm going to be speaking with you tonight about cancer research. About two-thirds of my time is spent in the research of cancer in the laboratory and in clinical trials. Roughly one-third of my time is spent in the actual clinical work involved in taking care of patients with cancer. My particular area of interest is in childhood cancer. Clearly, those two words should not go together in the same sentence, children and cancer. 

It's a difficult concept. When people meet me at a dinner party or on the street and they ask what I do and I say I focus on taking care of children with cancer, that's often a conversation stopper. People often ask, how can you do that? And the way I can do that is to let people know about what's shown on this slide here. I like this slide for a number of reasons. The first is it starts there in the year I was born. And it was at that time that the first chemotherapy was being tested to try and have an impact on cancer. And that work was being done in the area of childhood cancer, work led by Sidney Farber in Boston that began in 1949. And at that time, virtually every child with cancer was dying. Certainly with the most common cancers, such as leukemia, there was really no effective treatment and patients were dying within months. Now, you can see 60 years later, we have a cure rate approaching 80%. 

A lot of people are surprised to hear that, but these are real numbers. So for children that we diagnose this week with cancer, as much as it is a potentially fatal disease that is going to stress these children and their families in ways they couldn't have imagined a few days before, we can hold hope to them with this knowledge that we've got a likelihood of providing cure for eight out of ten children, and because we've been doing better every year, even for children diagnosed this week, we can say we're hopeful that the research we're doing this month might actually provide better results for them than what we got published up until now. So what about the immune system? You're going to hear more about it in a few weeks. Some of you have heard a fair amount about the immune system. But in more simple terms, our immune system really is the system of our body that protects us from foreign invaders. And the foreign invaders that our immune systems were really evolutionarily designed to fight are foreign pathogens, are germs. Germs like bacteria and fungi and viruses. And it's the white blood cells of our body that really make up the immune system, and there are a variety of different kinds of white blood cells. And this is a graphic picture of what some of these different cells look like, and I won't name all of them. But these different cells are involved in different aspects of protecting us from these germs. 

The immune system works in complex ways and there are literally millions of white blood cells in every drop of blood. And those white blood cells talk to one another and work together as a team in order to protect us. Now, the major accomplishment that some would say in medical research in the last century was the advent of vaccines to protect us from certain formerly deadly viruses, such as the Polio vaccine, established by Jonas Salk and others. That works through the immune system by exposing the body to certain molecules that might be part of that virus, your body makes an immune response against that virus and your body can remember those molecules that it was exposed to so that on any future exposure of that same virus, your immune system will remember what it saw initially and destroy it. So there's really only two systems in the body that have that kind of memory, our brains and our immune system. And the depth of memory and the depth of information that our immune system keeps within it is fairly comparable to that which our brain keeps within it. So the immune system, for the most part, is a very protective component of our body. At times the immune system can go out of control, and it can start attacking our normal tissues. That's a process called autoimmunity. Many of you know of this as arthritis or a disease called lupus and other various autoimmune disorders. 

So this is a slightly more complex picture of how the immune system works. It is a gross, gross oversimplification. The purpose of showing this is to help emphasize how many different kinds of immune cells there are, how many different molecules they need to make in order to talk to one another to work as a team to get an organized immune response that protects us from whatever it is we're exposed to, remembers what it saw and can make a very potent effective response against that same germ or pathogen in the future. So, specific recognition, it's the hallmark of the immune system. What it sees it remembers so it can do a better job against it in the future. How many of you recognize this picture? Okay. Now, how is it that we recognize this? We recognize this by a couple different ways. There are unique features that we see on this picture. Certain things that all of us are trained to look at when looking at a human face. It's been said that humans are best at recognizing differences in facial features of other humans, that we're programmed to be able to do so. 

So we remember seeing this face. Most of us never met this individual in person. But we've seen this picture or pictures like it in other contexts and can recall using various signals that allow us to remember this and to use that memory. Well, in different ways it's the same kind of process that our immune system has to use when it needs to remember that it's been immunized to Polio and if it ever sees that virus, it will recognize the proteins from that Polio virus and attack a live virus and kill it before it can cause problems. So not much was known about the immune system a little over a hundred years ago. And back at the turn of the last century, a very innovative researcher named Paul Ehrlich came up with this theory of how the immune system works. His pictures were based on microscopy work that he was doing using recent inventions and was able to come up with this theory. He could not see all of this under the microscope. So this was entirely fictional in his mind, but it was a program he thought might explain how the immune system works. So he supposed that when the immune system sees a foreign germ, a virus, that that virus would have some foreign molecules on it that he called an antigen, and he drew it in this picture like this, and that when the appropriate immune cell in the body saw that foreign molecule, it would have on it various surface molecules that might or might not be able to bind to that antigen. 

