Wisconsin Public Television University Place Wednesday Nite at the Lb  Treating Head and Neck Cancer Randall Kimple  Oct. 3, 2012 UP780  cc >> Welcome, everyone, to Wednesday Nite at the Lab. I'm Tom Zinnen. I work here at the UW Madison Biotechnology Center. I also work for UW Extension Cooperative Extension, and on behalf of those folks and our other sponsors, Wisconsin Public Television, the Wisconsin Alumni Association, and the Science Alliance here at UW Madison, thanks for coming to Wednesday Nite at the Lab. We do this every Wednesday night, 50 times a year. Tonight's topic is rather special and personal for me because my dad got sick in August of 2009 with a little thing that was in his throat. And I was like, wow, that's weird. I was talking to him on the phone, and he goes, well, I got something in there. He goes to the doc, and the doc says it will be all right, 95% chance it's benign. It wasn't 95% it was benign. 

And it was, we'll be able to take care of it. We think we know what it is. And then it wasn't that. Well, to make a long story short, my dad died in March of 2010 from, basically, a throat cancer. So tonight's topic has got my attention, and I'm particularly happy that Randy Kimple has agreed to give this talk because Randy's kids and my kids went to the same daycare, and this is how we get speakers for Wednesday Nite at the Lab. [LAUGHTER] Randy grew up in Holland, Michigan, and he attended Michigan State University. From there, he pursued graduate training at the University of North Carolina where he received both his MD and his PhD in Pharmacology. He completed his internship and residency in Radiation Oncology at the University of North Carolina Hospitals. He recently finished post-doctoral fellowship training at the University of Wisconsin and has now joined the faculty of the Department of Human Oncology as an assistant professor. His clinical practice consists of a general radiation oncology clinic, and his lab is interested in understanding the mechanisms underlying the altered response of human papillomavirus associated cancers to radiotherapy and to chemotherapy and to understand how these mechanisms can be used to improve treatment for patients. 

That's the part that I wish was a little earlier. So, please join me in welcoming Randy to Wednesday Nite at the Lab. [APPLAUSE] >> Thanks a lot, Tom. And thank you for being here on this evening when I know in about 50 minutes or so there's something on TV that probably many people will go home and watch. So I appreciate you coming out tonight, and those who are watching from a distance, I also appreciate you joining us. So, I wasn't actually sure what background Tom was going to give, and so I find at these types of events it's always helpful to give a little bit of a background on where a person came from to get to the point that they're at. So he talked about this. I grew up in Holland and I went to Michigan State and then to the University of North Carolina. At UNC, I worked with David Siderovski who, at the time, was a young professor in the pharmacology department, and we did a lot of biochemistry and structural biology and molecular biology. I had a really fabulous PhD experience there, and towards the end of my PhD I decided that I really wanted to be an oncologist, and, in fact, what I really wanted to do was to be a radiation oncologist. 

And so I ended up matching into the program at UNC where I spent the next five years. During that time, I did a fair bit of research studying signaling pathways that are involved in altering the response of cells to radiation. In particular, we were interested, at the time, in pancreas cancer and in breast cancer. And so as I neared the end of that process, I looked for opportunities and a place to further my training, and I came here to Wisconsin to the WIMR building, which is over by the hospital, and there I joined Paul Lambert and Paul Harari. Paul Lambert is a professor in the McArdle Labs here and is a papillomavirus virologist. Paul Harari is a radiation oncologist and has studied head and neck cancer for many years. And my goal was to take their two areas of expertise and be able to meld them together into an area of my own, and I think that I've been relatively successful in doing that in a short period of time. And so that sort of brings me to where we are today. And so what I want to tell you about tonight is a good bit of background on head and neck cancer, on how head and neck cancer has changed or how we now understand head and neck cancer has changed over the past 15 to 20 years, and how we think it's going to be changing in the next 20 years, and what we're doing in the lab, trying to understand how the different causes of head and neck cancer result in different outcomes to patients whose cancer is derived from these different ideologies that I'll talk about. And a little bit of the data and the avenues we're taking and really more of a focus we're using to try to answer these questions. 

