KAPOSI SARCOMA AND SOLID TUMORS

Giuseppe Grandaliano, Bari, Italy

Chair: Alex M. Davison, Ancrum, United Kingdom

Gerhard Opelz, Heidelberg, Germany

Dr G. Grandaliano Department of Emergency and Transplantation University of Bari Bari, Italy

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Slide 1

Doctor Opelz, Doctor Davison let me first thank the organising and scientific committee for the opportunity they gave me to share with you our experience with kaposi sarcoma.

Slide 2

Now kaposi sarcoma is an angiosarcoma, an endothelial derived neoplasia that was first identified by Doctor Kaposi more than one century ago in Vienna. The main feature of this neoplasia are being widespread in the body but as you can see there is also the possibility that this sarcoma will have a visceral involvement. Here you can see a lesion in oral mucosa, here there is a lesion in the GI tract and you can have multiple lesions also in the lungs of patients.

Slide 3

Now classic kaposi sarcoma was originally described as an indolent tumour of elderly men prevalent in Mediterranean areas or Southern Europe and Northern Africa and the Near East and there is an endemic form of kaposi sarcoma in some areas of Central Africa. The annual incidence, this is for the U.S. but basically it’s representing what the European incidence is too, it’s 0.2-0.6/pmp that makes the 0.02-0-07% of all cancers in the United States and even in Europe.

Slide 4

So now all of you will ask me why are we talking about kaposi sarcoma in this context? The answer is really simple, the incidence of kaposi sarcoma in transplant patients goes up between 400-500 fold over the general population with the same ethnic origin.

Slide 5

Indeed here you see an experience from a transplant centre from Northern Italy and you see the incidence of kaposi sarcoma around 1.1%. This is our centre in the South of Italy we are close to the 3% and if you ask some colleagues from the Northern transplant centre, they will tell you that most of these patients are coming from the South of Italy. Besides the clinical point of view, there is also a scientific reason to look for kaposi sarcoma, to go after kaposi sarcoma. We believe that kaposi sarcoma may represent a good model to look at the immunosuppression, the levels of immunosuppression on the development and progression of neoplastic disease.

Slide 6

Indeed, this kaposi sarcoma is strictly related with immunosuppression. It presents a viral aetiology, so it’s really at a crossroads because between viral infection and neoplasia and above all it’s an endothelial sarcoma. So if we are looking for neoangiogenesis or the effect of immunosuppression on neoangiogenesis of the range, on the range of endothelial function in neoplastic disease, this could be a very interesting neoplasia.

Slide 7

So if you consider from a clinical or scientific point of view the main question is, can we prevent or treat kaposi sarcoma? If yes how?

Slide 8

The answer to this question comes from very far away, from the Easter Island.

Slide 9

Easter Island was the place where they first isolated the Strept. Hygroscopicus that is the microorganism producing the first mTOR inhibitor known, rapamycin.

Slide 10

Now, rapamycin is an immunosuppressive drug acting on signal three, it’s an antiproliferative drug that stops clonal expansion of T cells.

Slide 11

So based on its antiproliferative effect already 10 years ago it was suggested as an anti-neoplastic drug as you can see in these in vitro studies.

Slide 12

This led us to our experience with the use of sirolimus of rapamycin in patients with kaposi sarcoma.

Slide 13

This is our population. We started with 15 patients, now we’ve completed our observation on 19 patients, CNI-based therapy as soon as we had the histological diagnosis the patients withdraw cyclosporin and introduced rapamycin with this loading dose, this maintaining dose aiming at this trough level. We performed skin biopsy at 6 months after the introduction of sirolimus.

Slide 14

These are our new patient population futures. As you can see, we didn’t observe any acute rejection after the switch. The renal function, the graft function was even slightly better. What I want to underline is that all the patients that are included in this particular population are with cutaneous form of kaposi sarcoma and as you can see, 100% lesion regression 3 months after the switch.

Slide 15

These were basically patients with kaposi sarcoma. This is a picture of the same lesion one month after.

Slide 16

Of course our experience was not the only one. This is a case report from Doctor Campistol’s group. They described 2 cases of kaposi sarcoma that regressed after switched to sirolimus.

Slide 17

Interestingly, Doctor Campistol also reported a case series of 7 patients with kaposi sarcoma that according to me added really precious information to our case reports, to our series. In our series all the patients stopped calcinurine inhibitors and started sirolimus. So we really could not say whether the beneficial effect was due to withdrawal of cyclosporin or to introduction of sirolimus. In this series instead, Doctor Campistol reduced cyclosporin first and when he did not observe any positive results then switched the patients to sirolimus with a clear reduction and disappearance of the lesion. So the results are due definitely to sirolimus.