And in this particular picture, this particular surface molecule, excuse me, this particular surface molecule would be able to fit like a lock and key with that antigen and provide specific recognition. And when that would happen, it would stimulate this immune cell to start making more of that specific surface molecule that could bind to that antigen and eventually this cell would start making lots of those. And that cell was making lots of those molecules that he called an antibody because they were fighting against whatever it is that this foreign antigen was on. So he really did this as a thought experiment because there was no technology for him to be able to understand what he was hypothesizing, but he was actually pretty close. And a hundred years later, through a lot of molecular biology and genetic experimentation, we know exactly what an antibody looks like. This is a construct of a picture of what an antibody looks like. Each of these circles, little tinny circles on it are different atoms. So an antibody is a fairly large molecule. It would take about a thousand of them put end to end to go from one side of a cell to another side of a cell. So they're pretty big for molecules, but they're still a lot smaller than the cells of the immune system. 

And they don't come in these colors. The colors are shown just for us to be able to tell what part of the molecule does what. And what's shown the yellow part at the end, that's the part of the molecule that's important in binding to the antigen. What's shown down here in the blue end, that's the part that lets the body's cells know they've got to attack the virus that's been bound to this antibody. So for many years it had been thought that the antibodies were the main part of the immune system. But roughly 1960 or so it started becoming apparent that this was only part of the story, that separately there were a variety of cells in the immune system that could stick to infected tissues and directly kill them. Some of the work that was done in this was done by my former mentor Fritz Bach. He was on the faculty here at UW. He joined the faculty in 1965, and I came to the UW as an undergraduate in 1968 and had the great privilege of starting to work in his laboratory in 1969 doing the equivalent of washing test tubes. But it was a terrific way as an undergraduate to learning what research was all about, and I became very interested in the research and started doing work with Fritz Bach and his students and ended up deciding that I wanted to stick with it, and this is where I got my PhD. 

So Fritz was very interested in the cells of the immune system and we know now that the cells that he was interested in working with are called T cells or natural killer cells. And each of these cells has on them receptors that let them recognize things that are interesting to the immune system, particularly things on cells that are infected or behaving in an abnormal way that the immune system should recognize as foreign and then kill. And so when either of these cells see those interesting things on the cell and say this is a cell we want to kill, the cell sticks to it and releases molecules directly at this cell it wants to kill. Those molecules are called perforin, and they punch holes in the cell and cause it to die. So Fritz developed these tests, and I apologize that this is a slide that's hard to see because it's faded, but I like showing this because this is actually one of Fritz's original slides from back in the 1960s, back before we had PowerPoint presentations and all talks were given back with a carousel projector. So he had a two-by-two slide of these tests, and he used it in virtually all of his lectures. And he had a couple copies of it, and he gave me one in the early 1970s. I probably used it several hundred times, and each time it faded a little bit more. When I switched to PowerPoint, I had it scanned and put into my computer. But what Fritz did was he determined that if you took white blood cells, cells from the immune system, from two separate people and put them together into a test tube, if those two people were identical twins, the cells would just sit there and not do anything. 

If the cells were from two unrelated people, they would react against each other and try and kill each other off. And he could measure this in a number of ways in the laboratory using radioactive chemicals to measure whether the cells were activated and using this assay down here to see if they were actually destroying one another. Using these assays, he was able to determine which genes are important in determining whether one person's immune system would reject another person's tissues. This was really instrumental in being able to do successful kidney transplants, that work was going on here at UW, and it was also critical in being able to pick who might be an effective donor for someone who needs a bone marrow transplant because they have a disease where their bone marrow isn't working right. So using that test, Fritz Bach and his team in 1968 published the first paper showing that effective bone marrow transplants could be done in people and would be curative. And this work came from the United States, here in Madison, out of this department of genetics. It was done at the same time as a comparable transplant was done using Fritz Bach's technique at the University of Minnesota. So this was in December of 1968, and it was exactly 12 months later that I joined Fritz's lab and started studying eventually bone marrow transplant immunology. Now, bone marrow transplants were done initially for children that didn't have an effective immune system because the bone marrow is where the immune system is formed. And if a child is born without an effective bone marrow, it meant they wouldn't have an immune system, they couldn't fight infection, and so the initial transplants were done for children that needed a bone marrow transplant to give them a healthy immune system. 