And then I'll finish by telling you about a new model system we've developed that I think is really exciting and that takes advantage of very generous patients who are treated at the University of Wisconsin Hospitals and some of the interesting and exciting outcomes that we're getting out of those patients. But what really drove me here was data like this. And so what you see here, this is a survival curve where you look at time in years on the X axis and the number of patients who are alive after diagnosis on the Y axis, and unfortunately with cancer, that number goes down over time. And what we began to see from about 2006-2007 on, this is data from a paper published in 2011, was that if you had a human papillomavirus related head and neck cancer, your outcomes were remarkably better. The prognosis was remarkably different from patients whose head and neck cancer was not related to HPV. And this was true even if you took patients who had the same stage of cancer, the same advanced cancer, whether it was resectable or unresectable, if you matched patients for all the other factors that might underlie who lives and who doesn't live after a diagnosis of cancer, this remained true. And so I was driven to work with Paul Lambert and Paul Harari to try to understand what the fundamental difference is and how we should personalize treatment for these sorts of patients. So, I want to give you a fairly broad background to try to get everybody at the same point before I talk about sort of the specifics of our data, but what do we mean by head and neck cancer? So there's a couple of things that when I talk about head and neck cancer that I don't mean. I don't mean cancers that arise in the brain or in the neural tissues. I don't mean cancers that arise on the skin. 

Although, there may be some relationship between skin cancers and human papillomavirus. But what I really mean are cancers that arise on the lining of the mouth and the throat and inside the nose. And, in particular, oncologists divide head and neck cancers up into a variety of different areas. There's the nasal pharynx, and these cancers are much more common in China and some other eastern countries. And they're related to Epstein-Barr virus. And we will not talk any more about those today. Although, there's a number of groups on campus who are studying those types of cancers. There are cancers of the nasal cavity and the tongue and what's called the oral cavity, which is sort of the lining that if you put your finger into your mouth that you could touch very easily. The area that I'm most interested in are cancers that arise in the oral pharynx, and that includes the tonsils, the area around the tonsils in what's called the base of tongue. The base of tongue is the area that if the doctor were feeling around in your mouth and put his finger all the way down the back of your throat along the back of your tongue and you started to gag, that's the base of tongue back there. 

There are also cancers that arise in the larynx. That's also called the voice box, and those share some similarities and we'll talk a little bit about lumping larynx cancers and tongue cancers together. But, really, the area that I'm most interested in is these cancers that arise in the oral pharynx because that's the area where we've seen a major shift in the demographics over the past 10 to 15 years. So, there are somewhere around 650,000 head and neck cancers diagnosed each year worldwide. About 40,000 in the US which makes it the 6th most common cancer in the United States. That fluctuates a little bit year to year, but it sort of sits right around that level. So this is an important problem that we're trying to address. Most people, if you say throat cancer or mouth cancer and you ask people what it's caused by, these are probably the two things that come to mind: tobacco and alcohol. And for years and years, tobacco and alcohol consumption and abuse really did contribute to head and neck cancer and still do contribute to head and neck cancer. So in this graph, what we're showing in these little dotted lines is the per capita tobacco use in the United States. 

And if we had extended this back another 30 years, that would have risen over time, and the incidence of head and neck cancer would have followed that by about a 15-year delay. What you can see here, and the bars represent head and neck cancer diagnoses, is that in the late 1970s as anti-smoking campaigns begin to ramp up and people begin to realize the adverse effects of tobacco use, the rate of tobacco use went down. About 15 years later, some time in the mid-1980s, the rate of head and neck cancers began to go down, which is great. This is a wonderful success of the public health infrastructure in the United States. But, of course, the story is more complicated than that. So if you look overall, if you look at cancers arising in the oral pharynx, again this is the tonsils and the base of tongue, the incidence of those cancers over that same period of time has been pretty steady or maybe risen a little bit. That's in yellow here. And in gray are the cancers that are related to tobacco and alcohol or other sort of carcinogens. And in blue are cancers that we now know are related to human papillomavirus, or HPV. And this, as you can see, has really increased over the past 30 years, and it really wasn't until sometime around 2000 that this was really first identified. 