Slide 18

How can sirolimus do this? How can mTOR inhibitors act on these neoplastic diseases? We have at least 4 points where the drug can act. Viral infection, neoangiogenesis, cell proliferation, cell differentiation.

Slide 19

Let’s start with the viral infection.

Slide 20

We don’t have any direct proof of the effect of this drug on viral infection in this particular disease but there are a lot of observations where they tell us that mTOR inhibitors can reduce the incidence on viral infection in transplanted patients. This is an experience in patients with anti-lymphocyte treatment treated with sirolimus, they do not show any kind of viral infection.

Slide 21

But besides this observation, I think that the most important observation comes from the use of mTOR inhibitors in the heart transplant. This is a big trial with everolimus, with Certican. As you can see here, the introduction of everolimus at different dosage prevents viral infection, cytomegalovirus, herpes simplex, herpes zoster, all herpesviridae infection significantly when compared with the use of azathioprine. So viral infection can play a role, we don’t have any data on this.

Slide 22

 

Neoangiogenesis.

Slide 23

 

Geissler group elegantly suggested in their Nature Medicine paper that the reduction in tumour growth was due to a reduction in tumour neoangiogenesis induced by this drug. They suggested VEGF as the main culprit.

Slide 24

We know that vascular endothelial growth factor interacting with its receptors on the endothelial cells can induce cell survival, migration and proliferation. 3 actions that are clearly directing the cells towards angiogenesis.

Slide 25

So what happens in the kaposi sarcoma cells? This is normal skin, very low expression of VEGF receptor. These are kaposi sarcoma, you see almost all of the cells are positive for KDR, for the VEGF receptor.

Slide 26

What happens with and without rapamycin? This is a normal skin, very low production of VEGF. This is still normal skin around kaposi sarcoma, you see the normal skin starts expressing VEGF, the normal skin is feeding the tumour. When we put the patients on rapamycin we went back at 6 months and looked for VEGF expression, the VEGF expression is close to the basal, so rapamycin is reducing VEGF expression, reducing the feeding of the tumour.

Slide 27

Cell proliferation.

Slide 28

I told you, rapamycin, I mean mTOR inhibitors are antiproliferative drugs, I mean their immunosuppressive effect is based on their ability to stop clonal expansion of T cells. So we can expect that the antiproliferative effect can be useful also in the neoplastic disease.

Slide 29

Now any growth factor you consider in the development of the neoplastic disease can induce proliferation through 2 intracellular signalling pathways. One intracellular signalling pathway goes through PI3K, AKT, mTOR, S6p70. This is the pathway directly involved in the effect of mTOR inhibitors. But there is also another pathway where RAS, RAF and MAPK are involved. As you can see, between these two pathways there is a cross talk, they are not isolated pathways. So let’s see what happens to these pathways when we introduce mTOR inhibitors in these patients. Let’s look at the final step. S6p70 kinase phosphorylation. This is what happens. In the tumour we have very high levels of Sp60k for phosphorylation. The pathway is extremely active in the tumour cells.

Slide 30

When we go back with a biopsy after the introduction of the mTOR inhibitors, I mean after the introduction of rapamycin, 6 months after you see the number of cells that present p60S6k phosphorylation is dramatically decreased it is almost back to normal so mTOR inhibitors are surely influencing this pathway but again this is the pathway where we expect they work.

Slide 31

What happens to this other pathway? The MAPK pathway. The MAPK pathway is involved too. This is normal skin. As you can see the active, the phosphorylating and then active MAPK ERK is in green, the total MAPK is in red. Almost all the MAPK present is red, I mean there is not a lot of activation in the normal skin. This is what happens in the kaposi sarcoma lesions. You see all the cells are green. There is a clear high activation of this pathway that induces proliferation.

Slide 32

What happens when we introduce sirolimus in these patients and we go on and biopsy the lesions 6 months after? Again, here you have something that is really close to the normal skin. All MAPK is non-phosphorylated, so mTOR inhibitors can influence also this pathway and cell proliferation not only the PI3K mTOR.

Slide 33

The final pathway that I’m really interested in is cell differentiation. I mean, of course, as always all the neoplastic disease, the future of the cancer is their differentiation. The cells lose their future, their mean future.

Slide 34

So there was already this paper that was published more than 10 years ago that demonstrated that if we use these melanoma cell lines, this is a culture of melanoma cell lines of course these cells cannot produce melanin, you see under basal conditions because as melanoma cells they are really undifferentiated. Now if we put on these cells sirolimus, rapamycin they start again to produce melanin. So sirolimus is inducing again a differentiation in these highly differentiated cells. This is also confirmed by the ability to induce TYR activity, TYR expression. This is a western blot, these are control melanoma cells. No TYR expression. When we add sirolimus, when we add rapamycin the cells come back to express TYR. So these original observations suggested that sirolimus may act on cell differentiation.