However, at least in mice, it looked like you might be able to use a bone marrow transplant to cure another important disease, namely, cancer of the bone marrow. This is what healthy bone marrow looks like under the microscope. Lots of different cells of sizes and shapes and colors that are all differentiating to become the various components of the immune system and the blood system, white blood cells, platelets, red blood cells. This is cancer of the bone marrow, better known as leukemia. All of these cells, at least to a hematologist, look ugly. They all look the same. They've taken over the bone marrow and we need to get rid of them. And at least in mice a bone marrow transplant was one way to get rid of the leukemia. And so after Fritz Bach did the first successful transplant in 1968, some other bone marrow transplant investigators started doing bone marrow transplants in an experimental way to try and treat leukemia. And it took about ten years to figure out how to do it so that it was making an impact for some people with leukemia. 

And, thus, it was in roughly 1978 that the number of transplants being done worldwide increased from virtually only a handful to many more per year. And it was at this point that effective bone marrow transplants were shown to make a difference for leukemia, and after that the number of transplants done around the world continued to increase such that now well over hundreds of thousands of bone marrow transplants have been done worldwide for leukemia and at least for certain kinds of leukemia it's the treatment of choice. Fortunately for most children with leukemia, we're now able to cure the leukemia without having to use a bone marrow transplant. We still need to use very strong chemotherapy, but we use the bone marrow transplant only for those children where the standard chemotherapy is not effective. So one of the questions is, how does a bone marrow transplant work to help fight leukemia? And this is some work that I had a chance to do in collaboration with the International Bone Marrow Transplant Registry. And the bottom line of this slide is that those individuals that got a bone marrow transplant for their leukemia and had an immune reaction where the immune system that was infused into them as part of the transplant started attacking their normal tissues but didn't cause severe damage to their normal tissues, a process called graft versus host reaction. Those people had a much smaller chance of developing leukemia again. While those patients that got transplanted from a twin or had no graft versus host reaction had a much higher chance of their leukemia coming back. What this says is that there was some immune reaction going on in these transplant patients, and it was likely the immune reaction that was fighting the leukemia. The question that our laboratory and several other laboratories began asking based on that and other results, was might this immune reaction that we could see in a transplant from one person to another, might we be able to understand it and control it and use it prospectively to try and help a patient's own immune system fight their own cancer without having to do the transplant? So let's take a moment to look at what would need to be going on in an immune reaction against a cancer. 

So cancer, of course, is made up of cells. Our own cells, not cells from some foreign germ that's gotten into us but our own cells behaving in an abnormal way, usually from the accumulation from a few different genetic mutations in those cells. But those cells start to grow, and one cancer cell becomes two and two becomes four and eventually after 40 cell divisions, you're up to a trillion cancer cells, that's a million times a million. A trillion cancer cells in a person weighs about a kilogram, about two pounds. So it's at that point that you've got enough cancer in you that something's not working right. And that patient goes to the physician because there's some symptoms that are identified. So that mass of cancer cells sits in blood vessels and sits in other cells called stroma, fibroblasts, and all of those could potentially be recognized by cells of the immune system. One population of cells I mentioned before are T cells. Another population of cells are cells that we call cells of the innate immune system. Some cells called natural killer cells, I mentioned. 

