[speaker001:] er on angiogenesis as an indicator of prognosis for invasive bladder cancer presented I believe by erm and representing er colleagues from Bristol, er Oxford and Sheffield. Thank you....
[speaker002:] Mr Chairman, ladies and gentlemen, good afternoon. The relationship between tumour and blood supply has been noted by surgeons and indeed some physicians for centuries. In his thesis on blood, inflammation and gunshot wounds, published in eighteen twenty eight, John Hunter stated that in disease in which there is an increase in the part, as in tumours, the increase in vessels is conspicuous
[speaker001:] [cough]
[speaker002:] Now, our understanding of this relationship has increased dramatically over the past few years and our knowledge of new vessel formation or angiogenesis has also increased. And it is has been shown that angiogenesis is in fact essential to tumour growth. Tumours er greater than two millimetres in size require their own blood supply. It is also essential for metastasis. Now John Hunter noted there was an association between the quantity of the vessels and the tumour and recently this has been er shown with micro-vessel quantification to be an important problems to indicator
[speaker001:] [cough]
[speaker002:] tumours.... Now, we've used this technique in invasive bladder cancer, and we've quantified the micro-vascularity in a group of invasive cancers and looked at the prognosis and metastasis. We found no res relationship between angiogenesis and metastasis but the rest of this paper will er concentrate on its role in prognosis....
[speaker001:] [cough]
[speaker002:] Forty five patients we looked at, age ranged fifty to ninety one, had the mean age of seventy three. Now follow up ranged between one and fifty one months.... All tumours were solid or had elements of er capillary and solid tumours and, routine sections were stained with a standard aminohistochemical technique, using anti C D thirty one anti-clotting. Now C D thirty one is an endophilial cell adhesion molecule and the antibody has been shown to be the most specific marker of vascoendophilia and consistently in studies has stained more vessels than other markers. We estimated the count er by looking at each individual section at low power and collecting the three most vascular areas. Then with a twenty five point chalky eyepiece graticule er the counts were made at high power. Now when you look down the microscope through the graticule what one sees is twenty five randomly spaced dots overlying your er section. If you rotate the graticule such that the maximum number of dots covers erm a stained and then you count those number of dots, that's the count. And we did that in the three areas, so we used the sum of three vascular counts in the analysis. Now we looked at both capillary tumours and solid tumours, but as this pictures demonstrates, we're not y er able accurately to determine the count of capillary tumours. This is the stain that you get, it's, it's black showing vessels. Now these are all vessels and these aren't the ones that we're interested in, we're interested in the small tumour induced vessels. Because we weren't able to define with any confidence areas of neovascularization in capillary tumours we've based the study on those tumours with solid er solid tumours or those with solid elements.... This is what you see when you do er the staining, on the left is an area of low count. And I don't think, if you can just see small areas of vessel stains, this has a erm only a few vessels present within the solid tumour. But on the right an area of a high count where a great deal more of vessels are present.... Of our forty five patients nine had T two tumours and thirty six had T three tumours. Thirty three had a solid morphology and twelve had a mixed morphology.... Now we performed uni-variant analysis looking at all the parameters shown on the left. We confirmed, which was j with a bit of luck, that T two tumours do significantly better than T three tumours, P value of nought point nought nought two. We also showed that grade two do significantly better than grade three, but most importantly perhaps is that we showed that patients with a vascular count that are less than twenty one do significantly better than patients with a vascular count of greater or equal to twenty one. P value of nought point nought one. We showed no significant difference between solid or mixed tumours and significant difference between tumours which had diploid picture or haploid picture on a D N A analysis. We then placed all these parameters in a multi-variant analysis. The only two that remained significant were stage and vascular count. In other words, vascular count is a significant, independent prognostic er indicator in this group of invasive bladder cancers.... Look at the survival curve of the vascular count, percentage of surviving up the Y axis and time in months on the on the X axis. [clears throat] These two curves are significantly different. The top curve, in yellow, is for counts less than twenty one, now the four year survival for this group is around forty three percent. With a median survival of thirty five months. Compared with those counts of greater or equal to twenty one, which is the white curve, greater or equal to twenty one, white curve, which is the lower curve. And this four year survival of only twenty three percent with a median survival of around nine months.... Now to calculate the hazard ratio on these two groups, those patients with counts of greater or equal to twenty one were two and half times more likely to die of their disease than those with counts of less than twenty one. In a paper that was published in nineteen eighty five, Journal of Urology, Shipley looked at prognostic factors in invasive cancer. The overall er five year survival for all, all invasive cancers are around about er twenty percent er twenty eight percent. But within this group he looks specifically at solid tumours and they only have a four year survival of twenty percent. So using vascular counts we are able to define a group of patients that do significantly better.... Therefore this simple technique of counting vessels as an assessment of angiogenesis does provide us with additional prognostic information and is good for patients.... And in summary, Mr Chairman, ladies and gentlemen, we would suggest that the assessment of angiogenesis in bladder cancer is an important and promising er prognostic indicator and has therapeutic implications. Not only in being able to select a group where perhaps we can justify more aggressive therapy, or even a group that perhaps we should just treat palatally but also in the future with the development of anti- angiogenic therapy.... Thank you very much.
