Level of Tumor Protein Indicates Chances Cancer Will Spread

Transcript
Media Availability

"Level of tumor protein indicates chances cancer will spread"

Presenters:

Y. Peng Loh, Ph.D, Senior Investigator, Section on Cellular Neurobiology, Laboratory of Developmental Neurobiology, Eunice Kennedy Shriver National Institute of Child Health and Human Development [Female speaker]

Karel Pacak, M.D., Ph.D, D.Sc, Head, Section on Medical Neuroendocrinology, Eunice Kennedy Shriver National Institute of Child Health and Human Development *[Male speaker with a Czech accent.]

Stephen M. Hewitt, M.D., Ph.D, Staff Scientist, Center for Cancer Research, National Cancer Institute

***

Operator: Excuse me, everyone, and thank you for your patience in holding. We now have our speakers in conference. Please be aware that each of your lines is in a "listen only" mode. At the conclusion of the presentation, we will open the floor for questions. At that time, instructions will be given if you would like to ask a question. I would now like to turn the conference over to Robert Bock, Public Information Officer for the Eunice Kennedy Shriver National Institute of Child Health and Human Development. Mr. Bock, please begin.

Robert Bock: Thank you very much. Welcome to the National Institutes of Health. We are here today for a media availability on an interesting finding coming out in the Journal of Clinical Investigation. The finding deals with an interesting marker for metastatic cancer. In a moment, I will turn this over to the principals. With us here today are Dr. Y. Peng Loh, Dr. Karel Pacak, and joining us on the conference is Dr. Stephen Hewitt. We hope you all made it in today and now I am going to introduce Dr. Loh.

Dr. Peng Loh: Thank you, Bob. So, good morning, ladies and gentlemen, and as you know, I'm Dr. Loh, and our team at the National Institutes of Health—which actually included a very talented young scientist in my lab, Dr. Saravana R.K. Murthy, and we worked together with a team from the University of Hong Kong headed by a surgeon, Dr. Ronnie Poon, who is involved in removing liver cancer. It is his specialty. So, together we discovered a new variant of the enzyme called, carboxypeptidase E, which is a protein initially involved in processing a hormone into processed insulin. So, we found that this new form of carboxypeptidase E, which I will refer to as carboxypeptidase E or CPE-delta N. This form is present in large amounts in primary tumors that have spread or metastasized. And as everyone knows, cancer cells break away from the primary tumor, pass surrounding tissue into lymph and blood vessels, and these cancer cells then travel through the body and form tumors elsewhere. So, metastatic cancer is a major cause of death and it's very important to know when a cancer is likely to spread, so the practitioners can actually contain the cancer early.

So, what is unique about our discovery? First of all, by measuring the carboxypeptidase E or CPE-delta N protein, or its RNA and coding this protein in the primary tumor that has been removed, we can tell from the levels whether (1) the tumor has spread and (2) predict whether the tumor is likely to recur in the same tissue or spread to other parts of the body in the future.

Currently, there are no accurate biomarkers that can achieve such predictions, and prognosis is determined by staging of the cancer using histopathological techniques. Finally, we have found that CPE-delta N is highly elevated in metastatic tumor cells from different types of cancers, such as liver, colon, breast, adrenals, and head and neck cancers and, therefore, this biomarker may be useful for many types of cancers. And I think that's very important.

So, just to give you an example—I think you all got the news release—I'm going to focus on the cohort of 18 patients with liver cancer, and these are the patients with Stage II cancer based on the pathology of the cancer that was removed. These patients will normally be told by their physicians that their cancers are not likely to recur and they do not receive further treatment after the surgery. Our assay showed that 13 of these patients had low levels of CPE-delta N RNA in the primary tumor, and 10 of them did not show recurrence of the tumor within 2-3 years after their surgery, indicating a 77 percent accuracy in predicting non-recurrence. However, more importantly, five of those 18 patients showed very high levels of CPE-delta N RNA in their tumors and four of them, that's 90 percent, developed metastasis within two years after their surgery.

So, CPE-delta N is a better indicator of the seriousness of the disease than current staging techniques. And, with more perspective studies—which we are currently doing in collaboration with the Liver Network in Taiwan—CPE-delta N will likely prove to be a very valuable biomarker for guiding the course of action and treatment for Stage II patients depending on the CPE-delta N RNA levels in their tumor.