Some other white blood cells called polymorphonuclear cells, sometimes called neutrophils or macrophages. In addition, there can be antibodies that might be made against the cancer that might recognize cancer cells better than those antibodies stick to normal cells. And all of these are being used in various clinical trials in different ways to try and have an impact against cancer. So our laboratory had previously been studying the T cell side of these lymphocytes, white blood cells that could kill abnormal tissues. And we started studying, in the laboratory, a molecule called interleukin-2, IL-2. IL-2 is a growth factor that makes both of these cells, both the T cells and natural killer cells, more active. But it was interesting that the natural killer cells seemed to respond much better to IL-2 than T cells when you do it in the test tube. This is a picture of white blood cells that are natural killer cells, they're the ones that are stained white here, that have been activated with IL-2 in the test tube, and after a couple days these cells become very active and sticky and they stick pretty well to cancer cells, and at least some of the time they'll kill the cancer cell, and that's what's shown in yellow, the white cell sticking to the cancer cell. So an innovative researcher at the National Cancer Institute named Steve Rosenberg started giving IL-2 to mice to activate those same cells and was able to show that at least some of the mice getting IL-2 and nothing else had their cancer progress, or grow, less quickly than the mice that weren't getting IL-2. So you can see here these mice on the left did not get IL-2 and they all died. 

This is a survival curve. While these mice that got IL-2, some of them died and some didn't. Based on that, the National Cancer Institute and a few other places started testing IL-2. Our research team here at UW was the first team outside of the National Cancer Institute to start testing IL-2 in patients. We started giving IL-2 to patients with certain kinds of cancer that were clearly not responding to the best of standard treatment at the time. And we took white blood cells from these patients to see if we were turning on those natural killer cells in the patients just like Rosenberg had shown was happening in mice. So we took blood and we tested them in a killing assay to see if their function would enable them to kill cancer in the test tube and the answer was quite clearly yes. This is the amount of killing they gave before the patients got IL-2, and this is the amount they gave after getting IL-2. So we clearly could turn the natural killer cells on. The more important question is were we doing anything beneficial for patients? And the answer is yes, but. 

The yes was occasional patients with certain cancer, particularly kidney cancer that had spread to the lungs or melanoma could be seen to have metastatic cancer. So what you're seeing here is an x-ray of woman that had metastatic kidney cancer, that means the cancer had spread to her lungs. And all of these white spots in the lung are places where the cancer of the kidney had spread to the lungs. And this is the same woman after she got IL-2, and most of those spots had gone away. This was really very important news because it was the first time that anybody had shown that one could take an immune-based treatment and get a patient's own immune cells to attack their own cancer in a way that could have a beneficial effect for the patient. That's the good news. The "but" part is that this happened only in about one out of 20 of the patients. For those patients that it happened in, many of those patients had the cancer shrink and then come back, but for every patient that got the IL-2, in order to give enough IL-2 to do this kind of anticancer effect in some of the patients, all of the patients were having side effects from the treatment. They were having fevers, they were having rashes. It was like having influenza but in a very severe form because how active their immune system was. 

So we wondered might there be some better way to use IL-2 to get a better effect. So we asked if we could make these natural killer cells smarter in some way so that they could stick better to the cancer cell and kill it more effectively if we were to activate them with IL-2. And in order to do that, we started working together with a researcher who was working out of the Scripps Clinic in La Jolla, California, an investigator named Ralph Reisfeld who had made an antibody by immunizing mice against human cancer and then taking those immune cells from the mice and fusing them into a hybridoma using a technique that Dr. --, who is here tonight, actually studied here at UW in his laboratory back in the '60s, but it wasn't until the '70s that the technique of doing this with immune antibody forming cells came about. The bottom line is these antibodies can be made and grown in a bioreactor, and through genetic engineering it's been possible to get large quantities of these antibodies to use for clinical purposes. Several of these antibodies are now proved for treatment of a variety of different diseases. So this is back in the late 1980s. And we wanted to use this particular antibody that Dr. Reisfeld had made because it could recognize certain tumors, particularly, it recognized melanoma, a skin cancer that most people have heard of, and neuroblastoma, a rare childhood cancer that most people have not heard of, but childhood cancer researchers are particularly interested in it because our progress in the treatment of this disease has been far too slow. This is a picture of neuroblastoma. It's the most common solid tumor of children that can occur outside of the brain. It's a cancer of the nerve cells normally growing inside the abdomen. 

