endoscopic ultrasound rectal cancer staging

this is the last traco lecture for 2015. so for those people interested in getting a certificates the examination will be posted on the website tomorrow, you can take it anytime between now and the middle of january. and you have to get 70% of the

questions right to pass. it's open book, open notes, there's one question derived from each lecture. and i'd like to acknowledge jonathan weisst for supporting this course, as well as anthony thomas, here in building 50, and up in frederick we have chris

graham. so today our first speaker is rena dobrovolskaia, she got her ph.d. in russia in 1998, subsequently came to the states, postdoctoral fellowship at nci as well as university of maryland, and in 2005 she started working at nci up at

frederick as a senior scientist, and now she's a principle scientist, the title of her talk "immunological properties of nanoengineered materials and their challenges." >> good afternoon. thank you for joining us this afternoon.

hello, everybody, who is watching us online. i'm very happy to be here this year again, sharing my knowledge and my experience with you. looking forward to your cooperation. is something is unclear feel free to interrupt me.

thank you for the invitation and introduction. i'll talk about immunological nanomaterials and challenges in would like to go with you over the definitions what is considered as nano. i will be talking about some examples of nanoparticle use in

daily life, i will give you some examples of nanoparticle use for cancer diagnosis and therapy, most importantly what benefits nanotechnology offers for cancer diagnosis and therapy. we'll talk about nanomedicines. i have to say definitions sometimes people call this

material nbcd, used in europe, in the u.s. we call it nanomedicine but you may see in the future one definition or the other in articles but you will know they refer to the materials with use in biological applications. i will tell you about ncl, the

nanotechnology characterization lab, briefly tell you who we are and what we are doing, and then the majority of my presentation will be devoted to reviewing information about the biological compatibility and particles of the immune system and their characterization.

what is nano? most people use this definition, nanoparticles as objects with a size and one dimension with one in one nanometer. if you look at the nanosize materials approved for clinical use, used in patients, most commonly this material is

100 nanometers, the united states food and drug administration defines any material including physical and chemical, biological effects, attributable to its dimension, ers, so f.d.a.imension exceeds considers up to one micro meter as nano.

in daily life, according to the web, there are more than 800 manufacturer identified products from 400 companies in 20 countries world wide. nanoparticles are used in clothing, wound dressings, washing machine liners, as lens coating in sunglasses, sporting

equipment, nearly all translucent sunscreen contain nanoscale titanium and zinc oxide particles, women should know perhaps products contain liposomes and emulsions, and some materials, especially carbon opinion based nanotubes are used for structural

materials for durability. what can nanotechnology offer to those who work in drug delivery? nanoparticles may improve solubility of hydrophobic drugs most common use of nanotechnology carriers is to deliver hydrophobic drugs, offers multi-functional

capabilities. you see the example of nanoparticle which has a core particle, has a drug bound to the particle, polyethylene glycol coating, prevention recognition by immune system. those that carrying drug and imaging agents are from the

combination of therapeutic and diagnostic. they reduce toxicity and sensing, computation, actuation and triggered. for cancer therapy, two different type of advantages. one is called epr effect, epr stands for enhanced permeability

and retention, what does it mean? it goes to the leaky vasculature of the tumor blood vessel, the blood vessel surrounds tumor, have fenestration, and nanoparticles due to nanosize may cross this leaky vasculature and into the interstitial space,

and this material tends to stay in the tumor space and release drugs bound to the particle. this type of targeting is called passive targeting because basically particle is utilized in nature of the tumor and the vasculature to enter the tumor space.

there's also so-called active targeting, this is then nanoparticles modified with a targeting agent, it could be monoclonal antibody, which recognizes tumor specific antigen, or it could be any molecule which binds to tumor-specific antigen.

it is important to recognize in order for the active targeting to work, basic targeting is needed. before a particle can utilize target it needs to cross vasculature and enter into the interstitial space. as i mentioned to you in the

beginning of the lecture, in europe a lot of nanomedicine, particles are referred to as non-biological complex drugs, nbcd, most common definition that you may hear is nanomedicine, so i will be calling these materials nanomedicines for the purpose of

my presentation. what you see on this slide is classification of the nanomedicines by category, and in categories we look at the medical devices and we look at drugs. there are two categories. one in the investigational

stage, so these are materials that given to each commercial stage, various stages of pre-clinical and clinical development, as you see majority according to literature, majority of materials are in medical deviceses category, only 17% is drugs category.