And so the studies, the numbers from back here are really retrospective studies where people go back into specimens that have been sitting on a shelf for 20 or 30 years and look at the incidence of HPV. So these numbers may not be entirely correct, but they're a pretty good approximation of what the rate was back then. It's estimated that within the next 10 years or so cancers related to human papillomavirus in the head and neck will be the most common type of cancer in all these different sub-sites of the head and neck that I showed. This is a major issue, or epidemic if you will, that's starting to face us. So what is the human papillomavirus? The papillomavirus is a DNA virus. It is very ubiquitous. Most people in this room probably have had a papillomavirus infection at one point in their life, and I'll tell you a little bit more about that in a few minutes. I don't want anyone to be scared that they're going to develop head and neck cancer because I certainly hope that that's not the case. And most infections of papillomavirus do not lead to cancer. It expresses a number of different proteins, and two of those proteins, E6 and E7, have particular importance in the oncogenesis. 

They also play important roles in the normal viral life cycle, and that's one of the things that the Lambert lab, who I worked with here for several years, has been studying is how this virus replicates in the normal viral life cycle and how that gets perturbed to cause cancer. Human papillomavirus has been pretty important, scientifically, over the last 30 to 40 years. So Harald zur Hausen actually won the Nobel Prize in medicine in 2008 for his discovery of papillomaviruses causing cervical cancer, which he first made back in the early 1980s. And so it's been, for a long time it's been known that papillomaviruses can cause cancer. For a much shorter time than that it's been really widely recognized that it causes head and neck cancer. There are now over a hundred different types of papillomavirus, and the details of this are not at all important, but, really, the important distinction is that they can be divided into low risk and high risk types of papillomaviruses. So when people, epidemiologists look at the incidence of papillomavirus infection in young adults, primarily, because that's a group that hasn't often been exposed to HPV, and this is a study that was just published a couple of weeks ago. In this study they actually did oral rinses. So they had people rinse their mouth with Scope and then spit that into a vial. And they looked to detect the papillomavirus DNA in that sample. 

They looked over time in these people. So they looked at patients who were, people who were negative for it, and then they looked at how many developed it over time. If they looked at what risk factors they could find that were associated with papillomavirus infection, what they found, and this is something that you may have seen in the news or seen reported in Time or Newsweek, is that marijuana use and sexual activities had a high at-odds ratio, which is how much more likely you were to have an HPV detected. So, in this case, low marijuana use, zero to three days per week, versus high, greater than 4 days per week or various open mouth kissing partners or sexual partners. So these sexual activities, certainly in young people, have a high relationship with papillomavirus infections or incidents. Now, most of these students or patients end up clearing the viral infection and never have any problem after that. This study didn't look at that, but there are reports that look at the incidence of clearance of the infection. This study actually didn't look particularly at the infection but just whether they could detect that DNA in the oral cavity. So that could just be that the virus is sitting there but hasn't yet infected anything. And so now we think that the causes of head and neck cancer have expanded, and it's not just tobacco and alcohol but also sexual activity has a relationship to or is a risk factor for the development of head and neck cancer. 

So, I said earlier that there's a lot of different types of papillomaviruses, and those can be divided into low risk types, which are the cause of very common and benign things like plantar warts and skin warts and other sort of diagnoses that are listed here but that are not cancerous. They may cause problems or be troublesome, but they're not cancer. They don't spread. The high risk papillomaviruses are the ones that are implicated in cancers. And it's now thought that essentially all cases of cervical cancer in woman are related to human papillomavirus. And this is what really has driven the development of HPV vaccines, and I'll have a slide in just a moment addressing that a little bit. Other anal-genital malignancies are also highly related to papillomavirus. And we now think that somewhere around 30% of head and neck cancers are related to human papillomavirus. And, interestingly, the anal-genital cancers are related primarily to HPV type 16. A number of other subtypes are also found in those cancers. 

