Why Does Treatment Fail? by Ian Tannock, MD, PhD

SLIDES & TRANSCRIPTS
Tuesday, February 15, 2000

Why Does Treatment Fail?
Ian Tannock, MD, PhD

Slide 1:

Dr. TEPPER: For our final talk of the morning Ian Tannock from the Princess Margaret Hospital.

Dr. TANNOCK: Good morning. This is the first time I have ever spoken at a GI conference. It is a new experience for me.

Slide 2:

I am going to talk about something a little different. If we look at the literature relating to why chemotherapy for solid tumors isn't very effective -- and let us face it, it isn't very effective -- about 99.99 recurring percent of the literature relates to problems related in some way to cellular drug resistance, be that the intrinsic or acquired resistance, be that the upstream or downstream events that Carmen Allegra was talking about, but that is where things have concentrated. It is my belief that that is highly inappropriate. I am not saying that it isn't important, but it is highly inappropriate in terms of the various number of factors that can influence the success or lack of it of chemotherapy for solid tumors, and I am going to talk about some of the tumor physiological factors that we have been looking at, problems specific to the micro-environment and there are various ones of them and talk about tissue penetration, and I am going to talk about this factor of repopulation between courses of treatment.

Slide 3:

Now, this isn't a GI tumor. It is actually human lung cancer, but this type of structure is found in many anaplastic types of human tumors. We aren't dealing with homogeneous collections of cells.

We are dealing with tumors that have an imperfect blood supply here running through the stromal tissue. We are dealing with situations where the cancer cells shown here in these cord-like structures may be at varying distances from the blood vessels with which we supply our tumors with drugs after intravenous injection or oral absorption. Here we see necrosis, and not only are there problems of penetration because of those distances, work of Rakeshjane and others have shown that the tumor blood flow is very variable so that some of those vessels are opening and closing all the time.

Slide 4:

One aspect that has been relatively neglected is this problem of tissue penetration. Most of the work on pharmacokinetics has talked about a tumor concentration, a tissue concentration, normal tissue concentration, blood concentration, but there is strong evidence that tumor concentration is rather meaningless, that there will be very large differences in concentration after an injection of an anti-cancer drug in different parts of a human or any other tumor,

Slide 5:

and just to give you one very simple demonstration of that, some old slides from a colleague, Bob Sutherland looking at the penetration of fluorescent doxorubicin, adriamycin into solid tumor spheroids showing at 1 hour that that fluorescence is around the outside. By 6 and 24 hours it penetrates more deeply but that is when you have the spheroid sitting in a bath of doxorubicin, and that is not what we do clinically.

Usually we are injecting it. We are seeing peak concentrations that fall rapidly, and penetration is going to be a problem, and if it works at all it is probably a little bit like peeling an onion, not from the outside of the tumor but peeling cells from around the tumor blood vessels.

Slide 6:

Until recently, we had no easy method of looking at tissue penetration, but there is a new technique which is remarkably simple. It was devised by Bill Wilson in New Zealand and in this technique tumor cells are grown on a coated Teflon layer. This is a porous Teflon layer. These are tumor cells, in this case MJA21 tumor cells of human bladder line growing on this membrane

Slide 7:

and we have done some work now to show that these multilayers -- here is the membrane again, and this has been stained. Using monoclonal antibodies, we have stained for various types of matrix protein, that they do produce collagen 1. They produce lots of laminin. They don't produce collagen 4. This is the xenograft, and this is the multilayer showing that these multilayers have many of the properties of the solid tissue in vivo, and therefore would seem to be appropriate for studying tissue penetration of drugs,

Slide 8:

and that is fairly easy to do with that type of model.

What you can do is grow your multilayer here. We can grow that to a thickness of about 200 microns which is very similar to the maximum distances between tumor blood vessels and necrosis in human tumors.

We can surround this by a support, and we can float it in a bath of medium. We can then add any drug we want to this compartment here, compartment one, and we can look for the penetration of that drug through the compartment two, and we can compare the penetration through a layer of tissue with simply the penetration through the membrane without a multicellular layer on top of it, and so we can do that for any drug we can assay. The simplest is to use radioactive drugs, but we have, also, used HPLC and other methods.

Slide 9:

This is my only deference to colorectal cancer. I have to show you one slide with 5-FU. 5-FU is a small water soluble molecule. It ought to be able to penetrate tissue pretty well, and compared with the other drugs that we have looked at it does penetrate tissue pretty well, but let me orientate you here.

This is the penetration of 5-FU as a function of time in hours through the Teflon membrane with no cells on it, and so this is the ratio of what you would expect under equilibrium conditions. By 6 hours you have got up to about 70 percent of true equilibrium between those two compartments. That is with the membrane alone.

