Transcript for:
สภาพแวดล้อมภูมิคุ้มกันในมะเร็งตับอ่อน

let's now move to the fourth talk of this track and it's my pleasure to introduce you to dr dana mustafa from the department of pathology humor immunopathology laboratory of the erasmus university medical center in rotterdam the topic of our talk is the new microenvironment of pancreatic cancer hello good afternoon from the netherlands i'm adil mustafa and i'm an assistant professor and the leader of the geomics facility at the tumor immune pathology in erasmus medical center um today i will talk about as the title indicates i will talk about immune infiltration and how that how the microenvironment of pancreatic cancer may influence the survival of the patients but before i start i'd like to first give my thanks to nanoshrink company for arranging this virtual oncology meeting and for giving me the opportunity to talk about our work in the lab i'd also like to thank the chair lady who introduced me a bit earlier so let me start by refreshing your memories about the bankiness parenchy is part of our digestive system it is in the abdomen just behind the stomach and it's two in one organ so it functions as a as a gland and it has two functions basically endocrine function that regulates blood sugar and excrement function mainly by producing enzymes to digest food the type of pancreatic cancer depends on the cell's origin if cancer starts from isolated cells its partner cells that they are responsible to produce insulin endocrine cells then the type of cancer is going to be neuroendocrine or pancreatic neuro endocrine tumor and it presents almost 10 percent of pancreatic cancer we lost some famous people for this type of cancer we lost franklin and we lost steve jobs for this type of cancer if that kinetic cancer originated from us in our cells we refer to it as pancreatic ductal adrenal carcinoma and this is a complete different type of tumor we lost unfortunately some famous people pavarotti and recently this year we lost we lost elf and han for this time of tuesday and kinetic cancer especially when kinetic doctor latino carcinoma i refer to it in my talk as pancreatic cancer but what i mean is pancreatic oxalatinocarcinoma which is the most common type of pancreatic cancer and it represents more than 90 percent of pancreatic cancer unfortunately it's a very bad disease it's expected to be the leading cause of cancer-related death in 2013. it's painful disease because the tumor cells invade nerves they also invade lymphatics and veins and they metastasize the five-year survivor rate is five percent and most patients die within two years the reason for that because it's an asymptomatic disease until the patient develops some symptoms and they come to the clinic to be checked the cancer is already there and it's in a lot of cases it's already spread histological level the cancer pancreatic ductal adenocarcinoma they are the granular or doctor differentiation so it's the epithelial neoplasm of granular differentiation it's a highly heterogeneous disease it's characterized by remarkable dismal place or fibroblasts in some cases dyspoplasia can represent 50 percent of the tumor or even more than have a very poor angiogenesis or a blood supply the dysmorblasia is a very important important component of pancreatic cancer and it's mainly composed of extracellular and matrix proteins fibroblasts a lot of fibroblasts various types of fibrocasts pancreatic stillated cells and immune cells immune cells or tumor infiltrated lymphocytes recently since five seven years ago they started to have a huge highlight because of the importance of these immune cells as a part of the tumor it's usually the balance between the tumor suppressor and the tumor progression that it is a balance in the immune cells that drive the tumor either towards suppression or progression so we usually refer to them as good and bad players not per se it's the balance between the good and bad players is the most important thing they can predict the response to therapy over all survival regression free survival it's very important to know not only the amount of infiltration cells not only the type of infiltration cells but also their location and their ratio relative to each other understanding the immune system may open therapeutic options like we see with immune checkpoint inhibitors now previously our group has published and studied the immune component of pancreatic cancer we have uh i'm referring to one of the publications and in this publication we did by immunohistochemistry we stained for several types of t cells the eighth d40 regulatory cells marked by fox p3 also some immune checkpoint inhibitors and what we concluded in that article pancreatic cancer or pancreatic ductal adenocarcinoma specific is low in cd8 t cell infiltration and high in immune suppressive myelin cells we also show an evidence that the cd8 to fox p3 ratio the high city a to fox b3 ratio is associated with favorable outcome like kabul meyer shows here if we look at the literature and research foreign kinetic cancer together with micro in your micro environment or micro environment we see number of publications increase in the last five to seven years and that is in relation with the energy point inhibitors and technical therapy that's been introduced to treat cancer patients in general so there is a demand and there is a huge need to understand the micro environment and specifically the immune micro environment not only invent kinetic cancer but also in other types of diseases and that is one of our focus on our understanding the immune micro environment of now several publications i'm referring to view here they um hypothesized and suggest the relation between tumor-associated macrophages and fibroblasts which is the desmoglasia this is where mainly we we see a lot of infiltration of immune cells some inhibit and some suppress the tumor cells there is recently a review and the meta-analysis trying to understand the prognostic value of genetic infiltration and trying to understand other publications are highlighting the importance of understanding the landscape of immune cell infiltration to predict the clinical outcome and to have an implication in regulated personality i'm listing here few examples the recent publications however what i like to highlight is most of these publications are focusing on results so the highlights of these publications is about the infiltration of diesels