So, on the flip side: the good news is that we found in 12 patients—these are liver cancer patients again—with Stage IV cancer: five of whom have very low CPE-delta N RNA levels, who did not show recurrence of their cancer two years post-surgery. Generally, Stage IV patients have little hope of survival. However, our assay, which can better determine the aggressiveness of the tumor can bring hope of longer survival and psychological comfort to those Stage IV patients with low CPE-delta N RNA levels. It will also encourage practitioners to actively treat these patients with chemotherapy, since they have a good chance of not having a recurrence. Generally they don't receive much attention if they are in Stage IV, at least in Asian countries. We have also now used our knowledge and looked at mouse models to show that when we actually suppress the expression of CPE-delta N, that the liver cancer cells will, when we implant them into mice, be inhibited from tumor growth and absence of metastasis.

So, this offers the potential for developing a cure for certain types of cancers using antisense o CPE-delta N to suppress its expression. So, now I will turn you over to Dr. Karel Pacak, who will tell you how we have had 100 percent success in using this biomarker to predict future metastasis in a small cohort of 14 patients with pheochromcytoma, which is a rare adrenal cancer prevalent in children and young adults.

So, Dr. Pacak.

Dr. Karel Pacak: Ladies and gentlemen, good morning. My name is Karel Pacak and I head the Section on Medical Neuroendocrinology here at the NICHD, NIH, and I've been focusing on patients with severe pheochromocytomama and paraganglioma for the last about 10-12 years. I should say something at little bit about pheochromocytoma and paragangliomas because those tumors are called endocrine tumors, and they are rare neuorendocrine tumors: we estimate that we have approximately about 1,000-2,000 new cases in U.S., so they are called rare neuroendocrine tumors. However, these rare neuroendocrine tumors can be very dangerous to patients for two reasons. The first reason is that these tumors, as neuroendocrine tumors, secrete hormones and we call them epinephrine or norepinephrine, or sometimes we say adrenaline or nonadrenaline. And these hormones can be truly devastating to our patients because they are affecting many organs and especially cardiovascular systems. So many patients who actually have these tumors, they may end up with myocardial infraction. They may end up with a stroke, or a very severe or lethal arrhythmia.

And I have to say that because there are not good markers—especially to predict the outcome of these tumors, including the metastatic spread— approximately about 50 percent of these patients in the United States (but also worldwide) are not well recognized and they usually die from the consequences of high catecholamine levels. These tumors are usually located in the adrenal gland, approximately 80 percent of these tumors. And approximately 20 percent of these tumors are located outside the adrenal glands and they can be found everywhere in the body, but the most common location is the abdominal area for these tumors.

So, in terms of histopathological examination, and especially if we send the patient to the surgical procedure to remove the tumor, compared to other tumors (and this is very important information) the surgeon, as well as pathologist based on his pathological examination, they cannot actually make the decision that the tumor is benign or malignant. This is in contrast to other types of cancers; for example, if you take breast cancer, colon cancer, or other types of cancers that, for example, what's called vascular invasion or so-called capsular invasion will support the diagnosis of malignancy. This is not actually valid and does not work for patients with pheochromocytoma and paraganglioma. Maybe I should say at the beginning that the paraganglioma is the pheochromocytoma, but only the pheochromocytoma that is actually located outside the adrenal gland. So this is why it was very important for us, especially in cooperation with Dr. Peng Loh's group, to look or to search after certain markers that would help us to predict prognosis of these patients, including if they have a higher likelihood to develop a metastatic disease.

Therefore, I think that it was very important, especially for the tumors, because the tumor is very unique compared to other tumors as I said before, to predict the metastatic disease or to predict the recurrence of these tumors and to provide the patient (especially in the future) with better information that will have a very important impact on their follow up as well as some treatment options.

Robert Bock: And with us on the line is Dr. Stephen Hewitt. Dr. Hewitt, do you have any remarks about the pathology efforts for the study?