completely different picture seen in the look at the materials that reach commercial stage. 67% of these are represented by drugs formulated through nanotechnology, 33% represented at you see from this slide isby medical devices. data from literature, different

between investigational and commercial materials. in investigational materials you see that the majority of these materials are liposomes. in commercial the dominant is solid followed by composite material. most nanomedicines are developed

for cancer therapy, so the primary market is cancer therapy, and this is on this slide, majority of nanomees are intended for intravenous administration and have size from 100 and 350 nanometers, less than 350 nanometers in size.

most of these materials are neutral, hydrocelic surfaces, spherical, important to prevent particles from off-target toxicity, immune recognition, and i will be talking about this and show you some case studies later in my presentation. there is increasing funding,

effort from funding agencies, dod and hhs, and we see increase in funding nanotechnology, starting in about 2001, up to 2015. right now we spent 1500 million dollars. the primary purpose is to develop nanopales for

medical application, or cancer specifically, but in drug development there is the definition, the so-called valley of death. what it is, valley of death refers to the stage in material development from the moment it was discovered and showed it may

have potential to cure cancer or benefit the cancer therapy, to the stage where this material actually ready to enter into clinical trials, because there is a lot of infrastructure, a lot of the knowledge which is invoked in translating this material from bench to the

bedside, that requires collaboration within academia and industry. lab was established in 2004 at interagency collaboration between national cancer institute, national institute of technology and f.d.a., the mission to accelerate the

nanotechnology concept from bench to bedside. from government organizations, big pharma, academia, in the years and also from other entities outside of the years, subject to rigorous characterization, looking at the physical chemical parameters and

in vitro pharmaco toxicology and immunology, and the final goal of this characterization is to provide materials to our clients, to support the regulatory findings, clinical findings of this material, also to inform the regulatory agencies such as f.d.a. about

compatibility to nanotechnology formulated drugs. nanotech expert and resources are brought together. we have been in operation for over ten years and in this time characterized a little bit over 300 nanoparticles, with majority of them liposomes, polymers,

metal oxides, metals, also other materials including dendrimers, emulsions, and other types of materials. it is now established from research in our laboratory and in the literature from publications by other laboratories that it is

nanoparticle physiochemical properties like size, charge, targeting and hydrophobicity, interaction with immune systems. on this slide i will show you some of the examples. you see in this case is increase in size in dendrimers, result in increase of particle activity

with membranes of platelets that causes platelet activation anding a -- and aggregation. size is important. but it is important to recognize particle size under pristine conditions, and in biological environments, are different things.

e look at this data. we're using here 30 nanometers gold colloid particles in pbs, no biological matter, we use three methods to look at size, traditional and dynamic light scattering, what you see in yellow is the measure size. so this is what instruments

measure.out 30 nanometers, in agreement with different methods. look what happens if we study these particles in human plasma, microscopy, look at the core, the solid core of the particles, show us the 30-nanometer size but when we use dynamic light

scattering which shows hydrodynamic, almost doubles, particle size increases to 76 nanometers. why does it happen? in plasma there are a lots of proteins, proteins can bind to particles, and the changes particle gets larger due to

protein coating, and this soft size is not detected by tam and afm but is affected by dls, this is the size at which this particle and their use in biological applications are seen by our body. other body does not see 30 nanometers, other body will

particle surface charge is also important in biocompatibility. again, i will use as example aggregation and my model particle in this case is dendrimer, this study was done in collaboration, activating platelets, very important aggregator, he used the chemical

magnification to mask 35% of particle surface, then another 25% and another 25% and so forth, as we decrease the reactivity was widely decrease. if we want to use this nanoparticle, if we want specifically to induce then we want to use

this particle, right? se we know this activates platelets, biological reactive, but if we intend to use this in biological applications, inject into animals, this material would be topic. we make it more biocompatible and less toxic.

composition of nanoparticles is important. this is example where we use dendrimers, artificial proteins, that if you think of a tree, this is the tree crown, spherical material, very soft, which may be made of different building blocks.