Whereas in the head and neck area, the vast, vast majority of cases are related to HPV 16, and there are very few cases that are related to the other types of papillomavirus. And that's important in what we're studying and the models we're using to study these papillomaviruses. So, almost certainly at the end of this talk, someone would have asked about the HPV vaccine, which has been talked about in our political debates and talked about on the news, and I think that this graph or this image tries to show the potential effect of universal HPV vaccination. So, cervical cancers could see a tremendous decrease in the incidence of cervical cancers. So the dotted lines are the cases that would develop in the absence of any vaccination. And the reason there's still some cases in all of these is that the vaccines that are currently available only target a certain group of these papillomaviruses. So they don't target all of the papillomaviruses. Some of these cancers may be related to other subtypes that aren't targeted by the vaccine. So cancers of the oral pharynx in the head and neck region could have a significant impact if everybody were vaccinated, but this impact, as I showed earlier with the tobacco use, the change in behavior takes 15 to 20 years to actually see a change in the diagnosis. And so even if we vaccinated everybody today, it'll be 15, 20, maybe 25 years before we see a change in the incidence because the people who develop these cancers in the coming years were exposed to these viruses over the past 10 to 15 years. 

So, this is sort of an opportunity to put forth an interesting, what I think is an interesting factoid that cancers that are actually caused by an infection are actually remarkably high. And so if you just put into your mind a number that you think of, a percentage of cancers that may be caused by an infection. The actual answer is it's about 20% of cancers worldwide are associated with or caused by an infection. Human papillomavirus is responsible for the second most of those, about 5% of global cancers. This 20% incidence is certainly higher in less developed countries where access to healthcare and antibiotics to treat things like H pylori are less commonly available. The hepatitis viruses that many people in this country are now vaccinated with are a major cause of cancer worldwide, and if those vaccines were available, that number would certainly drop. So, what I want to turn now to is to give you an idea of how patients are treated for head and neck cancer today and how that's developed over time. And so I showed you this data earlier that if you have an HPV-positive cancer you do much, much better than if you have an HPV-negative cancer, on average. These data are derived from trials where patients were treated with the same treatment regardless of what their cancer was caused by. Primarily because many of these patients were treated before we ever recognized that human papillomavirus causes head and neck cancer. 

So there are several what we think of as landmark studies that help define the treatments for patients with head and neck cancer, and there are a subset of patients whose cancers are easily resectable. A surgeon can go in and cut out the cancer and cure the patient. But many of these patients present with a cancer that is either too advanced to be removed by surgery or where the surgery is so morbid that it's not the first option for the patient or the first recommendation of their physicians. These patients are treated quite commonly with a combination of radiation therapy and chemotherapy. So here's two different studies that have looked and compared the treatment of head and neck cancer with radiation alone versus radiation with the chemotherapeutic drug Cisplatin. And it's pretty clear that if you could choose if you fit into this trial, that you would have preferred to be treated with Cisplatin. Likewise, this study on the right shows radiation versus radiation plus Cetuximab which is a newer generation of therapies. It's a monoclonal antibody. It particularly targets a protein called the epidermal growth factor receptor, or EGFR. And EGFR is thought to be at pretty high levels in head and neck cancer. 

And so this has also shown a benefit to the combined therapy. And these studies, and several others that show, essentially, the same thing, have really sort of set the stage for what the standard of care is if you were diagnosed with a head and neck cancer in 2012. But these studies really have not given us an idea on how HPV status affects this. So, these studies were done in the era before we routinely tested for human papillomavirus. In the case of this study on the right, the tissues that were collected as part of that study have been used to look at other things and so are not available to look for HPV and to know whether the patients who have an HPV related cancer would preferentially benefit from Cetuximab treatment. So there are a couple of ongoing studies, both in this country and other countries, that are helping to better define how patients should be treated. There are studies that are looking at how to best integrate surgery into the management of head and neck cancer, and in particular, into the management of HPV-positive head and neck cancer. And then there is, what I think is a really outstanding study, randomizing patients with papillomavirus related head and neck cancers to radiation plus Cisplatin or radiation plus Cetuximab, directly comparing these two treatments. And these studies are ongoing, but it will probably be three to five years before we have any real results from either of these studies unless there is a tremendous difference in outcomes between these. I think it's unlikely the difference will be 30% or 40%. 