Slide 35

So we decided to go to look at the mechanisms of cell differentiation. Now to do this we picked up a family of transcription factors. The Id family of transcription factors. This is a family of transcription factors that act through the inhibition of another family of transcription factors, the Helix-loop-helix transcription factors. Id proteins have been suggested to be involved as modulators not only of cell proliferation, this is the function of Id 1 but above all on cell differentiation and is the function of Id2.

Slide 36

Indeed if we take Id2 null mice, we can observe at the intestinal level a diffused loss of differentiation of epithelial cells. Indeed, this undifferentiation of epithelial cells will end up with a significant increase tumour incidence at the intestinal level. You see we have 96% tumours versus no tumours in the heterozygous or in the wild type animals. So Id2 is important in cell differentiation.

Slide 37

What happens in Id2 in our patients? This is Id 2 expression in normal skin. As expected I see Id2 expression in the basal stratum of epidermidis and in the dermal vessel, in the wall of the dermal vessel.

Slide 38

When we move to kaposi sarcoma you see all the red is gone. You don’t have any more Id2 expression. No more differentiation. You add back sirolimus and then again 6 months later Id2 expression is going back at the cutaneous level.

Slide 39

Now, of course, this could be due to withdrawal of cyclosporin but when we consider endothelial cells in culture and we add rapamycin for 6 and 24 hours, you see the gene expression for Id2 goes up significantly. That means that this transcription factor is modulated directly by sirolimus, by the effect of sirolimus. So with this background we demonstrated that sirolimus can influence, I mean, directly in human beings can influence cell proliferation, can influence angiogenesis, can influence cell differentiation. We don’t have that on viral infection but already this can be a perfect background to state that sirolimus may act also in other tumours.

Slide 40

Indeed this is the case. This was the first report from Barry Kahan. He compared his patients treated with sirolimus and he looked at the incidence of skin cancers, of skin malignancies. He demonstrated that in his patients the incidence of skin malignancies was 2.4%. He compared this incidence with the registry incidence of 7%. Of course, from the methodological point of view Doctor Opelz will tell us this is not extremely correct but this already gives us an idea on the reduction of incidence.

Slide 41

Something more sound from a methodological point of view comes from the work of Kaufmann. Also these are registry data. These are more than 30,000 patients. On mTOR inhibitors, either everolimus or sirolimus, mTOR inhibitors and calcinurine inhibitors or only on calcinurine inhibitors. If you look at all malignancies or non-skin malignancies, you see that patients with calcinurine you have a really high incidence of neoplasia here and here. If you look at patients with mTOR inhibitors alone, you see you have a significantly lower incidence of any malignancies and non-skin malignancies above all. But also when you associate mTOR inhibitors with calcinurine inhibitors, you have a significant reduction in the incidence of neoplastic disease. That means that the reduction in neoplastic disease is directly linked to the use of mTOR inhibitors.

Slide 42

If you look at these same data at multivariate analysis, you see you have 3 parameters that were independent predictors of development of neoplasia. History of previous neoplasia, of course, is a positive predictor. White versus non-white is a positive predictor. The use of mTOR inhibitors is a negative predictor.

Slide 43

The final proof we have it from a study from Doctor Campistol. This is the study where we participated also. This study is characterised by a group of patients that is randomised to stop cyclosporin after 3 months. So we have a group of patients with cyclosporin, sirolimus and steroids and we have a group of patients with only sirolimus and steroids after 3 months from transplantation and this is the non-skin cancer. The incidence of non-skin cancer.

Slide 44

Patients with cyclosporin and patients without cyclosporin only on sirolimus. There is a significant difference in the incidence of non-skin cancer.

Slide 45

We can move further and look at the incidence of all cancers; skin and non-skin. As you can see here, the difference between the two groups, sirolimus and cyclosporin and sirolimus alone is even more significant and again, here you are not comparing a group with calcinurine inhibitors with a group on sirolimus. Here we are comparing a group where you have calcinurine inhibitors and sirolimus with a group only on sirolimus. So this is very significant.

Slide 46

All these observations are also supported by the fact that different derivatives of new mTOR inhibitors, derivative of rapamycin are now in phase II trial for the treatment of neoplastic disease. You see here recurrent glioblastoma multiform. Here the use of temsirolimus, mantle cell lymphoma and also in locally advanced and metastatic breast cancer.