Dr. Stephen Hewitt: Yes, thank you. It's been a real pleasure to be a portion of this collaboration developing a new biomarker that identifies metastatic potential of tumors. We have a great deal of effort that focuses on the development of biomarkers that will help predict which patients will progress because if you can identify a patient in the early stages who's at high risk for progression of disease one can modify their therapy. My role in this study was to evaluate the use of immunochemistry as a potential tool for this. And interestingly in this study, we actually demonstrated that a reverse transcriptase PCR as they performed on the RNA was a better marker of prediction of metastasis than the immunohistochemical assay and I think that this assay and the development of these assays really foreshadow developments of how we're going to apply new testing in patient care in the future.

This study is nice because we were able to beat the current standard of care, stage, and grade. The challenge with stage- and grade-type systems is that typically staging and grading of a tumor occurs after the patient has had a surgical resection. You can only obtain some of the information at the time of initial diagnosis or biopsy. Tests such as this—where one can perform it on a small sample of the specimens without full resection—have the opportunity of allowing physicians to better plan patient care ahead of time, and direct the care in a more effective manner. So, this is an exciting opportunity for us to participate in because we see that it is where we're trying to focus aspects of cancer care in the future.

Robert Bock: Thank you very much, Dr. Hewitt. Operator, if there are any calls in the queue, any questions, the researchers would be happy to take them now. Reporters, we ask that you identify yourself and direct to your question to the appropriate researcher who's on the call. Thank you.

Operator: Thank you very much. At this time, we will open the floor for questions. If you'd like to ask a question, please press the "star" key followed by the "one" key now. Questions will be taken in the order in which they are received. If at any time you would like to remove yourself from the questioning queue, press "star two." Again, that is "star one" to ask a question now. Our first question comes from Miriam Falco with CNN Medical News.

Miriam Falco: Hi. Thanks for taking the call. This is for the first two doctors, especially Dr. Poh; I'm sorry, Dr. Loh, my apologies. These are very small studies; 18 patients, 12 patients. It's a very small patient population. And I realize it's exciting to find a new marker, but this seems to be very early in the research. Why are you coming forward with this now? Why not wait until you have larger trials to show that it really is that accurate? It just seems really early.

And the other question I have is: given what you were talking about— where the research you were doing—and I don't know which country the adrenal cancer patients were from, is this something that might be typical for a particular patient population? For instance, in Asian countries and may not be as indicative elsewhere in other parts of the world?

Dr. Loh: This is Dr. Loh. In response to your question about small numbers: actually I took those examples, but I believe you could get a link to our paper at the Journal of Clinical Investigation where the number of patients we have looked at is far greater than that. We've actually studied, initially, 99 patients for liver cancer. And we have Stage I, II, III, and IV patients, but I selected Stage II patients to illustrate the power and the usage of this particular biomarker, and also I picked out the Stage IV patients to illustrate that it doesn't mean that we only did 18 and 12. And, in that same paper, we've also assayed the biomarker using Western blot (which is the protein marker) of another 80 patients with the same accurate predictability.

So, altogether, we have done a total of 180 patients for this study. So, I think that is really a pretty good number to come forward with this. And…

Miriam Falco: But they're all in different stages. I mean. I guess, I guess. Is this practice-changing? Is this a foundation for more research? Or somewhere in between? I'm just trying to get a good sense of how big a deal this is. It sounds like a big deal, but as you break it down, it's 18, it's 12. Those numbers look very impressive when you're looking at the 12 patients. I'm not trying to be argumentative; I'm just trying to figure out what the message to people out there is.

Dr. Loh: Well, as I said earlier, we do have a total of 180 patients altogether and, if we looked at the 18… Well, what we were trying to do is to use different methods to come up with the same conclusion. We can even measure the RNA by PCR or we can do Western blot—which other people would like to know: that in fact you can do different methods of assaying for the same protein or RNA and come up with the same result—and I think that's very important. And basically, yes, this is a basis on which to go further.