in the kinetic cancer they protect better survival especially cd8 associated with a better outcome deregulatory cells or fox e3 t cells are associated with poor outcome but as you see most of these publications are about t-cells and they recommend that other types of cells like for example cd-20 which are b cells or natural rectangles should be studied original of what i'm presenting today is unfortunately most of pancreatic cancer patients they died in the first couple of years as i told you earlier however there is a very small subgroup of patients since they survive for many more years so there are factors to determine the long-term survival chip mainly and we hypothesize that the tumor micro environment and the immune cell infiltration may be the key to determine the long-term survival in patient doctor so based on this hypothesis our aim and the aim of what i'm presenting today is to reveal the immune and the microenvironmental pathway that may determine long-term survival in energetic cancer patients to achieve our aim we went back to the archive we have more than 20 we collected a big archive more than 25 years and we went to all these patients material and their clinical information you know the clinical evaluation and treatment and we selected the maximum number of survivals so we have 10 patients who survive for five years or longer and we compare that to 10 patients who survived for six months or shorter and that was our definition for long actual third term survival and the first thing we did is we immediately profiled them use using an encounter facility nanotechnology string technology and afterwards we selected subgroup of them to be studied in the geomets dsp and i'll come to back i'll come to that in details later our workflow we selected in this case in the first part of the study we selected fresh frozen material we enrich for tumor tissue so we section the tissue samples and pathologists and experienced pathologists would study them and then we enrich for tumor tissue meaning we enrich our tumor cell including dysmography or stroma that's part of the tumor that needed rna isolation we checked the quality and we used 200 nanograms per reaction which includes 5 microliters of rna good quality rna but fresh frozen material usually gives good quality rna and then we use the new profile panel from nanostring we hybridized for 17 hours we scanned 490 kilos of fuel and we used the in solver advanced analysis would you to analyze ourselves and then we validate it using the geometrics dsp now a technical detail i'm not aware of how familiar you are with an technology and the principle of uh of the world but just for a short notice we used a pancas the band cancer in your profile panel including 770 genes representing the innate and the adaptive immune system 40 housekeeping genes there are 24 different cell types and several pathways and the advantage of using their panels from from nanostring is you hybridize rna immediately with two drops so you capture you capture 100 base pair of the rna immediately without enzymes using two props 50 base pair each and each prompt at the end has a flag or a reporter prop of several six fluorescent dyes to present various genes so it will give account of the genes of your interest is a volcano plot of the differential expressions so in this side and the right side there are the genes that they are overexpressed or upregulated in the long term survivals and here are the in green and the green direction there are the genes that they are overexpressed in the short-term survivals please notice that the y-axis represents the look 10 p-value and the x-axis represents the look two voltage change so one here is two in linear so we have more than 14 differentially expressed chains at a p-value of 0.01 and the fold of a change of 1.5 in both directions if we take these 1414 genes in order to distinguish between the two groups remember we have 10 patients per group then there is a good separation we did principal component analysis and in gray you see long-term survivals that they are separated from the short-term survivals of course they overlap a little bit but the overlapping is not that big so we are very happy with these results then you can take the normalized data and you can do you can create heat maps or you can study for example i'm very much interested in the most differentially expression between the both groups but you are allowed to do all of that it's based on your taste what i'm also very interested in is knowing the differentially expressed immune sometimes so how can we utilize gene expression results by giving cell markers to identify immune cells i'm giving an example of a diesel of the definition of b cells we use four different markers then we calculate the correlation coefficient between these four markers and for us we define them based on or we trust cell markers that they have a correlation coefficient higher than point sex meaning like these cell markers behave in the same way across all the symbols and then you can trust them to give you a score for yourselves now if we look at the infiltrative cells defined by cd45 they are not different between long and short silver but if we look at the basal score then it's at significant different score which represent a higher score a score in long-term survivals as compared to short again the definition of the b cells in this case were represented by four markers this is cd20 cr2 cd22 and cd19 and this attracts our attention because actually the role of b cells in pancreatic cancer remains controversial it is suggested until now that it is a new it has a rule of the new submission there are three publications were published in 2016. they suggest or they indicate that b cells fuel pancreatic cancer pancreatic adductor adrenocarcinoma and they promote pancreatic tumor genes however a more recent publication in 2019 such as the opposite please remember that these applications are limited with the number of patients they use or say samples of patients they use they also are limited by the technique most of these publications are based on universal chemistry and a lot of them are based on using animal models we know that animal models they don't represent especially when we talk about immune cells or immune system they don't represent the situation but we have a we have opposite information in the literature our results shows that measles infiltrate more in the long term