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So I sort of have a little.

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So I'm delighted to have a guest speaker today, Simon Bouzouki, who many of you will know he is a colorectal surgeon.

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Quite a successful academic at Cambridge.

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I've come across him through a Cancer Research UK.

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He's a very good work, which, uh, made him win two awards from Cancer Research UK,

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one initial clinician scientist and then the second more recent and advanced clinician scientist.

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I won't steal his thunder, but he's got a very exciting research programme, clinical practise focussed on laparoscopic and colorectal procedures.

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And he was telling me yesterday that he also leads something which is new and important from the US,

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called out the digestive cancer programme and bowel well.

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So maybe he can tell us more about that. Um, he's he's lab programme.

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He's mainly about the um, the whole sub clonal interactions play on colorectal cancer cell identity and behaviour.

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Um, and we'll hear more about it. So I won't dwell on that.

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But a very warm welcome. Uh, Simon, and uh, please thank you very much, Prof.

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Thank you very much for the very kind invitation to come and speak.

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I I'm talking on net, which really are not the main focus of my clinical practise or my scientific focus,

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but I thought they were interesting because they're some.

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They're interesting from a surgical perspective, and there's a lot of very interesting science around net biology,

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which is being very, very poorly understood and poorly explored.

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So I thought I'd initially just give a little bit of a background to on my

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clinical practise in the scientific practise and the set up we have in Cambridge,

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just to put everything into somewhat of a perspective. So our unit is similarly sized to the unit.

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Here in Oxford, we have six contractual consultants who are doing purely colorectal colorectal surgery.

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We also have three emergency general surgeons who are also fully embedded into our department.

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So a quarter of their practise is EEGs work, and three quarters of it is colorectal surgery, including resection or what.

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So we fully embed them and then we have one full time academic myself.

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The department has a full spectrum of subspecialty practise.

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There's a pelvic exam, iterative service, which many of you in the field when I was led by Nicky Fern had supported by James Wheeler,

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who were both Oxford trainees originally, as well as John Moores and Justin Davis.

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Our pelvic floor services run by John Morton and Michael Powell.

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Early Rectal Cancer with Thames is run by James Wheeler and Nigel Hall, who have an anal cancer service,

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a newly developed endometriosis service together with our gynaecology colleagues and

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also the net service and the net service is really led solely by myself at the moment.

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We have a new colleague who will be starting next month, who will be joining me with Nat Resections,

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which will help because there's just not enough capacity for myself to do all of the net actions, as well as the standard cancer section of work.

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So similarly focussed, I guess, to what you have to wear based upon the Cambridge biomedical campus.

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And I don't know whether many of you been up to Adam Brookes recently, but it is expanding at an ever increasing rate.

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The biomedical campus is now the largest by health related biomedical campus in Europe and has a very strong cancer theme to it.

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Some of the new developments it is the hospital is based over here and so extends off the right hand side of this photo.

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There's a lot of new scientific developments and industry on site,

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so this building in the shape of a chromosome is the new laboratory of Molecular Biology, which opened about four or five years ago.

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This is AstraZeneca as worldwide headquarters for research and development, and this is Royal Papworth, which finally,

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after what seemed like an eternity, has finally moved from that village site in Papworth to being on campus with us.

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This is the senior UK Cambridge test tube, which is where most of my research has been done to date,

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and I'm in the process of moving into this building, which is the STEM Cell Institute, which is being relocated from being in town up here on site.

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There's also the MRC Hutchinson Cancer Cell Unit and the Cambridge Institute for Medical Research.

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There's going to be new children's hospital developed on this site over this site, which just got government support,

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and we're in the process of applying on this site to develop a cancer research hospital.

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But that will not have any surgery. Than it so you can.

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So there's clearly a very strong co-location of clinical and scientific disciplines on campus,

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which very much helps in interdisciplinary approaches to research.

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We have strong funding from the Biopsy and Cancer Research UK and we're seeing UK major centre and and as I say,

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with aims at being on site and MedImmune as well as outcome, we're having an increasing amounts of collaborations with industry.

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The cancer focus is interesting and worth mentioning,

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and that is there's not a strong GI focus on sight and this is historical based on the peers who are present there,

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and it's predominantly breast, ovarian and early detection. So my science so I said my focus is mainly on colorectal cancer biology,

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and it's about about understanding the interlinked behaviour between cellular identity and

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behaviour in cancer and understanding why cells transition from one state to another.

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And we're particularly interested in cells have received little focus to date differentiated cancer cells,

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but we've identified that subpopulations of these can acquire up to the capacity or so interested in tumour evolution.