And most children that are diagnosed with this disease are diagnosed with a large tumor in the abdomen. And some children, usually infants, can have a very large tumor that can be taken care of surgically and, with a relatively small amount of chemotherapy, be cured. But roughly half of the children that are diagnosed with this disease have a large abdominal tumor like this but also have had this cancer spread to multiple other sites. At the time the child's diagnosed, the cancer has spread to most of the bones in the body, to many of the lymph nodes and what you see here for this unfortunate child, he's got swollen lymph nodes that are full of this cancer. In addition, the bones themselves are swollen and that's why he has these marks around his eyes because of the swelling of the bones and the bruising. Unfortunately, for those children with high risk neuroblastoma, like that second child that I showed, their prognosis, their chance for cure is really terrible. For the chance for cure in 2009 to be this low only on the order of 25% or 30% of these children are being cured, we clearly need to do better. And so that's where we wanted to focus in our research with Ralph Reisfeld. So we wanted to take his antibody and use it in a way to help these natural killer cells kill the tumor. And we thought that it would make sense for this antibody that binds to the tumor but doesn't do much more, this antibody could be recognized by these natural killer cells because they have a receptor that lets them recognize the antibody. 

And these receptors, once this natural killer cell sees the antibody, would let them stick to the tumor and kill it. So Dr. Jackie Hank who works in my laboratory, I've been fortunate to have her working together with us for the last 30 years, tested this in our laboratory back in the late 1980s. We took Ralph Reisfeld's antibody, we took neuroblastoma cells, and we took white blood cells from healthy people, we mixed them together in the test tube using that same assay that Fritz Bach had developed to figure out how transplantation genes are important, and we asked whether the white blood cells would kill the neuroblastoma. And what's shown here when we take blood from a healthy person is that their white blood cells kill the neuroblastoma a little bit, but if we add both the IL-2 and the antibody, we did much better. Then we took blood from patients with cancer, and we didn't do as well. From this patient or this patient we did the same experiment, testing their white blood cells against the cancer with the antibody in IL-2, and we didn't get much of a response. This is in part proof that cancer by itself suppresses the immune system. It's why so many patients with cancer have difficulty fighting infection, and it explains why immunotherapy is hard to do in cancer patients. But these patients happen to be referred to our team to get IL-2. Like the patient I had shown previously. 

And when these patients got their IL-2 and then we took blood cells from them after giving them several weeks of IL-2, now we can take their white blood cells and show their natural killer cells could cause this great level of killing as long as we had both IL-2 and antibody there together. So based on that test tube work and based on comparable work being done in mice, we hypothesize that we might be able to make that same thing happen in patients if we gave them several weeks of IL-2 in a row to turn on their natural killer cells, and also gave them this monoclonal antibody at the same time for several days in a row. So we started doing the first in human worldwide studies of this using the mouse form of this antibody, then through genetic engineering, this antibody was improved by our colleague, Ralph Reisfeld, to be an antibody that had the mouse part that recognizes the human cancer. That's what's shown in blue at the top on the right, but it was linked to the human part of the antibody so it could stick better to the human white blood cells and cause better killing. So we did clinical trials here at UW in our cancer center for patients with melanoma. This was done together with Mark Albertini, my colleague who's the leader of our melanoma clinic, and separately this was done for children with neuroblastoma through an national organization called the Children's Oncology Group. I was able to lead those studies through this Children's Oncology Group. It's a consortium of over 250 hospitals around North America that together take care of more than 90% of all children with cancer in the United States and Canada. These initial studies showed that we could give these drugs safely, that we caused activation of the immune system, we could understand how they seemed to be working to activate the immune system, but the "but" part of this is most of these patients with cancer were not showing any improvement. We needed to go back to the drawing board and ask why. 

So in Mark Albertini's lab, we took some human cancer, some melanoma in this case, put it inside the abdomen of a mouse and then treated the mouse with that same antibody, and a day later took the tumor out and looked at it under the microscope. And we stained it in a way that wherever that antibody we gave the mouse stuck to the cancer would stain black. And wherever the antibody did not go would stain blue. And what you see here under the microscope is there's large areas of this tumor that are staining black. Around the edges and around the inside near blood vessels. But there's large areas of this tumor that aren't stained black, they're stained blue. And it means the antibody is not getting in. Now for a mouse, this is a large tumor. This tumor was three millimeters in diameter. That's a pretty small tumor for a patient. 