pamam and triazine. we see the same trend in both particles, the particle size increases, the density of surface increases, greater biological reactivity. we see these are less reactive, the way we can explain it, the density of the surface group is

lowered than in pamam dendrimers. nanoparticles may be good or bad depending on whether it is le or not. some nanoparticles are created to specifically interact to benefit, for use in cancer immunotherapy, autoimmune

disease and infectious disease therapy. however there are some nanomaterials that are mostly environmental materials which surface is not activated but biological membranes and immune cells and these materials cause undesirable immunotoxties, lead

be to hyper sensitivity, may lower body's defense to pathogen and cancer and suppress bone marrow and thymus function. what we are interested in, interested in the engineered nanomaterials that are developed for biological and medical applications, but not

specifically to target the system. we want to know these materials therapy to deliver cytotoxic drugs, would very also have undesirable side effects, anaphylaxics or other e consistencies for patients.

what we can see from our experience is that nanoparticles can be immunotoxic. however, there is no new toxicity attributed to their nanosize. nanotechnology-based pharmaceuticals are not more immunotoxic than other drugs in

current clinical use. there is increasing number of examples showing that incorporation of the traditional drugs into nanotechnology platforms helps decrease immunotoxicity. i would like to share two examples, one from small

molecule drug, one therapeutic protein. you will see on this side of the slide the traditional formulation of this drug, and on this side i will show you the nanotechnology formulated drug. doxorubicin is commonly used, the drug can activate

endothelial cells and cancer cells to express coagulant activity complex which serves as platform for assembly of plasma coagulation cascade, as a result blood clotsing, consumptive, specifically the side effect was described in acute leukemia, some types of blast cancers, a

nd in this case the therapy was discontinued. the same drug was encapsulated in liposomes, called doxil, no such toxicities described, we have studies showing induction of coagulant activity by doxorubicin. tnf alpha is cytokine, it is

expected for it to be protein may cause necrosis of the tumor, so no surprise, this protein was started in the trials for cancer therapy, but in the investigation of this drug was halted by systemic immunological stimulation, and toxicity, so in order to reach

the dose of this drug, enough to kill the tumor, the doctors have to administer so high doses of this material that the patients protein was discontinued. a company in rockville, maryland, used the same protein and attached it to the surface of cloydal gold nanoparticles,

poly acetylene glycol to mask the particle surface and protein this particle carries, this product is called aurimmune, ut reaching maximumical trials tolerated dose. all this nanotechnology formulated drugs are entering before this nanotechnology drugs

enter in clinical trials, they have to be rigorously characterized, the characterization involves multiple areas, including chemistry, which analyzes particle size, size, charge, shape, composition, surface coating, anything that is

related to chemical composition and chemical structure of the particle. there are a lot of challenges efficacy of the nanotechnology formulated drugs because in nanoformulations we have to not only look at the end result but we also have to correlate it

with drug load and stability, drug release, targeting. there are a lot of changes in pharmacology toxicology, including change in particle by distribution, i will show you example in my next slide. use of appropriate models, investigational toxicity,

mechanism of toxicity, a lot of changes in hematology and immunology, some of the common challenges include biological contaminants, impurities, which i will talk in my talk, and others i will cover in my presentation, predictive methods in development of predictive

mols to understand nanoparticle immunotoxicity. this is the example of the challenge of traditional drug and use of nanoparticle for delivery of this drug. again i'm using doxorubicin because it's a well-studied drug, many people are familiar

with it. it's a small molecule, typically distributes to bone marrow and heart. this drug was reformulated using liposomes, the distribution to bone marrow and heart decreased, toxicity is result, (indiscernible).

both materials are used in clinic for cancer therapy. this is experimeal pre-clinical formulation of doxorubicin using cyano nanoparticles, did not distribute to skin, researchers were happy they overcome, it did not distribute to bone marrow if

the dose is enough to cause suppression in heart tissue, so that mylar suppression is result, due to small size particles accumulate in liver and kidney, toxicity from skin to kidney. and this is very common change for working with formulang of

the drug, they basically have distribution. i mentioned to you that one of the challenges is associated with presence of excipients. we talk about formulated drugs, not only talking about drug toxicity and nanoparticle toxicity, we have to consider

other materials present in formulation, excipient, which could be detergent, to prevent particle from aggregation, which could be proteins, or some cryoprotective agents to prevent particles from aggregation and improve stability in life and storage.