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But today we're going to be talking about small intestinal net biology, which I became interested in when I started to do net resections clinically.

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The techniques we use in the lab are very much based on using primary tissue,

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and that's why it's key to have your science based on a closed location to where you're carrying out your clinical practise.

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We do a lot of organoid biology using CRISPR targeting on normal organoids.

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Singles out work so single cell RNA seq. We use a technique called lineage tracing, which I'll come on to shortly,

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and I'm increasingly interested in using game theory to understand clonal interactions.

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So nets to give you some background to them.

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They they are very rare.

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They used to be called carcinoid, however, of about sort of 10 15 years ago, the nomenclature changed to neuroendocrine tumours.

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And these are this is this sort of figure on the left shows the common sites below the diaphragm to find that the three common places really are long,

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small intestine and pancreas. And then to talking about the small intestine on.

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That's the reason I picture Steve Jobs up is many of you will know that he died from pancreatic cancer,

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but he seemed to be in the news about his pancreatic cancer for many, many years.

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And if he truly had a sort of pancreatic adenocarcinoma, he would've passed away much more rapidly.

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But he actually had pancreatic net, and that's part of the reason he lived for so long.

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I gather he didn't even have conventional therapy for that. So as I said, the very fact that they're rare, but they're also very, very slow growing.

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So these this table shows the five year survival rates for nets with both local and distant disease today.

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And we're talking about our small intestinal nets and you can see that even if you've got liver metastases with just standard of care treatment,

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there's a 54 percent five year survival rate. And this impacts very extensively your clinical decision making,

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and you can see that these vastly outweigh the equivalent survival rates for patients who've got adenocarcinoma with distant disease.

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So what do they look like? So this is a picture from a publication showing a typical net in the small intestine.

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I'd say that actually, in my experience, a lot of the primaries are much, much smaller than this,

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and you can't often even be able to detect them from the outside the small intestine.

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The only way you can actually identify them is through by manual palpation, and they are often multifocal.

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So you will have met acronis tumours throughout the ileum that you can detect by by Mannu, by, by Emmanuel palpation.

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You might think, Okay, well, that's pretty easy to take out, and these primaries are not the problem surgically.

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The problem is the mes and taric spread, and this is a resection specimen,

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not mind of a patient to use had the primary MI, but also with the mesenteric lymph node spread.

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And you can see here that all of these small bowel loops have been pulled into this and secularised into this large mesenteric deposit,

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which has significant plasma plays in making the resection of it challenging.

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It's also not uncommon to come across this upon opening up the abdomen and you find bowel that you

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would never leave in if you were closing and you think the patient was going to wouldn't survive.

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But they have chronic ischaemia with and you open up and you see that the boundless,

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unbelievably unhealthy and this chronic ischaemia causes significant bowel thickening and subsequent obstructive like symptoms.

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These tumours are really rare, so they, if you take in context about colorectal cancer, is going to affect what between one, 14, one and 15 of us.

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These occur in about one in 100000 patients. And because of this very slow and indolent growth, they often present very late with metastatic.

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Spread or mesenteric deposits? We probably all remember carcinoid syndrome from medical school, and they can also present with those symptoms.

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The the treatment of them is is complex and requires a very well-functioning MD.

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Clearly, surgical resection is is is the mainstay of treatment with curative intent.

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But also involved is somatic statton analogues such as octreotide and unretired and for liver disease,

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radiofrequency ablation, embolisation liver resection or even indeed, liver transplantation.

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Chemotherapy is only really of benefit when these tumours differentiate and start to become rapidly

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prolific because of the nature of chemo only really working on cells that are rapidly dividing.

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So the science, though, is absolutely fascinating, so unlike colorectal cancer or other adenocarcinomas, we know so little about these tumours.

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We don't know what the cell of origin is. We don't know what the driving mutation is.

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We don't know what the what the aspect of epigenetics is on that development or how these tumours evolve and progress.

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And there's lots of very low hanging fruit related to the science of these tumours

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that make them very attractive to study a little bit more clinical detail.

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Briefly, on how these tumours are present.

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So they said they present late, they have obstructive symptoms rather than problems secondary to the primary primary.

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The obstructive symptoms are secondary to ischaemia. I would say that live in actual pain isn't a common way that these patients present,

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but I have had one or two patients who presented with liver capsule pain and deranged liver function tests,

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which are then subsequently be investigated and found to have metastatic disease.

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And this demonstrates the characteristic flushing associated with carcinoid syndrome.

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We use enacts guidelines to manage, work up and develop subsequent follow up pathways for all our patients.