And, as I had mentioned in the introduction, we actually have a big study going on with the Liver Network in Taiwan where basically these liver cancer patients are much more prevalent in Asian countries and this is why we went [there]: our collaboration with the University of Hong Kong and China, as well as in Taiwan. They have a registry of 3,000 liver cancer patients and we're doing a prospective study with them right now, which has just begun to further test mostly with Stage I and Stage II, because we feel that their practice was "Okay, if you are either Stage I or Stage II, you really are cured and we don't treat you anymore." And I think that what this has done is to alert them, or alert the practice of doing this, that "Hey, there are Stage II patients who have very high levels of this marker and you better take care of them because it's an indication that the tumor is very aggressive and one should now take care of these patients instead of giving them the idea that they're fine, they can just go home and not come back again!"

And, likewise, I selected the Stage IV patients for illustration, and that is [because] those patients are generally are not even treated. They've kind of been told, "Well, you don't have much longer to live." But, we found that out of those patients, the ones with low CPE-delta N ended up not getting recurrence after removal of their tumor. And so, those patients with good treatment would really prolong survival. And they would have been missed if not for having some answers, or using this biomarker to further indicate the aggressiveness of the tumor that they are really dealing with.

Miriam Falco: So, you do believe it could be practice changing or should be?

Dr. Loh: It should be, yes.

Miriam Falco: But now or after the bigger trials are done?

Dr. Loh: I think that after a bigger trial has been done. And we are actively pursuing it.

Operator: Thank you very much. Our next question comes from Daniel Denoon with WebMD.

Daniel Denoon: Thanks for taking my question. I'd like to pursue this idea now that we have this test. Patients are going to be hearing about it. Could a patient who is diagnosed with cancer now get someone to measure their CPE-delta N levels and get some information? I realize this test hasn't been validated, but I think part of what Ms. Falco was getting at was when people hear about this they're going to want to know whether they can get this test. How available will this be? And how long will it take to validate the test?

Dr. Loh: I think the prospective aspect would take a longer time. I mean, liver cancer generally recurs in two years, so having started the prospective studies, we think that we will get our data in about 2-3 years and then it's a question of whether the patients are coming to the NIH, [to] Dr. Pacak, and they can get the test sooner.

Dr. Pacak: Yeah. For outpatients, I'm pretty sure (I would like also to connect it with the first question from the first reporter about the number of the patients). That's my feeling, but I think it's based on statistics as well. That if the largest center for patients with pheochromocytomama and paraganglioma in the U.S, this type of, this tumor, or this type of tumor is pretty rare and most of our patients are coming with metastatic disease. If they have a metastatic disease, and because those tumors are releasing the hormones, and I mentioned about norepinephrine and epinephrine, it's not ethical actually to remove any tumors or to do the biopsy because they can end up with a hypertensive crisis. So, to enroll patients in this study it was not absolutely easy because there are certain criteria that must be met before the patient can be enrolled in the study. For example, the tumor must be accessible. We have to remove the tumor. (Usually, those tumors are primary tumors because we are usually not allowed— except for certain exceptions—to remove the metastatic tumors.) And, of course, the material must be available for the study.

Despite that, I have to say that the results of this study—and I agree with you [Miriam Falco] that the number of the patients, and especially in the category of neuroendocrine tumors like pheochromocytoma and paraganglioma, the number was only 14 and somebody could question that the number of these patients is low… But, you know, the sensitivity and specificity of this test came [back] very high. Yes, they came back at 100 percent. And, I will be honest with you, that we would like to extend it to 1,000 or 2,000 patients, but never in medicine is something [a test] for 100 percent sensitivity/specificity. But it's still very high, and we are following up based on the data that we have—the data are valid and they are solid—but we cannot release those data [yet], or go outside and tell our physicians "Yes now we have a new marker and you should use it immediately in the clinic", because we have to actually start and to open, or to promote, these [future] studies.

And I think that this article will be extremely good to promote a new study: speed up the research about this marker for not only the start of these tumors, but also many other tumors. And, we hope, based on some of the preliminary data, that it will be a good marker that will help us to predict, actually, the outcome of the patients. And in the study, in 14 patients, if you look at one table, where we included patients with so-called [INDISCERNIBLE] gene mutation; it stands for [INDISCERNIBLE]. At least half of these patients end up with metastatic disease.