in this case using drug-loaded nanocapsules, we observed the material was hemolytic. chemical characterization using dynamic light scattering did not show any -- average particle size is 165 nanometers, expected, but transition through microscopy shows presence of the

small micelles. we found out in this formulation, brij 78 is used, and brij 78 is used to formulate the drug, it becomes non-reactive, but unreactive brij 78 forms micele, if diluted this micelle disappears. it is very well known in

detergent, cmc is reactive with biological membrane, unreactive bridge is causing hemolysis in this case. nanoparticle size and zeta potential are important in determinants of nanoparticle toxicity. i would like to say that despite

the importance, they are insufficient to discriminate within nanomaterials, displaying different biological effects. to prove this point i would like to use the case study, known as doxil. worldwide there are different formulations, doxil in the

united states, there is lipo-dox imported to the united states from india under executive order, for several months to provide this important drug to the patients. and now we have also generic version of the doxorubicin. in europe doxil is called

caelyx, and this is available in iran, lipodox available in india. i will be talking about these formulations, because they are available in the u.s. and we study this material. you see the size and use potential of what we call old

doxil, new doxil and lipo-dox. doxil was available in 2008 when the manufacturing facility had some problems with compliance with gnp, some issues resulted in shortage, temporarily unavailable, and many patients will undergo therapy with doxil, doxil gave better results, but

there was not enough doxil on the market. so under these conditions, the united states, the president issued the executive order to bring lipo-dox to temporarily cover this shortage, and meanwhile the manufacturing procedure and facility were

optimized to renew the production of this material, so then this material was produced in the u.s., produced also as doxil, called new doxil after shortage, and lipodox from the important message is the's no difference in the formulation in size and potential but let's

look at the biological reactivity. according to the literature, clinical use of doxil and studies of this model of carpa, many reported the old is inducing complement activation, in agreement with the dose of duration.

however, new doxil and lipo-dox did not have this toxicity, physicians that use doxil after the shortage noticed that lower incidence of carpa, and there is lower propensity of this material to induce complement activation. why does this happen?

we had the greater incidence of complement activated when the procedure was changed, new material was manufactured, there is less carpa induction and less investigation. this is not due to change in particle size and particle potential, but it's something

else. and we are currently investigatng the difference to understand the mechanism. another challenge related to particle characterization in pre-clinical stage is continuation with endotoxin, endotoxin is gram negative

bacterial cell wall, contaminant of nanomaterial, 30% pre-clinical nanoformulations. cat ionic materials are prone for endotoxin contamination, ionic binds to the surface of the cat ionic particle. the toxins may result in

erroneous data and lead to confound, confound efficacy studies, now results of increasing data showing some nanomaterials exaggerate endotoxin mediated inflammation and may cause undesirable potential problems.

alone the red line and blue line shows the effects of the same concentration in the presence of nanoparticle, concentration of nanoparticle is unreactive, we see we can look at the enhancement. the grand challenge in nanomedicine is detection of

endotoxin, accurate detection, because many nanoparticles interfere with one or more lal. you see two main types, one is inhibition, defined as less than 60%, another side is called enhancement, which is determined by spike recovery over 200%. what does it mean?

look at this data. this is endotoxin spiked in water. blue line shows the spike per milliliter of water, we recovered almost 100%, this is accurate detection. same in this case. but in this case, we're using

colloidal gold nanoparticles, now we detect about .1 instead of .4, this is inhibition, less than 50%. in this case polymeric nanoparticles, now detect 1.8 instead of .4, looks like this material contains a lot of endotoxin but polymer behaves

like endotoxin in the lal. enhancement is common for polymeric nanoparticles, the concentration of protein. the reason i would like to share with you next slide is to show how tricky it gets sometimes to distinguish within the presence of endotoxin in formulation.

what we do in our laboratory, we develop decision tree, we use the same type of nanoparticles, two different lals, chromogenic and turbidity. how do we know which one is correct? enhancement control which i showed in the previous slide was

acceptable in both states. we use biological test to confirm lal findings, basically what this test means, inject material into rabbits and you measure their temperature, if they develop fever then it indicates material contains endotoxin.