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And if you're interested in that and very much strongly recommend looking at these guidelines, which are available online at the link below,

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they need to have a baseline Cro-Magnon and a which is one of the best serum

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tumour markers to identify for patients for to might develop further recurrence.

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A 24 hour urinary five hydroxy in daily acetic acid is a prerequisite to identify and make the diagnosis.

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Staging radiological is key. But most importantly,

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in relation to identifying the anatomy of the vasculature that supply the the primary as well as the mesenteric

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disease octreotide scan is very important to identify occult peritoneal disease or liver metastases,

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but cannot on occasion be equivocal. And in those situations, a pet scan can sometimes give you the key in relation to surgical work up.

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An Echo is also important to identify or call carcinoid valve disease, which is again is not uncommon in these patients.

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And as I alluded to earlier, it's very important to have a functioning MDT with all of the associated subspecialty support.

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Staging is really quite simple. There are different staging types that have been put about.

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These are the innate stagings and as you can see that a tumour stage is based purely on size

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and invasion and the Anandan binary zero one and you have your stages of zero to four.

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So very straightforward staging. It doesn't really help, though, in the management, to be perfectly honest.

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So the general principles,

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as with any sort of solid organ malignancy for if they haven't got metastatic spread and they have limited nodal spread the game for for curative.

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The aim for curative resection is radical resection with purity intent.

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So resection the primary and radical launchpad.net to me to remove those mesenteric lymph nodes that are involved,

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there is some discussion as to whether this is safe to be done laparoscopically or open.

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The current recommendations and what I would agree with is that for anything other than respecting

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somebody who's got limited primary and mesenteric disease and they have metastatic disease in the liver,

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then only in that situation is a laparoscopic approach appropriate.

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And for all other approaches, there's this should be done via an open resection.

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And that's partly so you can make sure you get out all of the Mes and taric disease,

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but also due to the multiple Scality of the primaries and various techniques have been

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looked at imaging and endoscopic to identify whether to pick up these other primaries.

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But it's been shown that only by manual palpation has the highest sensitivity to pick up other primary,

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so you have to do it open if you've got metastatic disease.

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Things become really quite complex, and it's partly related to the tumour biology and how slow growing these are,

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and you have to tailor what treatment you're going to be offered to,

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how long the patient's likely to live with those disease, and they can live for many, many years with relatively stable liver disease.

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So there's various options for the liver, as I mentioned, and there are various palliative options for the primary the mesenteric disease.

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If these patients have obstructive symptoms, chemo, as I mentioned, is possible.

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Patients have highly proliferative disease,

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so this patient on the right is actually a consultant colleague who I operated on about three or four years ago,

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and she had a palliative laparoscopic right. And you can see she's got very extensive liver disease.

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She's now had stable disease, I believe, for about three years.

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And if that continues, then she would potentially be a candidate for our first liver transplantation for metastatic net.

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So this is a sort of, I guess, one of the key slides in relation to the surgical decision making,

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and these are the questions that go through my mind when I see a patient in clinic who I'm trying to work out for,

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whether they are, they can be resected with curative intent.

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So as with any patient, he first see, the key thing is, are they fit for surgery and are they going to engage with the process?

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And I think patient engagement with the decision making process is absolutely key.

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They've got to buy in to all of the disasters that are associated with radical mesenteric resection.

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They have to understand, albeit there's only a small chance of it.

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If you if you work at these patients correctly, that things can go wrong in the operating theatre, they could end up with shortcut.

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They could end up with a high output judge, an ostomy. They could end up on lifelong pain.

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And I don't think that you should embark on these operations where you're high up

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on the asthma and asking the if the patient hasn't bought into that whole process.

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So the other question is how symptomatic they are from their primary and these enteric disease.

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And then the question is whether or not this is respectable.

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Some studies have shown that if you there's invasion of the mesenteric disease into the retroperitoneal structures such as the duodenum,

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then that's a contraindication to surgery. I wouldn't say that's necessarily the case.

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I think there are patients as one to talk about next, where you can go ahead and perform limited duration activity associated with the resection.

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You can't pick up the doesn't apply to very easily on imaging. So that needs to be sort of taken into account.

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But the key bit here is that in relation to the number of proximal branches of the estimate that that are spared.

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So this is the Uppsala staging system, which I lifted from this chap's PhD thesis and the nice drawing he's got.

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And they describe these four stages of mes and disease one to four.

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Again, I think this somewhat oversimplifies the process, and I think you have to have a tailored, personalised approach to each each patient.

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Generally, as a rule of thumb, if you have three spare judging branches that should leave you with 100 centimetres of small bowel left.

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But apparently, these are the emoticons for grimacing. I didn't say that, but it can be.