As we have found in the table that they did not end up with metastatic disease because the expression of the gene, carboxypeptidase E was low. And, we can give them very good hope that most likely those patients will actually do well in the future. Of course, the shortest interval was two years; the longest interval in some tumors was up to eight years. But we have to extend this interval for the follow up, and we have to call for not only national collaboration (because these tumors are rare) but also for international cooperation, and I can tell you that we are working on that just now and we are trying to enroll many, many patients to speed up this type of research, at least in this part of neuroendocrine tumors and to be able to release the new data that I hope, or I'm convinced, will confirm the results of this study.

Daniel Denoon: Dr. Hewitt, is there any sense of—this is what people are going to want to know—is there any sense of when this test will be available? Or whether people with rarer tumors could go to NIH right now and get tested for this kind of thing?

Dr. Hewitt: Well, the test has only been demonstrated to be efficacious in the paricellular carcinoma and pheochromocytoma paraganglioma, so its extension to other tumors would be speculative at best, at this time. And as I think Dr. Loh and Dr. Pacak have demonstrated, really we're in the stage where we have a discovery with very good results, but it's time to validate this in other cohorts and expand these findings, including to the other tumor types. The nature of biomarker development is very much like the nature of drug development. It takes time. And sometimes you actually have to wait longer than with drug development because you're waiting for the patient's outcome to be determined, not a short order measure of response in a matter 8 or 12 weeks, did something shrink. You're asking the question: "Did the tumor metastasize or not?" And you have to be very patient to collect this information. So, no, this test is not something that patients can go and obtain through other sources at this time.

Daniel Denoon: Thank you very much.

Karel Pacak: [It’s] Karel Pacak. This is like the first phase, and we have to validate all these results. And, of course, we have to extend the number of patients because we are releasing—or we will release—really, very good solid information. And we have to have very good data because it's about the patient at the end. And the patient must know: what they can expect; and how good the test is; and what the test can tell them; and if this test will work for their disease. So, we are just now extending [the research] for the other types of cancer. It's already in collaboration with other hospitals, and I think that in the very near future you or the other medical community will hear about those other cancers. And I hope that we will put everything together in a positive way.

Operator: Thank you very much. Our next question comes from Richard Knox with National Public Radio.

Richard Knox: Thank you very much. I actually have three questions. Would you like me to ask them one at a time or all three at the same time.

Dr. Loh: One at a time, please.

Richard Knox: Okay. Sure. There have been other markers for metastases in the past, and people got very excited about them but they didn't work out very well. How is this one different, you think?

Dr. Loh: Well, one of the things is that there is a standard that has been put up by the various cancer societies, which is called REMARK criteria (or reporting of a specific biomarker). In order to get published in the Journal of Clinical Investigation, we have followed these 20 criteria and answered all of them, and showed all the statistics and so on. And so, as far as this biomarker is concerned, it has been put through the grind and it has made it through, at least for liver, the numbers of patients and so that have been used, it has made it through the criteria and so this is what the Journal wanted and quite rightly so.

Yeah, you're absolutely right. There were too many biomarkers that fell by the wayside and that's why this REMARK criteria was set up, and we followed every step of it, and they [the Journal] were very happy with it. So, to that extent I think we have done the best we can so far now in validating this biomarker at this point.

Dr. Hewitt: I'd like to add a couple statements. I think that that's absolutely correct, that we've followed the REMARK criteria and we've got actually a reasonable number of patients that we've applied this test to, in cellular carcinoma. But, I think an interesting and an important feature is also the fact that the biomarker has now been used in two very different and unrelated cancers: paricellular carcinoma (primary tumors of the liver) as well pheochromocytoma paraganglioma. These tumor systems are not closely related to each other. And I think that may be something that separates this biomarker from many of the other biomarkers that were previously identified, which are usually in a single tumor.

Dr. Loh: Yeah, I think you hit it on the nail in this respect! And this is why we chose these two tumors, one being to illustrate the neurendocrine and the other being epithelial-derived, as our first shot. And we certainly have different cancers in the pipeline now, including thyroid, and possibly we're starting a collaboration for ovarian cancer and maybe even kidney cancer.

Dr. Pacak: Yeah! And those tumors are really from completely different baskets. And if we find that this marker actually is useful for very different tumors, it's something which we feel is very, very novel. But, of course, there are more studies that are coming to validate these results and confirm actually those results.