survival as compared to the short-term survivors and that raises a lot of the questions i'm going to address two of them today one of the question is the location where do they infiltrate we talked that we i introduced the tumor as a heterogeneous tumor so i'd like to know where exactly they infiltrate and what are the other types of cells that they interact with and to answer these questions nanostring has developed the geomets digital spatial profile a profiler platform in my opinion it is a fantastic platform to answer our questions so now we take tissue sample and we stain the tissue sample with morphological markers so we distinguish between tumor cells in this case they are how we distinguish them with transatlantic marker in yellow and we distinguish alpha smooth muscle acting in green and any tumor cells which are positive for cd45 they are ignorant and what we did previously using the encounter platform is one sample we took a section we isolated one rna sample and then we did one measurement but now with the dsp one sample can be as many as we want we are focusing fan side to keratin cd45 and alpha smoothness effect in other words using this technology you are able to connect biology to morphology or expression how does it work dancing developed a neural oncology module in which it includes the core module to distinguish the most common types of immune cells as you can see city 20 cd3 cd4 ct45 and so on and so forth and then they are coupled with other modules based on your requestion like drug in your oncology drug targeting tubes or activation status suppressive status if status and across all types of tumor cells these are antibodies these antibodies are conjugated with oligo that oligo is odd uv clearable so in practice what we do is we take a section a tissue section of four or five micrometer paraffin embedded section and we stain that suction section with our morphological markers fancy to keratin adjustment muscle actin and cd4 and then we hybridize this section after thermal morphological markers with the cocktail of these antibodies leave it overnight and next day we are able to distinguish based on the segments we are able to profile each one of our segments severity and here is a real example this is one of the samples that we measure and each one of these boxes these are 12 boxes rois or asian of interest in each one of these regions of interest you see a circle and that represents for example circle number seven is full of the green color green is an accent please remember please notice that it is high area number seven is high in atherosclerosis actin it is very low in bad cytokeratin and it is very low in cd45 and now we provide i'll go to my target this is an example of a long-term survival symbol in which i chose this area this area is filled with tumors sorry with immune cells and when we look at the profile of this area they were very high in cd20 which is a marker for b cells i forgot to tell you that the data can be normalized in different ways what i'm presenting here the data were normalized using positive controls and housekeeping sheets so area number eight in a long term survival symbol was filled with or had a high expression counts of cd20 and here is another example of a short term survival in which i chose exactly the same morphological area this area is filled with immune cells it is low in alpha smooth muscle acting and it is low in fat cytokeratin this is area number 10 and if we look at the expression or the counts this area is filled also with 20 with cd20 which is a marker of these if we put these data together and we notice the value of the y-axis which is the count these two areas which is filled with immune cells are built with b-cells as well but in a complete different range long-term survival showed the range of 5 000 counts and short sears short term survival showed at a count of 600 now this is one sample versus one sample if you take six versus 6 and you put all the information together the long term survivals are much higher in infiltration with b cells as compared to short term survivals and i'm here presenting areas that they will fill within yourself i'm not presenting fan psychology in order to show it to you in a different way these are the areas of alpha smooth muscle active so the dysmoplasia and venkeretic cancer in which um the area in the right are overexpressed in short and the genes or the markers in them these are genes these are proteins in the left are overexpressed in long and please notice with me that cd20 is overexpressed with a good b value and a very high fold of a change twice as much in long-term survival as short-term and they are in a close approximately with t cells c4 cd11 c and cd68 which is macrophages and monocytes if we take the areas of pancytokitten which represent the tumor cells then we don't reach significant results in the long term survival we see the same trend but it is not significant however we see a lot of significance in box b3 cd regulatory cells and immune suppressive cells in the tumor cells themselves so putting this information together we saw that b cells infiltrated in the stroma of long-term survival and they are in close distance with cd4 cd8 c68 cd14 and cd11c cells b cells had gained a lot of interest at the beginning of 2020 in february 2020 three publications came in nature almost in the same way and all of them were referring to the important role of b-cells in the long-term survivor or in the good prognosis of various types of cancer like melanoma sarcoma and others but we are the first to show this role in pancreatic cancer so i'm now in the conclusions and i'd like to conclude with you to remind you that the immune infiltration is one of the keys that determine the survival and pancreatic doctor latino carcinoma they are infiltrated more in long-term survivals meaning patients who survived five years and longer as compared to patients who survive six months and shorter and b cells infiltration is associated with t cells into cells and these dc's dendritic cells the high infiltration of t regulatory cells was mentored or monitored in the small plastic tissue in the ductal tumor cells of fractal tumor tissue or pancreatic drug we have a lot of more questions and we actually have clinical data to answer our questions like to connect then infiltration with the clinical parameters we have or to study the band cytokeratin or the tumor cells a little bit more in