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It's not always. It's not always an exact science.

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So you so ideally you probably want more of those, but anything less than three,

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then I think you're on a hiding to nothing with relation to not having enough bowel related debunking,

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which is something we shy away from in our bowel.

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Adenocarcinomas can be used for patients who have got larger disease,

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so very much applying the principles of going to oncology to an intestinal tumour.

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So I'll give you two examples of patients who we've operated on just to sort of give you a flavour of some of the

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difficulties and in relation to clinical decision making in relation to patients in the surgical approaches that we use.

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So this this first patient was a 70 year old gentleman who presented initially

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with urological symptoms and then went on to have imaging for his haematuria.

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And he had a primary which have highlighted this is this amazing terek deposit in red.

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The primary was in his ileum, and you can see the bowel lips are tethered in some fat surrounding around the median direct deposit.

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And then green is his duodenum, which is of tented up, potentially involved in the and Tehrik deposit he had.

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He was pretty worked up quarantine ATS guidelines.

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He had his urinary levels measured and he would start on unretired, but he had no metastatic disease.

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And given that he was getting obstructive symptoms, we we looked at him with a view to radical resection.

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So I mentioned before that the key aspect with these patients is identifying the arterial and venous anatomy associated with the deposit.

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So this reconstruction of the SMA demonstrates, you know, you can see that the the the vessels here,

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the distal part of the asthma are all being pulled in to the tumour and the tumours have just highlighted by this little area of calcification.

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The tumours don't appear very clear on on on CT.

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This is the same imaging from the same patient cross-functionally and on the right.

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What I've drawn on here is highlighted actually the area that was involved by the Meis and Tehrik and primary deposit,

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which basically were just one in the same.

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And you can see here we got one to three, possibly four, but that's probably going to go in the resection margin.

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So three three judge panel branches and a duodenum that was involved, but we deemed him suitable.

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After getting a second opinion from Southampton for radical reception.

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So what are the what's the surgical approach these patients towards the latter end of my my training?

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I was fortunate enough to go to visit this gentleman and along and in Bavaria, which is a really beautiful part of Germany,

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I must say so it's a lovely place to go to this lovely university city outside of Nuremberg.

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And this chap here unusually tall and not that I'm small, he's unusually tall,

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is is is a chap called Verna Hamburger and he's the head of the department.

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And he developed this technique called complete musical excision, which is a very and he used it for adenocarcinomas of the large bowel.

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And it's a much more radical limp anatomy and a very close, sharp dissection along Ember Logical Planes.

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And he's published his outcomes in in various journals and improved five year survival rates for patients with,

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even with early stage, the old school duke's achievements to approaching almost 99 percent.

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It has come under some criticism more recently because of the morbidity associated with the procedure,

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but it was extremely useful to go out there and see him operate and start to learn the technique.

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So the technique that we use is applying say a bit for these NAT resections.

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And these two images from one of Hamburg is descriptions of Sammy describe the essential steps.

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So here you've got the caver in the aorta and this is the duodenum, not the Koran.

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So you do a very radical full cocker ization of the genome to bring it up out of the the wound so you can access on the right

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the root of the Atom A and the assembly to identify your whether you can actually reset this and leave enough bowel intact.

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So we mobilise everything. And then we go, obviously go above to then get access to this area here.

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So next case is going to describe this is again,

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sort of shows some of the interesting intraoperative decision making that you have to make and the importance of working well with colleagues.

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So this was a 63 year old lady who presented with typical features of carcinoid syndrome.

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She was investigated by our numerology colleagues and was found to have multiple liver lesions.

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Of note, her urine levels were extremely high. So that's a very high five level and again, a very high associated Cro-Magnon level.

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She got obstructive symptoms, however, from her primary and her actual primary, mesenteric disease was relatively minimal.

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But you can see on this opposite side scan, she's got significant liver disease.

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So because of her obstructive symptoms and otherwise good health, we plan to do a palliative laparoscopic right.

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Amick Let me say she because of our high, high levels.

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We gave almost all our patients have this. They have an octreotide infusion Perry optimally to reduce the chances of a carcinoid crisis happening.

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We gave her a bonus, followed by quite a high infusions.

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This is 10 times higher than what people have previously described as the infusion required and based on current literature,

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the incidence of carcinoid crisis with this degree of octreotide infusion is only about two percent.

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You can see what's going to happen. She got converted due to plays laser.

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As soon as we started manually handling the tumour,

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she had a pin drop to arrest and we felt that she'd had a customer crisis looking under the drapes.

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Everything a skin was bright red. The endocrine colleagues came into theatre and were in complete agreement.