That tumor probably has about a hundred million cancer cells in it. But it's too big for this antibody to penetrate. So based on this result, we thought the best way to use this antibody and IL-2 combination would be for patients that have less cancer than this in them. That would be a patient that's in remission, a patient that has already gotten their initial treatment such that we've gotten rid of all the cancer we can see. But statistics show us that the cancer is likely to come back in that patient, that we think would be the best time to use it. So how would we precede with this? I've talked about how the natural killer cells are activated with IL-2, a growth factor that activates those cells to bind to the antibody-coated tumor. There's another growth factor called GM-CSF. It stands for granulocyte-macrophage colony stimulating factor. It's a growth factor that activates these other white blood cells to also stick to cancer cells that have antibody on them and kill them. So we thought we'd try to get all of our ducks in a row to have everything stacked in our favor. 

We came up with a regimen that would allow us to use both of these growth factors, both IL-2 and GM-CSF in the same patient to try and maximize the number of white blood cells that could recognize tumor cells coated with antibody. So we came up with this regimen shown here. This is for children with neuroblastoma that have a very bad prognosis. Those high risk children first get six months of intense chemotherapy. After that they all get this ABMT, which stands for autologous bone marrow transplant. In essence, it's a way of giving them lots of chemotherapy and protecting their own bone marrow. After going through all of that, the whole process takes about nine months, the chances of that child still dying of this neuroblastoma are between 35% and 40%. Excuse me, let me say that differently. The chances of the child surviving are between 35% and 40%. In other words, close to 60% of these children are dying of the neuroblastoma even though they were in remission from all this therapy. 

So that's why we came up with this regimen where we gave them this antibody that Ralph Reisfeld provided, the Ch14.18 is the name of it, together with GM-CSF or IL-2 in alternating months. We did this initial trial in roughly 30 children. It looked like the results were better than what we expected. At two years it looked like 75% of these children were still alive. But that was early and some of them were alive but still had cancer. But it looked better than our previous results. But in order to really convince our clinical colleagues, the true test of whether something is making a difference is to do what's called a randomized clinical trial where you treat patients with the treatment that up until now everybody thinks is best and compare it to what you think might or might not be better. So back in 1998 we had a meeting at the O'Hare Airport with doctors that were involved in developing this concept and came up with a strategy that took us almost four years to put into place because we needed to convince all of the other doctors around the US that are treating children with neuroblastoma that even though our previous trials had not shown really dramatic results, we thought if we treated these children when they were in remission we'd be able to do better. So we started this trial then. All children got their intensive chemotherapy and then their bone marrow transplant, and then after their transplant, at this point where it says randomize, that's when these children would be randomized to get either the standard best treatment, which at the time is a differentiating agent called cis-retinoic acid,  a vitamin A derivative, versus that same vitamin A, plus the immunotherapy I talked about, the antibody plus GM-CSF plus IL-2. 

We started the study roughly at the end of 2002. We thought it was going to take us almost ten years in order to enroll enough patients with this rare cancer to have the statistical power to see if the immunotherapy was going to make a difference. We got a phone call in February of 2009 from the statisticians who were the only people that are allowed to actually look at the data and to see what's happening in the study because the study has to be done in a blind way so that the doctors that are looking at the data don't know what's what until we actually see a difference. And the statistician said we had to stop the study, we couldn't keep going anymore and they couldn't tell us why until we signed an confidentiality agreement to keep it secret until we could decide what to do with the information. And here's what they showed us. Obviously that secret didn't last very long because this was very important news. What they showed us was that the group of children that was getting the immunotherapy in blue was doing far better than the group of children that was not. So if you look at the two-year time point after treatment, the children that were getting the standard therapy, only 46% of those children were still alive without any cancer. While the group that got the immunotherapy, 66% of them were alive without cancer. And this P value at the lower left is what tells researchers whether we could have this difference by chance alone or whether it's real. 

So this says there's only about a 1% chance that this difference we're seeing is spurious, that it's by chance alone. There's a 99% chance that this is a real difference that if we started this all over again and did the same experiment we'd get the same result. So we're not going to start this all over and do the same experiment. As soon as we learned this result, that day, we drafted a letter to send to all the other doctors around the rest of the country. It went out about two weeks later letting everybody know that this blue treatment was now what we wanted all children to get. This red treatment was now a thing of the past, we weren't going to use that anymore. And so, thus, on that day the treatment for this disease improved in its cure rate by 20%. So that's not a breakthrough, but it's bigger than a small step. 66% at two years looks like we've got a plateau at this curve at about 60%. It looks like we're going to end up at a cure rate of about 60% of this disease. 