[speaker001:] [clapping] I'd like to start the question briefly with two points, firstly, you had forty nine patients?
[speaker002:] No. We had forty five.
[speaker001:] Forty five. Y you must have had many more than forty five
[speaker002:] Yeah.
[speaker001:] tumours [LAUGHTER] over the last
[speaker002:] Yeah.
[speaker001:] year or so [].
[speaker002:] Th we're limited to solid tumours or patients that have an element of solid tumour on histology. We did in fact have sixty eight patients, unfortunately erm thirteen of these insufficient biopsy material for us to do the count erm and
[speaker001:] Mm.
[speaker002:] erm we excluded all the superficial ones that had solid because it was insufficient er one patient we couldn't get the stain to work.
[speaker001:] Right. Just that multi-variant analysis on forty five leaves you with some very tiny subgroups.
[speaker002:] Yeah. I it does. We weren't erm we weren't able to do a multi-variant analysis with the grade because we didn't have a sufficient number of, of the grade to do it. But we er the, there were sufficient for all the other.
[speaker001:] Microphone five, please. a question. Mark from Liverpool. The way that erm one might have effective treatment for muscle invasive bladder cancer, if one uses radiotherapy, is maintain a decent oxygen supply to the tumour and if one uses chemotherapy to deliver the blood to the tumour. Have you got any information about the efficacy of the treatments that were used in those?
[speaker002:] We haven't we haven't analyzed the results according to the treatment that the patients received. Erm we know that three patients had cystectomies and one er that then the remainder have radiotherapy but I haven't analyzed the results dividing them into curative or potent radiotherapy.
[speaker001:] And none had chemotherapy?
[speaker002:] Erm, I think one had, one had chemotherapy. There, we hadn't got enough in the group to, to make that erm analysis.
[speaker001:] Microphone five, again. Er, Glouston. I noticed your survival curves tend to er come together er in about four years, and you're not attending in that direction.
[speaker002:] Well, then all survival curves will eventually
[speaker001:] Well, of.
[speaker002:] come together won't they?
[speaker001:] Now did you, did you find that er were you ab were you able to repeat any of the biopsies and see whether in the er in the er group with the less vessels more, more vessels were er accumulating? Er?
[speaker002:] We haven't looked at any of the repeat biopsies, no. We didn't look at them, we just chose death as an end point because it's the easiest end point to choose. So we looked at just the,th the histology of the patient and related it to the prognosis. We haven't looked.
[speaker001:] Microphone two, please. Er Joanna from Middlesbrough. I just want to ask you about the actual erm methodology, which sections did you examine? And you said you just chose the three most vascular, could you tell me a bit more
[speaker002:] Yeah.
[speaker001:] about that?
[speaker002:] The section we chose were the ones that were chosen by the pathologist because they were the best representative sample of that tumour.
[speaker001:] In what respect?
[speaker002:] In, in being able to stage or grade them.
[speaker001:] In what respect?
[speaker002:] I don't know, what do you mean by in wh
[speaker001:] I mean th there are pretty clear cut guidelines for doing morphometric analysis of erm tumour erm er tumour structure. You know, cells, erm vascular tissue, connective tissue matrix. Which particular technique did you use? Because you just seem to have chosen the things by looking, not by actually randomly sampling from the tumours.