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She'd had to interrupt her carcinoid crisis. So we were then left with this very awkward surgical dilemma of an otherwise fit

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lady who has had an arrest on the table would mobilise the bowel by this stage,

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but hadn't actually divided any vessels or got the tumour out with obstructive symptoms and really imminently about to obstruct.

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So what options were available? We could take her to ICU, bring her back, then stabilise her, bring her back the next day.

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But the concern was as soon as we start mobilising this tumour again,

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we're going to be in exactly the same situation we could the function of bring a loop out upstream of the primary,

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which is one option we could close and not do anything and just stick around assays and treat it punitively.

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So lots of things to think about and lots of discussion with with with colleagues,

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with endocrine colleagues, with anaesthetist, with critical care support. And in the end, we did that.

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We did a very quick smash and grab right Hemi colectomy and got the tumour out within five 10 minutes.

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With with with relative stability and obviously didn't join her up and gave her an end.

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I lost me and she actually did very well following this, and she's gone on to have treatments for her liver disease.

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However, the moment this person, this patient crosses the threshold into Addenbrooke's, she seems to develop a carcinoid crisis,

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you seen that even looks at like a medical instrument, and she has a constantly crisis.

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They try to do an RFA on her, and even with the local anaesthetic, she started about constantly crisis like symptoms.

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So she sees me back in the clinic and she's desperate to be joined up, but I'm less keen.

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So in summary of our clinical experience, I'd say that is really key to work as a team.

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You need a dedicated and experienced in these tests. You need a lot of endocrine support, octreotide infusions and infusions.

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And really, I think open surgery is the key unless you're doing a palliative, a section with minimal disease.

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We use CMMI resection temporary slinging of proximal vessels to adapt to be absolutely certain that you're not going to lose a lot of small bowel.

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This is my friend. The ligature impact I think it's really useful for for with the decimal places in the country,

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and you also need to have, aside from the ligature, you need to have good friends available for when things get hairy.

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So moving away from this, well, moving away from the clinical side,

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I'm now going to just touch on some of the biology, which I said is really fascinating.

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And if there's anything you don't understand to ask me, it's all really unknowns, as I alluded to earlier,

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and this is partly due to the paucity of tissue for analysis and how rare these these tumours are.

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So most of the larger scale studies have only really been approaching around about 100 patients.

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The genomic studies of the very early ones identified that there was some somatic copy number aberrations and there was a loss of chromosome 18.

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But interestingly, when they looked at chromosome 18, where the usual suspects, such as samat four are located, none of these genes have been mutated.

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The probably the biggest study, which was this one published in Nature Genetics,

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which used whole exome sequencing on a panel of 100 small intestinal nets identical in the keys.

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In the title, they suggested that CDK scan will be with a was a commonly lost trunk or mutation in nets.

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But actually, when you get into the details of this paper is not common,

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and it was only actually present in around about 10 to 15 percent that tumours.

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So I wouldn't say that it's a it's a driver of of nets the the role of epigenetics,

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but methylation histone modifications microRNAs is largely unexplored.

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There have been a couple of studies that have looked at methylation identified this global hypermethylation associated with nets,

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but again, no specific genes and microRNAs.

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Like most microarray papers, they come up with a list of four or five that they think are involved.

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But really, that's that's about it. I've been interested in it in relation to the phenotype associated with nets and that the characteristic

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secretory phenotype of the hormones and the relative quiescence of the cells that are present.

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And in particular, we've been interested in trying to understand what the cell of origin of nets are.

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And this paper from this this American group was published in Gastro about three years ago,

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and shows give some very interesting circumstantial data, which agrees with what we think about what the cell of origin of nets are.

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So I'm just going to just show you one of the figures from that paper.

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So this is some immunohistochemistry and in situ hybridisation, which of two imaging techniques to identify what genes cells are expressing.

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And here's your team on the light, right? And they've got tryptophan hydroxylase staining in dark blue.

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And here they've just countersigned it in a red will change the colour to a red.

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So you can see that this is a crypt which is present in the in the tumour, and they've got high tryptophan staining,

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as you'd expect in the net, but also in association with this in the same cells.

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You've got very high expression of algae, all five and algae. All five is this ubiquitous stem cell.

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Marcus, if you're a stem cell biologist, the moment you hear out your five, your ears prick up.

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The conclusion from this paper was that they felt that these these familial nets arose from a subset of intra chromatin cells,

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which are essentially enter endocrine cells that express reserve intestinal stem cell markers.

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So this is this is very much in agreement with some of the data I'm going to show you next.

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So I can show you a mixture of published and unpublished data.