details to know what types of cells infiltrate in the short-term survival in that case but with that i would like to conclude my presentation and i hope i convince you that vessels play an important role in [Applause] determining the survival shape of pancreatic octal carcinoma i'd like to thank people who participate in the study from the tip lab or the tumor in pathology lab at erasmus medical center and that lab is headed by professor egg i'd also like to thank the bioinformatics team from nanstring that helped me to analyze the data generated by dsp and i very much appreciate the involvement of the patients the donation of the tissue that enabled us to to study the tissue and i'd like to thank support kaspar for the financial support and i would be very happy to answer your questions thank you very much dr mustapha for the nice talk i have few questions for you too can you please expand on how one can identify immune cell subside using gene expression of genes and actually i received this question quite a lot i'd like to go back to my slides and i showed you the example of t-cells at identification of pieces so we use different um gene markers to identify an immune cell type and actually for pancreatic cancer we there is a publication in preparation right now we use the cancer genome atlas data and we can calculate all the definition of cells by calculating the correlation coefficient between the genes so using public data which is from the cancer you know the cancer you know atlas we calculated the correlation coefficient between the different types of cell markers and we kept we trusted those that they have 0.6 and higher to define the types of cells in that way we are able to identify various types of cells in a very confident way so the default of nanostring or in solver advanced analysis the default gives various types of cells but we were able to increase that for example nanostring gives the b cells we were able to increase it to plasma b cells regulatory b cells and memory b cells in our dataset we also do the same for t cells you do the same for dendritic cells for any type of cells you distinguish or from the literature you know the markers the gene markers to identify types of cells and then check the correlation coefficient in between the genes in order to determine if you trust them in your data set or not that's how you define immune cells using gene expressions there are other softwares that they do that one of the famous one is a cell sword or a super sword so it's a very well known technique thank you since in your study you have used both the bulk technology platform as well as the tissue imaging platform which kind of advice will you give to researcher about which one to use when the goal of your research is to discover difference between two group of interests two groups um it is based on your personal taste and the time money and effort number of samples that you have in the study the scientists want to do i think or the scientists want to perform um the way i chose is to profile the samples using encounter and then validating using in geomex national spatial profile the reason for that is because i think it's a little bit faster and cost effective if you do it this way you will study more samples with the encounter and then you can validate your results using a smaller cohort of patients or of samples however you can do the same you can do also the discovery and the validation using geomix dsp it depends on the symbols from the number of symbols included in the study the dsp will make will allow you to study the symbols in much more details as compared to the income the encounter is usually i refer to it as a suit so you isolate rna and from the whole sample and then you measure the expression of cells or the you measure the expression of genes not knowing what gave rises to these expressions in the dsp now you know exactly where you are located in the tumor you study their population that you want to study it and you can do that at two levels you can do that at the protein level using antibodies quantitative conjugated to oligos or you can do that also at the rna level which i think is a fantastic method to study secreted cytokines i hope thank you what is the function of b cells in pancreatic cancer very good question um we our data show that it is they are higher in patients who survive longer as compared to patients who survive shorter it's a relative expression so i'm assuming that there or i am confident that that b cells in pancreatic cancer they play a good role they actually suppress the tumor however we still need to validate or not validate to discover the factors that facilitate b cells to infiltrate to the tumor cells to that human areas in long term survivals and the function of b cells is still not known in pancreatic cancer we are working on it we are studying samples in much more details um but i cannot really give a precise answer for that question thank you very much dr mustapha for the presentation and the discussion so at the end of this presentation i hope i convince you that um b cells play an important role in the long term survival shift in pancreatic active carcinoma but i hope i bring a little bit bigger message which is the important of studying the tumor micro environment again i'm concentrating here in pancreatic cancer but we study more tumors tumor microenvironment is gaining a lot of attention lately and the reason for that is because it is it may open new therapeutic options for patients who suffer from this bad disease so um i think the effort the international effort that we all put together in understanding the human micro environment whether it's pancreatic cancer or other types of cancers is it will be great to put all this effort together and to collaborate to connect our databases so to identify uh or to discover more about the tumor microenvironment the more we are we understand the tumor in general the more we are going to be successful in winning the battle against it with that i'd like to conclude and once again i'd like to thank um that you are in the pathology lab at erasmus medical center um and i'd like to tell you that we have the geomix dsp we installed it just before corona era and i cannot wait to start working with it i also would like to thank lannistering for hosting and for arranged organizing this virtual conference and if you have any question i have i'd be happy to answer them at any time