[speaker002:] W we cut,th the path pathological er er the where the blocks come from, they're graded one, two, three, four. One is the best example from the tumour that came from the reception.
[speaker001:] So that was a na er that was a naked eye examination?
[speaker002:] Well we know that from the repo the initial report
[speaker001:] Mm.
[speaker002:] done by the pathologist. He'd designated that was the best block. We cut that section, we stained it and then looked at it under low power and selected out from that block the three most vascular areas. We could have chosen the three least vascular areas but then the counts wouldn't have been, would have been much more difficult..
[speaker001:] Yeah, but why wouldn't they have been much more relevant? Because y you, obviously you've presented different information in your talk this afternoon, you've given us more information that was in your abstract, so the figures are different, where you, in your abstract you just talked about differences between G three, T three tumours.
[speaker002:] Well in the abstract we were loo we, I bought them and decided whether they are high or low counts, we now have a way of quantifying that, we have a way of counting each individual er vessel. So we're can no we can number them as such.
[speaker001:] Well, ha, there's no point counting things unless the counts are relevant to the condition you're looking at. If you can just look at the naked eye appearance and choose three areas that you think are most vascular from a one-off block.
[speaker002:] No. They're not it's not just choosing th they're stained
[speaker001:] It's not going, going to be representative.
[speaker002:] the areas are the most stained. They're the most strongly stained areas, they have the most vessels, therefore you choose to count them.
[speaker001:] Yeah and I think were not, we'll perhaps talk afterwards. Can I suggest you do so? Yeah. [cough] Microphone one please. Er in use of other you should have the difference in survival between the patients that had more than twenty one or less than twenty one but actually it er you didn't show us the survival curves for [cough] reaction against the survival curves er with the G er pathology grade and the G with the tumour and to see actually the difference which we can, in curves which we can deduct from counting the vascularity.
[speaker002:] No. We chose just to show this one curve of vascular count cos that's what we think is important.
[speaker001:] Cos if we have seen the, the same survival curves according to the er pathology so it'll have been er more that G three is worse than G two but er there is a difference in your curves between the grading quality and the vascularity. We know now that it's really needed to count the vessels because we will get definitely more information.
[speaker002:] Well the uni-variant analysis and the multi-variant analysis shows that i it is, adds additional information. I didn't show you the, the grade and the stage curves because we all know what they look like but what I'm saying is that by doing the count provides us with additional information on this group of patients. More than we can gain from looking at the grade and the stage.
[speaker001:] I think the real nitty gritty of this question is, given a T tumour can you pick out the good from the bad using this technique? And can you give us the figures to support that?
[speaker002:] Erm... from a specifically, no, what I we can pick out the bad solid tumours and the good solid tumours.
[speaker001:] Yes but you accepted that T staging was still the strongest prognostic indicator
[speaker002:] Yes, it is.
[speaker001:] we will all continue using that. If we add to that your technique
[speaker002:] Right.
[speaker001:] will it benefit us and the patient?
[speaker002:] From this pilot study I cannot say yes or no, but what I can say is that this pilot study suggests that it will
[speaker001:] It may.
[speaker002:] It may. A and numbers in our, a s in a stage group too small to say definitely that we can pick out those T two tumours badly or well on the vascular count. But what we can say is that we can select out from the group of solid tumours the ones that do badly.
[speaker001:] Can we look forward to your poster next year with a couple of hundred T two patients? [LAUGHTER] [LAUGHTER] [] Mm. Have you another question? Yes, I was just wondering, er raise one point. The C D thirty one staining, is that just identifying blood vessels? Or is it possibly staining blood vessels that, that are under attack by vascular invasion?
[speaker002:] Erm... I can't answer that. Erm because I, I know that er cell adhesion molecule C D thirty one is present in all erm vascular, vascular endophilia whether it's, it's erm er expressed in more erm vessels average...
[speaker001:] Thank you very much indeed. Thank you. Oh, we've got one more quick question. Oh, two. Okay. Microphone one please. Come on. We've got just er two minutes. . If this is another exhibition of the intercapillary distance in er progno in defining the prognosis