It's more like to be a 5% to 10% difference if one of these is a winning arm of that study. And so I think it's important that we continue to study and understand how cancers that are related to HPV respond to therapy and how we can optimize therapy for those particular patients. So what I've shown you so far is that head and neck cancer is caused by tobacco and alcohol and also by human papillomavirus, that there's better survival for patients with HPV-positive head and neck cancers but it's not clear why, and that it's unclear, at least at this time, how to personalize therapy for patients based on the cause of their cancer. So what I want to turn to is some of our work in trying to understand how HPV-positive cancers respond to therapy, and then we'll talk about the new model system that we've developed and how that will help or we hope will help advance this process. So I mentioned the two HPV proteins that are involved in oncogenesis that are called very simply E6 and E7. And E6 and E7 degrade two very important tumor suppressor proteins, P53 and RB, that help control the response to DNA damage. And so as cells generate DNA damage, these proteins are supposed to allow the cells to repair and not develop more and more mutations, where E6 and E7 really degrade that and perturb that normal response. This is an incredibly simplified diagram of that response. In reality, E6 and E7 interact with dozens of proteins within the cell and have multiple different effects, but I don't want to go into too much detail on that. What I do want to talk about is our work looking at the sensitivity of HPV-positive versus HPV-negative cells to various treatments, to chemotherapy or to radiation therapy. 

So here, this is what's called a clonogenic survival curve. And so we've plotted out increasing doses of radiation here on the X axis, and the number of cells or the fraction of cells that survive a given dose of radiation on the Y axis. You can see this is a logarithmic scale. So these are big changes as you go down the scale. And when we looked at eight different head and neck cancer cell lines, four that are HPV-positive and four that are HPV-negatives, the HPV-negatives in blue and the HPV-positives in red, what we saw was that there was a pretty good stratification where patients who have HPV-negative head and neck cancers or cells that are derived from HPV-negative head and neck cancers are less sensitive to radiation than cells that are derived from HPV-positive head and neck cancers. This sort of fit with some of the implications from some of these clinical studies that suggested that patients whose cancers were treated with radiation had better outcomes, but this was some of the first data that we're aware of that really looks at whether there's actually an inherent difference in the sensitivity to this therapy. So we also were interested, despite the fact that I'm a radiation oncologist, we're also interested in chemotherapy, and if chemotherapy is the better treatment, then that's great and we should pursue that. So we looked at sensitivity of these cells to Cisplatin or to Cetuximab, and what you can see is that, although I didn't identify the cell lines individually, there's a great overlap in the sensitivity to both drugs such that we can't say that one type of cell is more sensitive or less sensitive than the other to either of these drugs. And these are the two drugs that are most commonly used in the treatment of patients with head and neck cancer. So, what I've just shown you is that HPV-positive cancers are more sensitive to radiation than HPV-negative cells but that they show similar sensitivity to chemotherapy. 

But we're interested not just in seeing if they are but understanding why they are. And so that's a lot of what we've spent or we're spending time on and our efforts on now. So, one of the things that can affect sensitivity of a given cell to radiation is where they sit in the cell cycle. As cells divide, they have different phases as they replicate their DNA. Cells that are irradiated in different phases have different sensitivities to that radiation. So we thought, well, perhaps HPV-positive cells are sitting more often in a very sensitive phase of the cell cycle compared to HPV-negative cells. But in fact, what we saw when we looked at these cells is that they were in very similar distributions. So that probably was not the explanation for this. We saw that this is looking at the cells that are in the G2/M or mitotic phase of the cell cycle, so after they've replicated their DNA and before they've divided, and what we saw was that radiation induced or caused the HPV-positive cells to stop in that G2/M phase, suggesting that there's something within the cell induced by the radiation that's causing this particular change and it's very different from what we see in our HPV-negative cancers. We used western blots to look at proteins within the cell that also support the cells stopping in the G2/M phase. 