the material was pyrogenic, we sent this material back to investigator from whom we received this material, they conducted purification, we verified by lal, repeated the test and found material is not sometimes it is desirable to conduct biological tests, but

the test cannot be conducted in rabbits. we used monocyte activation test, this test is conducted in monocytes, and induction of pyrogenic cytokine, interleukin 1 and interleukin 6. different formulations of nanoparticles, abelcet and

abraxane. ata between different lal, but for some formulations the result is inconsistent and depending on which assay you use, you may either overdose endotoxin or be safe. this data can be verified by monocyte activation test, but

this nuance deals with the drugging that the particles deliver. this recovery is acceptable. nanoparticl stabilization represents another challenge, stabilization methods traditional used can change change integrity of the

particles, in this case study i reviewed ethics of gamma radiation on gold and silver colloids. both particles have this shape respect after gamma radiation the dispersian in shape of gold colloid does not change but silver colloid disappears.

instead we have this material form. gold colloids are sterilized by gamma radiation. silver colloids, not only the shape of the particle changes integrity of this particle is affected but biocompatibilityis affect the.

after sterilization, as we increase the dose of gamma radiation, these materials change and become reactive, we have to be careful in selection sterilization method which would not affect integrity and which would not harm drugs and which would not change biological

purpose of this material. the likelihood of finding toxicity increases with progression, and correlation between in vitro and in vivo method is a hot topic because high cost and ethical issues and low through put of in vivo tests limits application.

on the other hand in vitro tests have high throughput and lower cost but the struggle is whether or not pre-clinical in vitro tests are predictive. i would like to step back from nanotechnology area and look at the biotechnology, what lessons we learn from biotechnology

therapeutics, as example i will review this product, tgn-1412 manufactured by the european company tegenero, it's a monoclonal antibody that activates cd28. antigenic cell presents antigenic peptide, receives signal one, there's an antigen,

t cell will not start proliferating until it receives signal two, signal two delivered by interaction between cd28 and the antigen presenting cells, cd80/86, developed to overcome energy of the t cell, stimulate t cells under immunosuppressive conditions.

and then this antibody was studied in pre-clinical studies in non-human primates and rodents, everything was fine, there was no cytokine storm. however, it was a tragedy in europe, six out of six volunteers, the human patients, who received this drug developed

multiple organ failure and experienced cytokine storm, what you see on this picture is inverse and many patients had necrosis in toes as well, due to the overproduction of tumor necrosis factor in other inflammatory cytokines. interestingly, the material was

investigated, it was found that this material passes all pyrogenic tests in terms of lal, not endotoxin, not chemical or biological contaminants but the material itself induced cytokine storm. apparently in non-human primates and rodent models were

insensitive to this change. it tells us in some cases you always need to use animal models because especially with nanotechnology, animal models allow to look at biodistribution of materials, in vitro do not test for biodistribution. it may help overcome this tragic

accident. when it comes to nanotechnology we know from studies that common markers for nanoparticles toxicity are hemolysis, complement activation, thrombo genicity, cytokine induction. most of these toxicities can be rapidly assessed in vitro prior

to more resource and time-consuming in vivo studies. i summarize on this slide the states with good predictability, means if you see positive response in vitro, likelihood of experiencing toxicity in vivo is high for toxicity. usually based on one in vitro

it's difficult to conclude because models do not allow to account for particle by for example, cfu-gm allows us to look at bone marrow toxicity, correlates with mylar suppression, in vitro there's bone marrow cells, if we inject in vivo it may not be

in the marrow. this is a case study showing how nanoparticle induced cytokines correlate with animal models and in vitro in pbmc. we have identical metal core composition, different surface coating.

formulations, formulation 1 and 2, was very similar but these materials studied in rodents, in rats and rabbits, then one formulation was very toxic, and congestion in multiple organ damage similar to septic shock, however both formulations tested for endotoxin they were found to

be endotoxin free. the plasma samples from the animal models displayed congestion in spleen cytokine cells. again, this is confirmation perhaps among other in vitro, (indiscernible) are sensitive to predictive of cytokine storm

reaction, and this is why i would like to spend the rest of my presentation to the other example speaking about cytokines. what we found, we found very interesting observations, by studying cytokine response in nanomaterials.