Richard Knox: That leads to my second question, which is that you do mention that you have looked at cells from liver, breast, colon, head and neck tumors and found that the most aggressive tumors had the highest levels of CPE-delta N. Can you just talk more about that work, and how many tumor samples? And what do you mean by "most aggressive," and how important is that finding?

Dr. Loh: Let me just correct something. Those are tumor cells, so they are cell lines derived from those various tumors. We haven't actually taken tumors right out of the patients as we did with the liver and with, in fact, with the sarcomal cytoma. We've also done some with colon cancer directly from the patients and that is in our Journal of Clinical Investigation paper, in a supplementary where it wasn't used for prediction but it was a validation that a cancer is really metastatic. So, from the point of view of those cell lines, we have taken at least 2-3 different cell lines which are metastatic, and 2-3 cell lines which are low metastatic from each type of tumor… and have found that the correlation was extremely good.

Richard Knox: And, finally, do you have any information on how CPE-delta N works to promote metastasis? Do you think it's directly involved as a metastasis promoter? Or do you think it's a bystander, or what mechanism do you think might be at work?

Dr. Loh: Oh, that's a very good question, and I think that's also in our news release, a little bit, but it's very well laid out in our Journal of Clinical Investigation paper. And the way it works is that normally carboxypeptidase E as an enzyme would go through the regulated secretory pathways, which means that it goes through a secretory pathway into granules. And that's where the hormones are generally stored, and that's where the enzyme plays its role as a processing enzyme. However, this splice variant—which means that the gene is being read a little bit differently with an alternative fight—and so it was actually missing the end terminal part of the molecule which contains the signal for directing it into the secretory pathway. So now it's no longer going into the secretory pathway; it came out in the cytoplasm. And what we found [is] that in high-metastatic cells, that something is sending this molecule into the nucleus and, inside the nucleus, this molecule is binding to a histone deacetylase enzyme which is involved in modulating gene expression. And we actually found that it modulates and up regulates a metastatic gene, and that's well established—called Nedd 9—and that was published itself some years ago So, the mechanism is that it actually turns on metastatic gene Nedd 9 and actually probably some antipeptidic genes as well as we have preliminary results for.

So, the mechanism is well worked out and I invite you to read our paper in the JCI.

Richard Knox: Thank you.

Operator: Thank you. Again, ladies and gentlemen, if you'd like to ask a question, please press "star one" at this time. Our next question comes from Jennifer Corbett with the Wall Street Journal.

Jennifer Corbett: So, thank you. My questions have been answered. Thank you.

Operator: Thank you. Our next question comes from Peter Aldhous with New Science Magazine.

Peter Aldhous: Hi. In the future, if I'm a liver cancer Stage II patient and my physician finds out that I have high levels of the splice variants of the enzyme, how might my treatment be different from that of a patient with low levels of the enzyme? And, as well, just on the question of the variety of cancers this might apply to: I know you need to test that, but is it clear from what you know about biology whether there are likely to be any solid tumors to which this doesn't apply or potentially could this apply to all solid tumors?

Dr. Loh: Well, should I take the second question first? In fact, I think that there will be some tumors that it may not apply to. We actually have screened a lot of databases where there are micro-array data available for some 14 cancers, and we have actually published a review article on that. Now, what we found was that out of those 14 there were a couple, mainly the oligodendroglioma cancers, that there wasn't a big correlation between CPE messenger RNA and the metastatic state of that type of cancer. But it did with some 10 of those other ones; there seems to be a correlation.

Now, I have to say that this is work that has been done previously, some five years ago, and when they do a micro-array analysis, you can't tell whether it's the CPE's splice variant or the CPE itself. Now, we took one of the studies with the colon and found that actually they were really measuring splice variant, so we guess right now that maybe what they are all measuring is actually a splice variant and not CPE itself. But based on what they had in the literature, or what has been done in the literature regarding the CPE RNA, I think that this could be applicable to a lot of cancers, but not all cancers.

