So, our next speaker, we'd like to introduce Andrew Golodin. He is the winner of the Supervascular Section Best Scientific Paper, and he's going to be giving a talk on vascular targeting causing thrombosis in arteriovenous malformation animal model. Congratulations. Thank you, Mr. Chairman. I have no disclosures, and this research was funded by the National Health and Medical Research Council of Australia. So although many AVMs may be treated successfully with microsurgical resection or radiosurgery, approximately one-third have no effective treatment, and it's for these that newer treatment modalities are needed. One potential treatment modality is that of vascular targeting, which has shown some promise in cancer research. Vascular targeting is the method by which we identify a potential target on the luminal surface of the endothelium. We then administer a protein or antibody to bind to that target and deliver a treatment-effective payload. Now this is very easy in cancer research due to the inherent molecular differences of cancer endothelium to normal systemic endothelium. It's not the case with AVMs, which as far as we're aware, there are no discerning molecular differences. A way around this that is proposed by our group is that by establishing a volume with radiosurgery, we can create the presentation of certain targets on the endothelium. We can then target it with our protein or antibody and use this to deliver a prothrombotic compound causing thrombosis and ultimately vessel occlusion within the AVM nidus without affecting normal brain vasculature or the draining vein. The added advantage of such a technique is that we can also do away with the long latency period that can complicate radiosurgery. In order to study these techniques, our group uses an animal model formed by the anendocyte anastomosis of the external jugular vein to the common carotid artery. The resultant arterialized vein in micro-vessels is very similar to that of a human AVM from a hemodynamic and morphological perspective and also as best we can tell at an ultra-structural and molecular level. Using this model, we've been able to identify a number of potential targets. One of these is phosphatidylserine or PS. PS is an apoptotic marker of phospholipid that normally is located on the internal leaflet of the phospholipid membrane. What we have found is that in response to radiation, it translocates to the external surface which in the case of the endothelium means it presents itself as a marker in the luminal surface of the vessel. We've demonstrated this both with immunohistochemistry as well as in vivo immunofluorescent imaging where we have found that following radiation of our model at three weeks, there was an increase in PS translocation which became more pronounced with time. Interestingly, we've also discovered that there was a baseline level of PS translocation in the non-irradiated animals which presumably occurs as a result of the change in hemodynamics of the anastomosed vessel. So it's clear that PS may be a potential target and we've used this with a non-ligand approach with tissue factor and lipopolysaccharide and we've been successful in causing small vessel thrombus formation but as yet no large vessel occlusive disease. Furthermore, we can't give LPS and tissue factor in the human setting due to its inherent toxicities. So our group proposes that with radiosurgery and specifically ligand vascular targeting of PS, we'll be able to create avium thrombus formation and occlusion. And so our aim was then to assess the thrombotic effect of combined radiosurgery and vascular targeting. Now we could use an antibody to target PS but an alternative is the protein Annexin 5 which binds with a high affinity to it and we've conjugated this in-house to the prothrombotic molecule thrombin. So to recap, PS is normally on the internal leaflet of the phospholipid membrane. In response to radiation, it translocates to the external surface and we then systemically administer our Annexin thrombin conjugate to bind to PS and cause thrombosis and ultimately vessel occlusion. So following a six-week period of maturation after surgical formation of the AVMs, the animals undergo gamma knife radiosurgery with a marginal dose of 20-grey given to the model AVM. The targeting agent is then administered and finally an angiogram is performed and the tissue analysed. So in order to assess our Annexin thrombin conjugate, we performed a six-arm study to look at its effect both with radiation and also without and to compare it to the control arms of thrombin and saline with at least eight animals per treatment arm. This angiogram is from one of our control animals and the tip of the catheter is located within the common carotid at the point of anastomosis and it demonstrates a flow of contrast retrogradely along the external jugular vein, ultimately draining into the opposite side. Now obviously what we want to see is blockage of this system and that's exactly what we saw in a number of animals. In this example, the tip of the catheter again is in the common carotid but it demonstrates no flow in the external jugular vein but rather a distal physiological pattern of flow within the common carotid artery. In other animals, the complete occlusion was less clear but what we did see was the presence of stenotic areas or filling defects within the anastomosed vessel which on inspection were revealed to be large calcified thrombi. So I think no one would argue that we're clearly seeing an effect of our conjugate here with the only animals demonstrating angiographic abnormalities of occlusional stenosis being those treated with the Nexen thrombin in either the GKS or SHAM group and accounted for roughly 60 to 70 per cent of animals and these were statistically significant when compared to the control arms but the question remained over whether we could substantiate these findings with histological data. And certainly when looking at large vessel thrombus formation such as the EJV, there was evidence of thrombus within both conjugate treatment groups and the GKS thrombin group matching nicely with the angiographic data and these were statistically significant when compared to the saline controls. A much more diffuse appearance of microvessel thrombus was observed present in all of our treatment groups to the exception of the SHAM saline group but this was not unexpected and is believed to be a combination of a result of either the radiation effects or the prothrombotic compound that we are injecting. So it's clear that we do have an occlusive effect of our conjugate however in the data so far we do not have a difference between the GKS or the SHAM group and this may be because of the baseline PS translocation we observed in the non-irradiated animals. So our group postulated that perhaps with dose modification we might be able to differentiate between those two groups. So we conducted a further two experiments to look at halving the dose per weight of our conjugate per animal and giving it as a multiple dose regimen in line with other targeted cancer treatments. And what we found was a statistically significant difference between the GKS arms and the comparison SHAM arms and in fact in the double half dose conjugate 80% of animals had evidence of occlusion or stenosis compared to none in the SHAM comparator group. We also found evidence of thrombus formation within all four of those treatment groups so that was not unexpected from our earlier research. So in summary we've got clear proof of principle here of vascular targeting causing occlusion of our model AVM but furthermore we actually have evidence that dose modification allows for increased selective targeting of only that endothelium that had been pre-treated with Gamma Knife and this is actually the first example of thrombus formation and occlusion in a high flow AVM vessel with vascular targeting. Now we are one step closer towards clinical transition but further work is needed. We need to better understand what the distribution of PS is in human AVMs and perhaps consider other targets. But in conclusion GKS and an exanthromic conjugate is effective in causing flow cessation in our animal model of AVMs and ligand vascular targeting with GKS may be a promising new treatment in brain AVMs. So in the clinical setting when we are faced with these large deep high grade AVMs where microsurgery has too high a risk and radiosurgery may be ineffective, perhaps our new missing modality is vascular targeting. Thank you. We will again allow for one question while the next presenter comes up. Really interesting work. So I'm intrigued by the at least partial thrombosis you saw without the Gamma Knife in your sham AVMs, correct? Yes. So I mean why not consider even using this treatment without Gamma Knife radiosurgery in some cases? We've certainly considered that, particularly with the dose. But I suppose the concern again is the distribution of PS translocation. So with our further work we do need to be careful that PS is only translocated within the AVM nidus. Right. That was a question I had. Was it outside the AVM nidus in your animal model? So externally we saw no evidence in our initial dose escalation studies of systemic thrombus formation, which is reassuring, but we're still not aware of the extent of PS translocation. Okay, thank you very much.

vascular targeting

thrombosis

arteriovenous malformation

AVM

treatment options