That's means 40% of children are still dying. We've got to do better. But we've made an improvement and so all children now around the US and North America and soon in Europe and going to be getting this form of immunotherapy. So other people had published some work with this same antibody but without the IL-2 and without the GM-CSF and they didn't see any improvement. It doesn't prove that the improvement we saw was because we used the two growth factors, but it sure suggests that that's what's going on. We think this might be important, not only for neuroblastoma but it's got a chance of making a difference from other cancers. Cancers like breast cancer, like colon cancer, like head and neck cancer, and like lymphoma. The reason I mention those is that there are other antibodies that are already being used with clinical benefit in those diseases. But in those diseases those antibodies are being used by themselves. There's not been a study in those other diseases using the antibodies that react to those cancers where those patients get the antibody together with IL-2 and GM-CSF at a time when those patients are in remission. 

So our team of doctors that are studying a rare childhood cancer are sharing our data with doctors that are studying those more common cancers in adults and seeing if we can work together to try and have them start testing this concept in those patients. Time will tell whether it gets tested and whether it provides any benefit. So I want to finish with the next several slides talking about where we go from here. So this was a good step forward. But we're still left, even in this disease neuroblastoma, with only 60% of these high risk children being cured. 40% of them are still dying of their cancer. Is there something more or better we could do? So collaboratively we're working with a research team that has taken that antibody that we've used up until now and genetically engineered it to try and make it more powerful. I don't have the time to go through how that genetic engineering takes place. I just can show you what it is we've ended up with that we're very excited about. This is a picture, schematically, of what this new genetically engineered antibody does. 

So what you see in this picture up in the left is part of the cancer cell. What you see here is the antibody shown in red. It's now an entirely human antibody except for these tiny strips that are left from the mouse part of the antibody. That's the part that lets this antibody recognize what's on the tumor cell as making it a tumor cell and not a normal cell. That's how this antibody can know to bind to cancer cells and not to normal cells. What's interesting about this, though, is what's going on at the other end. What's down here at the end of this antibody that normally binds to white blood cells is a molecule of IL-2. So I mentioned before how we give IL-2 and the antibody together and they help but they're separate molecules. Now we've got a molecule of IL-2 attached to each part of this antibody. They're fused together in what's called a fusion protein. 

It means wherever the antibody goes, the IL-2 has to go. And they're some reasons in the laboratory we're studying as to why this should work better than the antibody and IL-2 as separate molecules, and here's some proof that it works better in mice. If you give neuroblastoma to mice by injecting the mouse with neuroblastoma on day zero and then you sacrifice that mouse one month later on day 28, all of those mice have neuroblastoma spread throughout their bodies. You can look at their liver and count the number of neuroblastoma nodules on the liver, and what's shown here on the left each of these bars shows the number of melanoma spots or metastases in the liver of a different mouse. And this is on the left shows the number of metastases that you get if you don't treat the mouse with anything. What's shown in the middle is the improvement you get if you treat the mouse with the antibody and the IL-2 as separate molecules just like we're doing clinically in the large clinical trial. What's shown on the right is what you get if you use the same amount of antibody and the same amount of IL-2 but linked together as a fusion protein. It's a dramatic difference. We gave that data to the Food and Drug Administration. We got permission from them to start the first in human, in the world, studies of this molecule here at UW in patients with melanoma and in children with neuroblastoma. 

We had to do the standard kind of approach starting with patients that have failed all of their other treatment. The problem with that last mouse experiment, though, is that those mice were getting their immunotherapy only one day after the cancer was put into them. Now, no mouse model is an exact replicate of what's happening in patients. However, starting treatment only one day after the animal gets cancer is very different than what we think is going on in a patient that has their cancer diagnosed and then needs to get lots of treatment to put them into remission. So we did this experiment in mice. We gave all these mice cancer. We gave each mouse five days of this immunotherapy and we sacrificed the mouse a month later and counted the number of spots in their liver. What's shown on the left-hand bar is the number of spots, over 200, in the mice that didn't get any treatment. What's shown here are the number of spots in mice that got five days of treatment but starting at different times. These mice got treated starting on day five. 