And we also see that HPV-positive cancers undergo more apoptosis following radiation, and apoptosis is a way of cells dying. A very organized way that cells can die. So we think that that may be involved in the increased sensitivity to radiation, the fact that these HPV-positive cancers are dying at a higher rate through this organized mechanism called apoptosis than the HPV-negative cancer cells. We have data, actually I just got data today that I haven't put in here, but it's suggesting that there's differences in how these cells are repairing DNA between HPV-positive and HPV-negative cells, and that fits with the fact that radiation is causing a significant difference in sensitivities in these cells. So this just sort of summarizes what I just talked about. So we are also using global gene expression analysis. So here we're using affymetrix chips to look at both HPV-positive and HPV-negative cells and to look at all of the RNA that's being encoded in those cells and looking at how those RNA profiles change over time. So we not only have our different cell lines, both positive and negative, but then we harvest the cells with no treatment and then at four and 24 and 48 hours after a dose of radiation so we can look at changes over time. What we see is there are a great number of genes that change following radiation. So this is in the HPV-positive cancers, and you can see the red are genes that are elevated and the green are genes that are decreased. 

What you can see is that as you go over time, there are a large number, each of these little lines represents a different gene, so there are a large number of genes that change over time. If we look and compare cancers or cells that are HPV-positive to those that are HPV-negative, we see significant difference in which genes are up-regulated and down-regulated, suggesting there's fundamental genetic or genome level differences in these cells that is perhaps critically involved in this response. So we've begun to validate these what are really more screening studies by looking at particular cells. So these are cells that are involved in the regulating the cell cycle that are all induced by the protein P53, one of those targets of the HPV oncogenes. And so we see that after radiation, these RNAs are all increased, presumably in response to P53, and we're looking at that as we speak, hopefully, whether if we can further decrease P53 in these cells, does that sort of change the sensitivity to radiation. So what I've shown you here is that using these sort of genetic approaches, we can look at or we see increases in P53 target genes following radiation, particularly in the HPV-positive cancers. We believe that's occurring because the papillomavirus, while its proteins degrade P53, it doesn't result in mutations in P53 that are incredibly common in other types of head and neck cancer. So the P53, we think, is still able to act normally, it's just kept at very low levels so that in response to the radiation induced stress, P53 is activated and preforms its normal functions to protect the cell, causing a rest and causing apoptosis. And so we're sort of pursuing strategies to try to better understand that and better define that response. What I want to turn to in the last 10 or so minutes is what I think is a really exciting model system that we've developed in the past few years for studying head and neck cancer. 

Most, in fact all, of the studies that I've shown you thus far from our lab have been performed on cell lines that are grown in petri dishes and have been around for sometimes decades, oftentimes many, many years, and so there's certainly a concern in the community that these cell lines don't necessarily represent the cancer that they originated from 15 years ago in Germany or 10 years ago in Pittsburgh. And so we've been trying to develop a new system to help us to give us better tools to ask questions and that may be more relevant to actually what's happening in the patients. And so what we've developed is a system that a number of other groups have developed in other types of cancer, in breast and pancreas cancer, and it's termed a tumor graft. We term it a primary tumor graft. So we take biopsies from patients who consent to donate their tissue. They're undergoing surgery as part of their cancer treatment or having a biopsy done as part of their initial workup, and we take those small chunks of tissue directly from the operating room in a sterile container and on a bucket of ice, and take them up into the lab. We chop them up into very tiny bits, and we inject them into immunocompromised mice. And this mouse may be hard to tell but growing right here, not obviously in the head and neck of the mouse, but under the skin of the mouse is a head and neck cancer. And we can also take those cells and grow them on a petri dish and generate very early passage cell lines, or what are termed cell strains, that we can do the same types of experiments that I showed you earlier with the cell lines, looking at sensitivity to chemotherapy here with a cell strain derived from patient 24. So, to date, we've been successful in about 75% of the cases, which was about 70% higher than my original expectation. 