we observed, among particles that induced cytokines, over 60% of these materials induced interleukin 8. do we have anyone who does immunology research in this audience, raise your hand if you do. so then i ask you, normally when

we speak about bacterial contamination, polysaccharides, you add this to your cell in in vitro cultures in animals, you see multiple cytokines, right? interleukin 1, il-6 and 10, you don't see in response induction of il-8 without tlf and il-1. with nanoparticles, we came

across nanomaterials that induced interleukin 8 without inducing tnf and il-1. about 50%, the rate of 53% of this materials, exclusive in use of il-8, which means using interleukin 8 without induction of tnf and il-1 and the common denominator was their

composition. in my group, we're excite to show this induction of interleukin 8 is related to oxidative stress, which did not involved protein expression from the three existing mrna, oxidative stress, stabilization of the lower levels of mrna

that mono nuclear cells maintain, and resulted in expression of the protein from this mrna, which was very interesting. why it is relevant for us to look at the cytokine storm, because i think by now you're convinced it is easy to predict

and common toxicity, some therapeutic products, for example, therapeutic oligonucleotides are known to induce cytokine storm, type i and type ii interferon. we have nanocarrier, if we use that, induce cytokine for delivery of therapeutic, what do

we have? cytokines used by the final product, and this is example. you see we tested liposome and nanoparticle platform, the (indiscernible) by the final product, then api, nucleotide, did not induce interleukin 8 but

the platform did, and again the final product is interleukin 8, induces interferon and that led us to create the takehome message for the drug delivery used in nanoparticles. this message is very simple, that immunotoxicity of both drugs and the nanocarrier have

to be considered, and using immunologically reactive carrier when immunomodulation is wanted, avoid such platform when immunoreactivity is undesirable. to identify common or overlap in toxicity, for example, both carriers and drug induce cytokines, and if we use shall

it - if we intend to use this formulation for systemic administration, which we don't want any immune reactivation, we have to stop and reformulate the drug using different platforms, but if we are talking about vaccine, for example, delivery or some immunostimulation,

overlapping cytokine induction by platform in a drug may be desirable, right, so we can proce. also there is plenty of data in the literature that some nanoparticles may by composition and through physical process may for example inhibit and prevent

effects, these processes can be leveraged for systemic administration of drugs that are not intended for immunostimulation because they will reduce immunotoxicity. in summary, i would like to emphasize several points. each platform is unique.

and therefore comprehensive characterization is very important, not just dls and zeta but potential. key factors in pre-clinical development are important to be understood including particle size, charge, composition, functional properties such as

stability, drug load and drug release, targeting. structure-activity relationship, why we need to understand structure-activity relationship? if for example we develop nanoformulation, and we show that it passes all the tests except let's say cytokine storm,

and we have cytokine storm to the presence of certain moiety on particle surface, if we determine this, we may optimize compositional particle, deliver this drug, by distribution, change the property to overcome undesirable side effects. endotoxin and sterility are

important, depyrogenation is important, very important in predictive in vitro models to save time and optimize formulation before we enter pre-clinical animal studies and before material is progressed to clinical trials, with this i would like to thank my

colleagues at the lab, the director, i'm great follow for sharing expertise, data that i shared with you today was generated by these, and other scientists on the team. i would like to thank michael from f.d.a. and eric from texas christian university.

thank you all for your attention (inaudible). >> it's very good question. the question was about what is the current stage of the f.d.a. review of the nanotechnology formulation, how many of them are on the market. i cannot give you percentage,

like a number of formulations. i can tell you for example doxil is approved for clinical use for cancer therapy. (indiscernible) is also formulation of anti-cancer drug, abroxone, non-albumin formulation of paclitaxel, ambisome, liposomal, and other

formulations with anti-bacterial products, also imaging agents (indiscernible) so there is quite -- like at least several of these formulations that already are approved and on the market. i cannot speak about the f.d.a. procedure to approve these

materials but i can say that right now this is a very hot topic in this area, many nanoformulated drugs are (indiscernible) patent, so f.d.a. has nanotechnology passports, huge in the method to understand bioequivalence and similarity within the brand name

nanoformulation and generic nanoformulations. i hope i answered your question. >> one more question. the question is cytokines in organ failure. it depends. human necrosis factor, from the name of protein you know induce

necrosis, right? the mechanism is cell death for induction of necrosis, they induce hypotension and hypertension, basically cytokines damage pathogen, and in the absence of the pathogen it will do the same damage to the normal tissues, in the area