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But before we go into the details of it, I'm just going to remind you of the cellular makeup present within the gut.

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So within the small intestine, we have crypts embedded into the wall of the bowel.

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We are villi projecting into the lumen of the bowel colon exactly the same.

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There are no variety within the bottom of these clips is the stem cells, and which we known as the stem cells aimed for donkeys years,

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30, 40 years and within the bottom of these, there are some undifferentiated cells, which are these slender green base columnar cells.

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There's also an undifferentiated, very slowly cycling cell termed the labour retaining cell in this characteristic +4 position from the

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bottom of the crypt and then cells that are also present in the bottom of the secretary Paneth cells.

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And these are only rarely found in the small intestine in the colon that you occasionally find pretty,

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that they're very essentially uncommon in the colon.

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And then you have absorbed of anthracite and lots of secretory other secretary cells, a goblet, cells, top cells, an enter underground cells.

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And these are the cells that are really very prominent within our necks.

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So, so what is a stem cell in the small intestine? And this is the seminal publication from Hans Cleves, this group who's based in Utrecht in Holland,

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and he used a technique called lineage tracing to very clearly identify the age of five.

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Mark was was the bona fide mark of homeostatic stem cells in the gut and lineage.

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Tracing is a technique which you do in mouse models or animal models,

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and you develop the animal model whereby you give the mouse an injection of a drug and that drug cause the

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activation of an enzyme and that enzyme cuts and pastes in your DNA or the DNA of the cell that it's activated in.

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And you can cause it that enzyme to be activated only in specific cells because enzymes driven by gene reporter.

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So you, let's say you're interested in saying, is our five a stem cell marker?

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You're on the back of the algae, all five gene. You get activation of this enzyme called CRE recombination only algae.

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All five expressing cells activate the enzyme.

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The enzyme then cuts and pastes and causes the expression of a of a reporter that you can actually visualise now because that's a cut and paste.

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So it's a permanent genetic rearrangement or the daughter cells of that cell will inherit the activation of the reporter.

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So if it sounds a stem cell,

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then the daughter cells and the cells of the daughter cells and therefore they're on will all activate will express the reporter.

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And if you then stain for it, you'll see clones of cells expressed in the report and that will show that there is a true stem cell.

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So that's the technique that Cleaver's used, and he very clearly showed that indeed, these ourjob five cells were stem cells.

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So the question was what what are these other cells? What are these?

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The undifferentiated, slowly cycling cells in various publications suggested various markers BMI one empty at Hop X and Hourigan,

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as marking these undifferentiated, slowly cycling cells, which might be more likely to be the the cell of origin of the Nets.

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And Cleaver's came back and then said, Well, actually, that's all well and good, but all of your other markers actually overlap with our Channel five.

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So in fact, you're just describing our original five cells.

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So rather than using the technique the Cleaver's used,

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we decided to approach this problem in a different way by isolating the slow cycling cells based upon the nature of them being slowly cycling.

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So we developed this mouse model where you've got expression of yellow fluorescent

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protein tied to a histone that's driven from an intestinal specific promoter,

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but as a pulse of expression. So here's a cross-section through the mouse gut.

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We've given the mouse, the injection and all of the anti-north.

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All of the cells of the intestine epithelium express the yellow for protein, but it's just a pulse of expression.

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It's not on all the time. So with time, as the cells migrate off the villi and are lost or apoptosis and died, the signal becomes lost.

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But by three weeks, you can see at the bottom of the crypts there are still these so-called labour returning cells,

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which retain the label and are therefore quiescent or dormant and haven't divided.

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And then you can use various techniques to pull these cells out and interrogate them for what genes are expressing,

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whether they grow in culture and the like.

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And we, in collaboration with a group who are now based in Denmark, we could pull these out using two other a combination of two of the markers CD24,

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which marks the bottom of the crypt and a lectin called EULEX leptin, which would rule out us pulling these long Paneth cells.

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You might remember I mentioned earlier on,

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so the combination of these two markers allows us to pull out our labour, retaining cells with very high affinity.

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So here are some actual images of the cells that you can pull out here on a

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label retaining cells which are CD24 positive wifey positive leptin negative.

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Here are the rapidly cycling cells because they've lost in the bottom of the crypt

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that see 24 positive wifey negative leptin negative and then the pain of the cells,

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which are double positive for the Moon. And as a result of using this mouse,

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we we had sort of three different publications describing the nature of the labour retaining cells and actually what was happening in the bottom,

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the Crips. And to summarise what we read about, so eight years work on one slide, we were able to show that indeed these.