Dr. Pacak: I think regarding the first question; it was a very interesting question: if we have a patient or if you would become a patient and we would find those three regarding carboxypeptidase E in a high number, how it will actually impact the patient, or what are we going to do as a physician, and what we will give to patients in term of some suggestions. And I think that there are two—actually I would say—two options or two steps that we should follow. And let's say that we are correct [that the current study’s findings are accurate] and we will confirm the first study on a larger number of patients with various cancers, and we will find that this carboxypeptidase E marker is working, so what am I going to tell the patient? The first thing, if I see that there is high expression [of the biomarker], I know that the patient is in trouble: the patient is in danger and something may happen to the patient. As a physician, I will approach the patient with a sensible discussion, explain what's going on, and then I will know that the metastatic disease spread may come or the recurrent tumor may come in the near future.

So, what is most important is to do a very regular and very good follow up, because today we have very good techniques (and we don't have the time right now, and this is not for the discussion today to talk about those techniques) but I can tell you that those techniques are extremely good and I'm not talking about the classic surgery. I'm talking about, for example, radiofrequency ablation or other approaches that can be very successful if we find just one tumor (a solitary tumor/solitary metastatic lesion) that can be removed, then the patient (practically in 24 hours or 48 hours) is going home.

So, the regular follow up—much better [more often] than we do for other patients—because the other follow up is sometimes between three to six months, but we can see the patient every three months and we can do some imaging studies, depends on the type of cancer.

And the second, if we know what is the mechanism—and Dr. Peng Loh suggests it, and actually not only suggests it but she confirmed and found it, and it is in the JCI article—that how actually this carboxypeptidase E is working through different aspects of [INDISCERNIBLE] and to the expression of Nedd 9, we have a very good drug on the market and there are even new ones right now that are called, histone deacetylase inhibitors, and they can actually prevent expression of these genes and may actually be very useful in some patients who will demonstrate the high expression of carboxypeptidase E.

So, I think that we need to do teamwork, but if we have some marker that we feel that is really very solid, valid and works in some cancers, the teamwork to find a good approaches and therapeutic option would be really good. And this is what I would recommend.

Peter Aldhous: Thanks.

Operator: Thank you very much. At this time, we are holding for questions. If you would like to ask your question now, press "star one." We have a question from Daniel Denoon with Web MD.

Daniel Denoon: Thank you for taking the question. I'd like to ask about the possibility of this knock-out of the CPE-delta N that you demonstrated with antisense. Would this or some other approach really seem to be a promising way to be treating metastatic cancer, or cancers that threaten to metastasize?

Dr. Loh: Well, I think some work has been going on with this type of so-called reverse gene therapy approach. I think that, like gene therapy, is moving slowly, but I think the time will come where we can introduce some antisense through the use of adeno-associated viruses into the liver; actually people have done it by injecting it into melanoma tumors directly since it's on the outside. But this antisense approach is up and coming—and there's a lot of work being done it—and it eventually I think would be usable.

Daniel Denoon: Any sense of some kind of small molecule or something might also be used to inhibit this CPE-N? I'm just trying to get some idea of whether this is a promising target for therapeutics.

Dr. Loh: Well, the thing is that it's not working through the enzyme, but through enzyme activity. But we are actually studying what regulates the expression of this gene. And, if we can find what can shut it down then we can develop some small molecules that would actually shut down the expression of this gene some other way, besides antisense.

Dr. Pacak: Yeah, and this would be very important to be done in in-vitro studies as well as on animals. And to study the pathophysiology of these processes and look at cells and see what kind of, for example, what kind of tumerogenesis pathways are actually involved and based on that, to pick up or to suggest maybe the target that would be most promising and then, of course, to translate into clinical research, which would at the beginning –could start—with so-called mini trials, and then we would see how we would work… and then, of course, it would depend on the availability of some drugs that we would obtain from outside, especially even developing our institutional medical center to work together with other institutions or companies.

Dr. Loh: I think the bottom line is that it is a potentially good target.

Daniel Denoon: Thank you. That answers my question.

Operator: Thank you. We are again holding for questions. Please press "star one" now if you have any final questions. We're showing no further questions at this time.

Robert Bock: This is Bob Bock. If there are no further questions, we will end this briefing. Just want to let everyone who's holding know that if they need anything; materials connected with the paper or graphics, etc., please feel free to call our office, 301-496-5133 and we will have a tape of this briefing on our Website in 24 hours. Thank you very much.

# # # #