If you intersperse a tissue layer of either the murine EMT6 cells or, in this case, the human MCF7 cells, we haven't found major differences between the different cell types we have used in this multilayer -- you can see that 5-FU penetrates at a rate that is about 30 percent through that membrane.

There is likely to be a very strong tissue distribution even of a drug like 5-FU between the cells that are seeing a lot of drug, by analogy those close to blood vessels and those which are further away.

Slide 10:

This is what you get for doxorubicin. I will show you a bit more in a moment but here is the doxorubicin penetration which is at a level, the same cell lines of about 5 percent of the penetration through that Teflon membrane alone, a huge barrier to tissue penetration.

The title of this section is why treatments fail,

Slide 11:

and we have one paper that is in press that tells us a little bit about why P-glycoprotein reversal has failed, and let=s face it, failed it has. To me that whole exercise was an example of how not to do science.

Basically you found out that P-glycoprotein was an important drug efflux protein. You showed that reversal agents such as verapamil and some of the more modern agents are wonderfully able to reverse cellular uptake to improve cellular uptake in P-glycoprotein expressing cells when you do that at 10 to the 5th cells per ml in tissue culture.

Every animal experiment that I know of using established tumors failed to show an effect of P-glycoprotein reversal unless you did the rather stupid experiment, I think, of implanting the cells on day one and treating them on day two.

P-glycoprotein reversal did not work in solid tumors in animals, but it didn't stop us doing multiple clinical trials including at least four randomized clinical trials to look at it in human tumors. It doesn't work because the effects fall off with tissue concentration. Here is another reason that it may not work. Here are P-glycoprotein expressing cells, AR10. We have done it with two or three different ones, and now this is the index of tissue penetration. I have taken out the bare membrane, and this is showing, and I am sorry these colors don't show up very well, that this is the wild-type cell. Penetration is poor. P-glycoprotein-expressing cells, because they pump the drug out of cells, allow more drug to be available for tissue penetration. Penetration is better through P-glycoprotein-expressing tissue, and if you then come along with verapamil and reverse that you move penetration back to a level where it was before.

P-glycoprotein inhibitors, yes, they may allow more drug uptake in cells proximal to blood vessels, but they also markedly decrease penetration, another reason why that probably hasn't worked.

Slide 12:

We are not interested really though in why things haven't worked. What we would like to do is to delineate properties that are amenable to modification, and I think this is amenable to modification.

Strategies which might improve tissue concentration by anti-cancer drugs, tissue penetration by anti-cancer drugs and hence more uniform concentration B one is to inhibit intracellular uptake. We have shown we can do that, say, for standard ways of inhibiting methotrexate uptake. We have shown that P-glycoprotein will do that, but that is not really likely to improve therapeutic index. One way that might improve therapeutic index is if you can stop the drug sitting in compartments of cells where they are not going to their target, usually DNA< and secondly where you have more drug that is available for tissue penetration.

Slide 13:

This is an idea we had relating to basic drugs, and many of the effective drugs we have, including doxorubicin, mitoxanthrone and most of the members of those families, they are weak bases and sitting in cells' endosomes, lysosomes and other intracellular particles that are acidic. Their pH is around 5 and there are methods of measuring that using fluorimetry.

That acidity is maintained by proton pumps in the endosomal membrane which are similar but not identical to the proton pump that we have in the stomach. Now, what happens to a basic drug? If the pH is 7 in the cytoplasm and 5 inside, that basic drug will be concentrated in a ratio of 100 to 1 inside endosomes, well known, for example, for the drug chloroquine used to treat malaria. The argument that we had was, suppose you could decrease that pH gradient, decrease the sequestration of drug, the basic drug there? Then you would have two effects. One is the drug would be more able to reach its target, which is usually DNA. Second, you have decreased net cellular uptake while increasing cytotoxicity. So more would be available to penetrate tissue for which we have evidence is largely extracellular, and we tested that using two agents in this model. One is omeprazole, which inhibits the proton pump and two is chloroquine, which is simply a competing weak base that can be used in high concentration.

Slide 14:

Here is the work of Dave Callen in my lab looking at the effects of chloroquine, relatively low concentration. This is penetration of doxorubicin through the bare membrane. Here is that very low rate of penetration through the multicellular layer, and as you add chloroquine in increasing concentration you do, indeed, increase penetration.

Slide 15:

Here, the same effect for omeprazole, and we have shown that for doxorubicin. We have shown it for mitoxantrone

Slide 16:

and this is a confocal microscope picture of net fluorescence of doxorubicin in the absence and the presence of omeprazole showing a decrease in cellular uptake. I don't have time to show you all the data, but we have shown that this does change the pH in those endosomes, and that it does decrease net uptake using radioactive drugs. Paradoxically, but predicted by this model, we decrease net cellular uptake but increase cellular toxicity and increase penetration.