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al-Aziz expressed LG five. But they also importantly expressed this other marker called PEG.

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Three or one? They fell into a population called the site population, so the site population on this graph is marked by this, by this black box.

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And you can see all the cells present in that grey colour fall into the box.

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And the side population is a technique that's been used in blood to identify stem cells in the blood.

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But it's also clinically important because it's the same population is shown to overlap with the extrusion of DNA binding dyes,

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so thereby chemotherapy resistance that they are potentially of interest from a clinical perspective.

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We found that during normal homeostasis, these cells weren't stem cells, and they were actually enter endocrine and Paneth cell progenitors.

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And we did this by using short term lineage tracing. I won't go through the details in this graph, but you have to just take my word for it.

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But interestingly, when we put these in culture into organoid culture, which is when you can grow sort of mini organs, in addition three dimensions.

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These cells were able to then grow into organoids, implying they had some degree of stem cell potency still latent.

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And then we finally resolved all of this by generating another mouse model whereby we could lineage trace,

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as Cleaver's did but direct, based for the first time in the mammalian system,

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based upon how long a cell lived and were able to show that indeed, when you injured the epithelium and this in the epithelium as generating these

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cells changed fate from being a secretary progenitor to being a stem cell.

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So that was all very good. We've gone on to collaborate with a group in Barcelona led by Adwa Patil and identified

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two new markers of slowly selecting cells in the colon and the small intestine.

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With that whereby seven and Max 3a, they can both generate multiple image clones.

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And they also both form organoids high efficiency.

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But the interesting thing about our labour retaining cells is the relationship to enter endocrine cells,

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and this red markers Kramer in which is remembers the serum marker we used to,

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you know that patients and these labour retaining cells are destined to become endocrine cells, as well as the Paneth cells.

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And more recently, after our publication, there's been about sort of six or seven other publications describing similar features of cells.

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So cells that are destined to become one type. But if you push them and injure the epithelium, they then acquire stem cell capacity.

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But of all these other populations are, alas, these are the only ones that are entering into crimes.

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Our progenitors and combined with the very slow cycling dormant state, leads us to believe that they may well be the cell of origin of gnats.

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So are they? So this is unpublished data. Here are our losses.

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First of all, I mentioned that P.W. one was a very specific marker of the labour retaining cells.

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And indeed, we've gone on to validate that in lots of ways and become very interested in P.W. one, which is also known as Peg three.

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It's an imprinted gene. The P.W. an expression overlays very nicely with with our alliances and looking at the whole gene transcriptome data.

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So looking at what genes or the genes that are expressed by our labour retaining cells and then comparing it to previously published datasets.

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This is called gene set enrichment analysis and the fact that this Green Line is all

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pushed over to the left hand side and you can see the bunching up of the black hair this.

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This is basically overlaying all our genes, which are the signature in our labour retaining cells amongst a previously published

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data set of enter endocrine cell progenitors showing that indeed they have very,

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very similar gene expression profiles.

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This is some single cell data whereby I'm identifying where cells that express PD one lie in relation to all the cells that are present in the gut.

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These are the enter sites which I haven't highlighted, and then you can pull out genes in these single cell data.

384
00:39:50,940 --> 00:39:57,060
So these are individual single cells where these different populations lie, and you can just see up here.

385
00:39:57,060 --> 00:40:00,930
All of the red dots are present within the enter endocrine cell pool,

386
00:40:00,930 --> 00:40:06,510
showing that the P.W. one cells are indeed enter into growing and in the end trend declining cell population.

387
00:40:06,510 --> 00:40:12,660
But in fact, they ended. They're actually within the progenitor population subpopulation of this group.

388
00:40:12,660 --> 00:40:16,680
So if you then take it to nets and then you say, well,

389
00:40:16,680 --> 00:40:21,990
all the genes that are expressed in our labour retaining cells overexpressed in nets, you see the same thing.

390
00:40:21,990 --> 00:40:30,330
So genes, that enrichment analysis demonstrates an enrichment of the signature within nets and turning that question on its head,

391
00:40:30,330 --> 00:40:35,250
looking at all of the genes that are defined that define the gene expression profile of nets.

392
00:40:35,250 --> 00:40:40,710
Again, these are all overexpressed significantly within our labour retaining cells.

393
00:40:40,710 --> 00:40:51,450
So we have a very strong suspicion that the labour retaining cells are indeed the cell of origin of the Nets and the finals of science lead is also.

394
00:40:51,450 --> 00:40:58,020
One is of real interest because it doesn't just appear to be a marker of introductions out progenitors.

395
00:40:58,020 --> 00:41:04,500
It seems to control their fate. So we've got a knockout mouse from a collaborator in Paris.