Slide 47

To conclude the introduction of these drugs in our immunosuppressive regimen may allow us to reach the ideal objective. We have always to keep in mind that we need to preserve allograft survival, of course, to allow a good a long-term renal function but we have also to remember that the ideal immunosuppressive protocols need to avoid morbidity and mortality that is usually due to common immunosuppressive protocols.

Slide 48

Now just let me thank the laboratory and clinical team at the University of Bari and the University of Foggia.

Slide 49

And I have also to thank another team that I think you should have know in the last month. Thank very much for your attention.

Slide 50

 

Chairman: Thank you very much for this very nice presentation. I have two questions and one comment if I may? My questions are first of all, there was a report recently from a Swiss group in Transplantation reporting on a patient with kaposi that did not respond at all to treatment with rapamycin. As you know, as we all know, negative reports are usually not reported. Are there other cases? Do you know of other patients who did not respond or is this a real exception or how often does rapamycin work and how often does it not work? Secondly, if it is so effective, you would think that patients who are non-transplant patients who develop kaposi nowadays would be treated with rapamycin. What is the experience there?

Dr Grandaliano: Ok, for the first question I have to answer that, of course, there is no therapy with 100% success and we are well aware of this. Actually, yes, I know another patient who is one of our patients. I showed you our experience of cutaneous kaposi sarcoma. We had some experience even if with fewer patients with visceral kaposi sarcoma. With visceral kaposi sarcoma we always treat the patient with chemotherapy and we switch them to sirolimus. One of these patients presented a relapse in the GI tract. So, of course, I mean I’m expecting patients not responding to sirolimus. Coming to the second question actually, since the paper was out we had a lot, of course, of question from oncologists as to treat or not the kaposi sarcoma. Of course neither sirolimus or everolimus so far are licensed for this kind of treatment and I think that as for other tumours the two pharmaceutical companies should go and try to start a phase II or III trial of course.

Chairman: Well my final comment is I have to a little bit dampen the enthusiasm here. I think the kaposi data are quite solid. I agree with everything that has been said about kaposi. The other data I think is very soft.

Dr Grandaliano: I know.

Chairman: Although it’s presented as strong. I have to tell you, first of all the Kaufmann paper, although as you have said there were 30,000 patients but they were not treated with sirolimus. Only 2000 of those 30,000 were treated with sirolimus.

Dr Grandaliano: And only 500 treated only with sirolimus.

Chairman: Exactly. I have to tell you that in more than 3000 patients that we have studied in the Registry we do not see a reduction of any tumour. So that I think the last word is not in and we’ll just have to wait and see, it’s a little bit too early to alarm everybody that rapamycin should be used to avoid tumours because we see a tumour rate that is identical to all the other patients.

Dr Grandaliano: I mean, of course, as for any other kind of evidence, I mean to have the best evidence we should wait for control trials.

Chairman: I agree.

Dr Grandaliano: I absolutely agree with you.

Chairman: I don’t think this supersedes everything else, I’m just saying even skin cancer we see the same increase in patients on sirolimus than in the others.

Dr Grandaliano: Now there is a study, there is a multicentre trial on skin cancer. So let’s wait for the results it’s already ongoing.

Chairman: I agree. Yes please.

Question: Congratulations first of all. Just a question. There are papers suggesting an interference of mTOR inhibitors on hypoxia inducible factor, which could be another mechanism, which can explain the anti-oncogenic activity. My first question is whether you have any study or are planning any study in this direction? The second question doesn’t concern exactly the cancer rather polycystic kidney disease. Have you looked at the growth of cysts in your transplant patients with polycystic kidney diseases treated with mTOR inhibitors? Because as you know, there are experimental studies showing that these agents can impede the growth of cysts in these particular diseases?

Our experience if 1-alpha is limited so far and is mainly linked to the DGF, to delayed graft function, to the ischemia reperfusion injury and there you can easily see that indeed, mTOR inhibitors can influence the expression of these transcription factors. By the way, these transcription factors, HIF-1 alpha is a helix-loop-helix transcription factor, so it’s activity can also be influenced by Id 2. So that’s an extremely interesting area like the polycystic kidney disease too. Now we have a fellow in the United States working on that signalling pathway and also in polycystic kidney disease such as also in diabetic nephropathy but what I don’t remember really, we didn’t do a review on our polycystic kidney disease there is a really good suggestion and I believe that this will be the first thing I do as soon as I’m back in Bari. Thank you very much Professor Ponticelli.

Chairman: Thank you very much I’m afraid we have to move on because we’re running out of time. This was a very stimulating talk. Thank you Doctor Grandaliano