The next group on day seven. The next on day nine. And you can see the later the treatment starts, the less effective the treatment is. What we conclude from this is what we had said previously, namely this kind of immunotherapy is going to work best when you've got the smallest amount of cancer. So we've started this approach in children with neuroblastoma that has already come back. So these are children with high risk neuroblastoma that have already failed all of the best therapy. Or a better way of saying it is our therapy failed them. Their cancer has come back and their prognosis is pretty close to zero. Nobody has any therapy that they think has really any great hope of making a difference in children with neuroblastoma once it's come back. So that's where we started these experiments. 

And we had two separate groups of children with neuroblastoma that came back. One group are those children that had neuroblastoma come back in a big way, big enough for us to be able to see by standard x-rays. The other group were children that had neuroblastoma come back, but there were less of it. We needed a special kind of a nuclear scan to see it or we needed to look in their bone marrow under the microscope with a biopsy. So we did the trial giving this experimental agent to these two groups of patients, and as we predicted, none of the patients with large bulky neuroblastoma showed any difference. 13 patients got treated, none of them improved. But of 24 patients that had the neuroblastoma come back in the less bulky way, there was still a lot of it, these patients probably had a billion or more neuroblastoma cells in them, five of those patients went into complete response, which means we couldn't see any neuroblastoma no matter how we looked, and two of them showed very significant improvement. So when we asked the question whether this group, group two, is actually doing better than group one, when we include those seven patients that either had a complete response or improved, we've got a statistically significant difference showing that this kind of immunotherapy really is making a difference for patients that have less cancer in them. This is what we had hypothesized from the mouse studies, and it's very reassuring when what you do in the test tube or in the mouse ends up showing you what you actually get in the clinical trials. It gives you faith that your mouse research should continue to direct what you're doing. 

So what are we proposing to do next? I showed you in the first two-thirds of this talk how if we give this chimeric antibody from Ralph Reisfeld, this Ch14.18, plus IL-2, plus GM-CSF, how that's currently the best standard treatment available for neuroblastoma. I've now shown you that this molecule that links the antibody to, directly linked it to IL-2, how that is far more effective in mice than the antibody and IL-2 separate molecule.  And it's safe to give to patients and it looks like it's helping some patients with relapse neuroblastoma. So the next thing that we need to do is a randomized trial where children with neuroblastoma in the future get put into remission with their chemotherapy and their transplant, and then we give them either this regimen, the current standard regimen, or give them that same regimen where we substitute for the antibody and IL-2 this improved fusion protein and do a direct head-to-head comparison to see if the improved molecule really is getting us any further in our search to try and do better than 60% cure rate. So I'd like to end on that note. I've mentioned some of the research coming from our laboratory and some of the collaborative clinical research. I've had the good fortune to be part of this since 1980, but there are many people that I've worked with over these 30 years, many of them that are working with us still today are listed on this slide, and unfortunately I don't have time to mention all of them. But clearly a large team of clinicians and laboratory workers, immunologists, molecular biologists, all working here at UW. As well as collaborators around the country in this consortium of children's cancer centers, as well as our collaborators at various universities and at the National Cancer Institute, as well as very important support that we get from our National Cancer Institute to provide the grant funds to enable us to do this work. So I'll end practically where I began. 

I'm a believer in research. And I showed you the slide at the beginning showing the improvement in cancer survival of children with cancer, how back in 1950 the word cure wasn't used. Even in 1980 when I joined the faculty, even for those children that were in remission for a long period of time, we weren't sure we should tell their parents that those children are going to be cured of their cancer because we still weren't sure if it was just a matter of time before their cancer would come back and they would relapse. But now because of research, we're really curing eight out of ten children with cancer. So our pediatric oncology program here at UW has a patient list of roughly 1100 children with cancer that have been cured of their cancer. Every five years we invite them to come to a reunion. And I'm surprised that about one out of five of those kids and their families do want to show up. One of the reasons they might want to show up, it's hard to see, but in the very middle of this picture is the sponsor of this event, Cindy Crawford, a super model whose brother sadly died of cancer here at UW in 1975. She was only nine years old at the time. She thought she wanted to go to medical school but ended up picking a different career. 

But she's been an incredible benefactor to cancer research here at UW and a spokesperson for childhood cancer research nationally. She sponsors these reunions, and every five years these kids come back. And these are our former patients. So I will end there. [APPLAUSE]