396
00:41:04,500 --> 00:41:08,520
These are the control slides, so this is one expression. It is dark.

397
00:41:08,520 --> 00:41:14,100
You have to believe me. It's dark right at the bottom where the alliances are. There is some low level expression on the fly,

398
00:41:14,100 --> 00:41:22,450
and we can knock out the expression of it using tamoxifen to to eliminate the gene and doing transcriptomic data.

399
00:41:22,450 --> 00:41:29,220
So looking at what genes change when you knock out the gene? What happens is you lose all of your mic targets,

400
00:41:29,220 --> 00:41:37,620
which is basically saying that everything stops proliferating and you lose progression through the G2 m checkpoint of the cell cycle.

401
00:41:37,620 --> 00:41:46,080
So again, things appear to slow down. This is validated using some staining for the RTU, which is basically a synthetic nucleotide,

402
00:41:46,080 --> 00:41:51,540
which we can give to the mouse and is incorporating cells that actively dividing.

403
00:41:51,540 --> 00:41:55,530
Here's the quantification of that, where you can see that in the wild type mouse,

404
00:41:55,530 --> 00:41:59,490
you've got higher levels of biology and corporation compared to the knockouts.

405
00:41:59,490 --> 00:42:08,260
But what is most fascinating is that when you lose expression of P.W. one that a large number of the cells change into an trendlines cells,

406
00:42:08,260 --> 00:42:16,200
you get much higher numbers of Cro-Magnon positive cells throughout the crypt, Phyllis Axis and also tuft cells as well.

407
00:42:16,200 --> 00:42:23,760
So it's a very interesting gene. It's unfortunately very difficult to work with because it's expressed at very, very low levels.

408
00:42:23,760 --> 00:42:31,710
My collaboration Paris has had three goes at trying to generate a mouse model where we can lineage trace from the P.W. on cells, all unsuccessfully.

409
00:42:31,710 --> 00:42:36,690
But we're having another go at that ourselves, but it's a particular interest.

410
00:42:36,690 --> 00:42:41,820
So the summary of where we are with the net biology, we're still very early stages.

411
00:42:41,820 --> 00:42:48,510
They the clearly have shown the the Aloisi work is very well published and very clear,

412
00:42:48,510 --> 00:42:53,220
but the work with PD one is still somewhat early and ends in its stage.

413
00:42:53,220 --> 00:42:59,790
But I think it's a very it's a very interesting gene, largely unexplored in the gut.

414
00:42:59,790 --> 00:43:07,710
So what are the future directions for net work in in Cambridge in relation to the surgery and the science?

415
00:43:07,710 --> 00:43:14,190
We're in the process of applying for a centre of excellence status. As I said, I've got a colleague who's joining the department next month.

416
00:43:14,190 --> 00:43:22,980
[INAUDIBLE] be I'll be training up on net surgery and will be joining me in helping out with getting through all our cases.

417
00:43:22,980 --> 00:43:25,950
I mentioned we're going to hopefully try and generate from the scientific side

418
00:43:25,950 --> 00:43:31,350
this P.W. on creating two mice to enable lineage tracing and PD one cells.

419
00:43:31,350 --> 00:43:38,490
We're generating a big biobank of organoids derived from the tumours and the normal matched normals,

420
00:43:38,490 --> 00:43:50,310
and also trying to model net development by using CRISPR to target candidate genes from using this more from the small intestine.

421
00:43:50,310 --> 00:43:55,530
So thanks very much, everybody for listening. Obviously got to do my my thanks.

422
00:43:55,530 --> 00:44:04,140
I have to thank Doug Winton, who's my scientific mentor in Cambridge and is an absolute fantastic, world leading stem cell biologist.

423
00:44:04,140 --> 00:44:09,690
My previous all raise various core facilities in my old institute colleagues,

424
00:44:09,690 --> 00:44:13,800
most importantly patients for their samples and consenting to our studies.

425
00:44:13,800 --> 00:44:19,890
Ruth Casey, who leads on Team Paul Roe, who's my anaesthetist for the difficult nets.

426
00:44:19,890 --> 00:44:29,160
Simon Harper, who's the ah, HPV net surgeon who we often do combine cases with and my various collaborators throughout the world,

427
00:44:29,160 --> 00:44:34,950
and funding bodies Cancer Research UK and asks for funding my fellowship on Christmas Eve.

428
00:44:34,950 --> 00:44:39,390
Core funding support from the Wellcome and the MRC at STEM Cell Institute.

429
00:44:39,390 --> 00:44:45,689
Thank you very much for listening.