We have over 20 of these patient cancers that we are passaging in mice and we're able to do experiments on. These cancers are not just derived from human papillomavirus-positive cancers but are derived from the tonsil and the mouth and the tongue and the base of tongue. So these two, base of tongue and tonsil, are the areas most likely to be related to human papillomavirus, but we trying to develop this system not just for HPV-positive but also for HPV-negative cancers because, certainly, advances are needed in that group of patients as well. Most of these are developing from primary cancers. Although, about a third are coming from patients who have a recurrence after some initial therapy, whether that be radiation and chemotherapy or radiation alone or surgery. And so we're getting a good diverse group of tumors that are growing out. About two-thirds of ours are HPV-negative. About one-third are HPV-positive. And we've also seen a really nice spread of differentiation where we see both well differentiated and moderately differentiated and poorly differentiated. And the reason I show you these last two slides is to say that we're not just, our technique and our approach is not just selecting the very worst cancers to study and that we're really getting a broad spectrum of cancers that we're able to studying using this system. 

We hope that makes it more broadly applicable and gives us better insights into what's actually happening in an average patient. So if there's any pathologists in the audience, this slide is for you, and it shows the patient's tumor from the pathology lab and then the tumor that grew out in the mouse. And what you can see is that, even if you're not a pathologist, you can see that there's a great variety in these and they all look very different. There's not just one sort of common type of cancer that grows, and what you really see is that the primary tumor and the cancer that grows in the mouse have a lot of similarities. There's a great stability between what we see in the patient and what grows out of the mouse. And in those tumors who we've been able to look over multiple passages, we see stability between multiple passages. So, from the first and second out to the fourth passage, these tumors retain the same characteristics. So there's something inherent in the cancer that's causing them to form a well differentiated tumor or a poorly differentiated tumor, and whatever that factor is, it's not left in the patient when the tumor is taken out of the patient. It's actually inherent within the tumor, and we think that there is potentially a lot of exciting possibilities from studying just that aspect of these tumors. So we use these tumor grafts or we can use these tumors grafts, we are using these tumor grafts, to look at therapeutic response. 

So how do these tumors that are months from being in a patient, how do they respond to various therapies? So here's the growth rate of two different tumors. On the left is one that grows very, very quickly. As you can see, the scale is only just over a month, and the tumor goes from very small to very large very, very quickly. This tumor, the scale is almost two-thirds of a year, about nine months, and so although the slope here looks similar, it's actually a much longer time period. It takes 30 to 40 days before the tumor is up at a similar size. If we treat these tumors with radiation, what we see is that their growth rate slows following radiation. This tumor still grows very, very quickly, but it does slow considerably. This tumor has a pretty good response to radiation. And if we look at the addition of radiation and Cisplatin, one of those studies that I showed you earlier or sort of mimicking one of those studies I showed you earlier, we see that in this cancer, in this particular cancer, we see a much better control with radiation and Cisplatin. Although, as soon as we finish treatment, that tumor grows about as fast as it ever did without any treatment. 

And here, in this cancer, we see that there's really no additive effect of radiation and Cisplatin. In fact, really it overlies the growth to radiation almost perfectly. If we look, on the other hand, at Cetuximab, the other drug that was used in that large randomized study in patients, we see that there's a small effect in this patient on the left to radiation and Cetuximab but a much better effect and much better control in the tumor on the right, suggesting that if you go back and forth or if you think of these treatments that were radiation and Cisplatin where the left had a good response and the right had very little response, and this treatment radiation and Cetuximab, that response is almost inverted. That would suggest that there's some factor in these cancers that is really, if we can figure out what it is, can help us to personalize therapy for these patients. And we can pick the best therapy for patient A or patient B, and hopefully, that's really the goal of all this work is to able to improve outcomes and treatment options for patients. So we currently have somewhere around 20 to 25 of these tumor grafts. We have an ultimate goal of about 50 head and neck cancer tumor grafts. We have a request or a grant application out in which we would preform what we've termed a co-clinical trial where we would mimic one of the ongoing clinical trials in our mouse patients, if you will, and try to predict the outcome of those clinical trials by using 40 or 50 of these tumor grafts. Each tumor graft, although unlike a patient, each tumor graft can be put on to both arms of a trial, and we can really, we hope, learn an awful lot about which patients respond in which ways to which therapies. So we are interested in that. 