Slide 17:

So there are ways that tissue penetration is open to modification, and we hope to take that. We are now looking at animal models and, if they bear fruit, we will take that into clinical trials.

The other thing I want to talk about very briefly is this question of repopulation between courses of chemotherapy.

Slide 18:

In radiation therapy, as Joel said in his introduction, we know that repopulation between courses of radiotherapy, between fractions of radiotherapy is important. This has been referred to as the dog-leg diagram from papers of Rodney Withers and others where you look at the dose of radiation to give a given effect, in this case controlling a portion T2, T3 larynx cancers. As you extend the time over which the radiation is given there are some centers in Europe that use short periods of time up to a month. There is very little difference, but as you then increase the time you have to increase the radiation dose to give the same effect, and there is substantial analysis to show that that is likely due to counter the proliferation of surviving tumor cells between fractions of radiation.

I have recently written a review of repopulation in chemotherapy. I could not find one clinical paper that has even mentioned that process.

Slide 19:

There are about four studies in experimental literature on repopulation between courses of chemotherapy, but again, as Joel said, we give radiation fractions once a day. We give chemotherapy fractions typically once every 3 weeks. This is likely to be a more important process between courses of chemotherapy. It is, of course, the process whereby the bone marrow and other normal tissues recover.

Slide 20:

What is the effect of repopulation between courses of chemotherapy? This is the simplest modeling you can do. Here I have simply taken a mock survival curve, 3 weekly fractions of chemotherapy and assumed in these two diagrams that either 80 percent or 95 percent of the cells are killed by each course of chemotherapy, not an unreasonable level of cell kill, and then assumed that repopulation, that is the increasing slope here where the surviving cells are repopulating, either takes place with a doubling time of a week, which might be rather rapid ,although rather more rapid rates of repopulation are recorded in human tumors on radiotherapy or of a month, and you can see that if you kill 80 percent of your cells with each fraction and you assume simply that between fractions of chemotherapy between courses, that you have repopulation with a doubling time of a week the tumor will grow despite that cell kill.

Slide 21:

It obviously isn't as simple as that. What evidence there is in the literature? As I said, there are only about four studies that I know of, and we have done a search looking at experimental tumors that the rate of repopulation as you tend to initially shrink a tumor and kill cells presumably is nutrition improved, that the rate of repopulation can increase, and it can increase at remarkably rapid rates, also during radiotherapy.

What we have done in this modeling is to consider a tumor where in this case we kill 70 percent of the cells per cycle and where initially the repopulation takes place with what is about the median doubling time of the growth of human tumors, 2 months, and then accelerates to 1 week.

In this modeling we have allowed that there will be some period after chemotherapy where there will probably be cytostatic effects, and we have allowed that repopulation doubling time to increase or to decrease, the rate of repopulation to increase to a doubling time of 4 days which is the calculated rate of repopulation of relatively slow growing head and neck tumors at the end of a course of radiation therapy. With this modeling you can see that if you took a solid tumor where you had a moderate cell kill from chemotherapy and, without any induction of drug resistance at all, there is no acquired or intrinsic drug resistance here, you would expect that tumor to shrink and then regrow purely on the basis of repopulation. That is obviously what we see in that 20 percent of your tumors that you manage to shrink with 5-FU and in other solid tumors as well.

The assumption that tumor shrinkage and regrowth is due to the selection of intrinsically resistant cells is not necessarily the case. Clearly it will be the case in some tumors, but you can have other tumor physiological effects, including those that I have described today, and they are not exhaustive, that can lead to a similar reason or similar rate of failure of therapy,

Slide 22:

and is repopulation amenable to modification? Absolutely. No repopulation of tumor cells between courses of chemotherapy seems to me might be selectively inhibited by biological agents.

Biological agents haven't gone very far on their own thus far at least, but many of them are cytostatic. Many of the tumor cells express growth factor receptors that are not expressed, say, in bone marrow, and using biological agents that inhibit those, put cells out of cycle might well improve the effectiveness of chemotherapy.

It is going to be complex because you will want to stop them before the next cycle so that the cells are again proliferating and that you are going to make them more sensitive.

Slide 23:

I think these are additional factors that are open to modification, and the conclusions are that these are neglected. I think there is no question that they are neglected in the literature as important as intrinsic or acquired cellular resistance in my opinion. We don't know the balance between those processes in limiting the effectiveness of chemotherapy, poor penetration, accelerated repopulation, and these are modifiable. I hope that in the future we can look forward to meetings that, instead of spending 95 percent on cellular factors, we can pay more attention to some of these other things.

Thank you.

(Applause.)