We're interested in how genes change and gene expressions change following radiation or Cetuximab or Cisplatin and how we can use those changes to sort of look backwards and find a profile that would suggest that a particular patient would respond to one treatment versus another. So, I want to give just a couple, that's really the end of the data. I want to give you a couple of conclusions and then tell you who's been contributing to this work and thank them. So, very simply, human papillomavirus causes head and neck cancer. I think that's an important take-home point, and that's important because I think, as an oncologist, it speaks to the need for vaccination and the need for education of people because this is a cancer that we can prevent. It may take a long time, but it is something that we can prevent. Nothing would make me happier than not having any patients left to treat. HPV-positive cancers, we believe, are more sensitive to radiation than HPV-negative cancers. Some of the mechanisms of that are there, but I think the bigger question is, does that mean that patients should be treated with lower doses of radiation which would be less toxic than higher doses? Or does that mean that they don't need chemotherapy because they do well with radiation alone? And these are questions we don't yet know the answer to, but we're hoping that some of our work will give insights into that over time. And HPV-positive head and neck cancers are sensitive to combination therapy with Cisplatin or Cetuximab, and it doesn't appear, at least from our initial data, that if you have an HPV-positive cancer that one of these two treatments is better or worse for you than the other, but that there's some other factors that are probably important in which treatment is best for which patient. 

And this tumor graft system, we feel is a really outstanding, robust system for studying both HPV-positive and HPV-negative head and neck cancers. And we hope that over the coming years will enable us to make important contributions and help to design the next phase of clinical trials or the next group of clinical trials after the ongoing ones are finished. So, I have, actually, two pages of acknowledgments. The first one are the groups that I've been working with over the past few years as a fellow here at the University of Wisconsin. Paul Lambert and Paul Harari and the members of their labs are too long to list here. No one would be able to read anything on this slide. But we've also been working with Greg Hartig is the surgeon who's provided most of these samples. His patients have volunteered and donated their tissues for use in research purposes. Paul Ahlquist is a professor here who with whom we're pursuing some of the genomic analyses. Michael Newton is the biostatistician we work with on our microarray studies. 

Some of the work that I actually didn't show tonight but Al Klingelhutz at the University of Iowa has been a great collaborator over time. Of course, the patients, without whom certainly the last part of my talk would not have been possible. And I was supported during my time here by a Kaye Fellowship by a generous donor and his wife through the Head and Neck Cancer Research Fund. And some of the work with the tumor grafts was supported by an investigator initiated clinical trial award through the UW Cancer Center. Funding through AACR and RSNA. Currently, I have funding through the National Cancer Institute. So this is the growing Kimple lab. A number of undergraduate students, Molly Smith and Grace Blitzer, have been with me for almost two years now. -- and -- are postdocs and a lab manager who started over the summer. Davia and Ebony are two new UW undergrads who started just in the past couple of weeks, and Tyler is actually a medical physics graduate student who is collaborating with us on a number a projects. 

Although I didn't show any of his work today, he's an important member of the lab too. And then there are former lab members. Bob Yang is now an ENT resident studying to be an otolaryngologist and be the surgeon who operates on these patients at the University of Minnesota. Alex Torres is actually in the audience here. She's now a graduate student here at UW. And Charlie Pete is a former postdoc that did some work with us as well. With that, I'm certainly happy to take questions here. I'm happy to take questions by email. I'll try to get back to you as soon as possible if you send me an email. And I want to thank you for your attention, and thanks for having me here tonight. 

[APPLAUSE]