where the high level of cytokine is. thank you. >> okay. and our closer is dr. hussain, ph.d. in india, postdoctoral fellowship in switzerland, then came to the university of maryland school of medicine in

baltimore, nci was smart enough to hire him, initially he was a staff fellow, now a tenure track investigator. he's head of the pancreatic cancer unit, the title is pancreatic cancer, current understanding and future

challenges. thank you for the introduction, and what i'm going to do today to give you a -- i know it's very hard sitting after 5:00 for the one-hour session, i will finish in 40 minutes so don't worry. what i'm going to do today is

briefly give you a short overview of pancreatic cancer, some current advancement, see the challenges that are needed to achieve the better outcome, disease outcome in this tiny little malignancy. let me start with the slide which shows disturbing facts

that are associated with this cancer. it's fourth leading cause of cancer death in the united states. median survival is less than six months, and there is estimated to be 48,960 new cases, and 40,560 deaths in 2015.

if you look at the number, if a person is diagnosed with pancreatic cancer, he will most probably die within one year of diagnosis. and this is because there is no effective treatment. and disturbingly, it is estimated that it is going to be

the second -- i think this is too -- it is estimated to be the second leading cause of death due to cancer by 2030. by 2030, which is more disturbing. so what are the risk factors for developing -- (inaudible)

yeah, both, what is happening in last several decades, if you look at the statistic, what happens is incidence is increasing but at the same time there's no effective treatment strategy that has been developed. so both incidents and the deaths

will increase. and there are several risk factors, and some of the risk factors that are mentioned here, smoking, diabetes, pancreatitis, smoking is the well established one among the risk factors, there are several genetic syndromes, and the alteration in

associated genes that enhance the risk of developing pancreatic cancer, and one of the most striking examples is hereditary pancreatitis. what is the status of treatment and as i mentioned, there has been disappointing progress in

the treatment of pancreatic cancer, and i will give you a brief overview how the treatment strategy has developed. more than two decades ago, gem cytabine was found to be better, since that time gem has become the standard.

and arlotinib was given in combination, enhancing survival only by a few weeks, about two weeks, but the situation was so desperate that f.d.a. approved that combination drug to be used in clinic, and then recently in 2011 folfirinox, a combination of four chemotherapeutic drugs,

showed much bigger survival, relatively speaking, of about 11 months as compared to gem cytabine alone, associated with a lot of toxicity and not all the patients would sol rate this regimen. interestingly, in 2013 as the speaker pointed out about

abraxin or albumin bound cycloparticulars in, with gem cytabine was enhanced by a couple months, much less toxicity than folfirinox, became a first line treatment now for the pancreatic cancer patient, and i just change-- inserted some new slides because i just

found in like a recent issue of "lancet," included here, not in your handout, so if you're interested i can e-mail you the powerpoint presentation, and here liposomal bound irinotecan in combination with fluororacil extended survival in patients treated with gemcitabine.

this enhanced median survival to 6.1 month as compared to 4.2 months, and this is approved recently to be used. so as we know, the dismal outcome is because of the poor therapeutic response and late diagnosis of pancreatic cancer, and there is no breakthrough as

yet, and i will give you one or two examples of the early detection, but there is no reliable marker so far that can be used clinically to detect pancreatic cancer earlier. so what happens that about 18 to 20% of these pancreatic cancer patients are de detected earlier

and qualify, go for surgery, resection is performed, and but it's still in the resected case, the median survival is less than two years, and about 80% of of the resected cases, they comekin about two years. but about 10 to 12% of these resected cases, they do survive for five years, some also

survive for ten years. and there are many clinical prognostic factors that are associated with survival. for example, what is the stage at the time of resection, grading lymph node spread and resection margin, is resection margin negative or do you have

the positive margin? but none of these are consistent and we do see variable outcome. for example, there are many patients stage 1 resection margin zero, perfect surgical removal, but some die within a month, within two months, some go for five years.

so that means there is some distinction, like molecular makeup, they are different tumors, although they are stage 1, or histologically, but it seems that there are different molecular makeups and they are responding differently to the surgical resection, and also to

the adjuvant therapy. so the question is that are there molecular differences in tumor determined patient outcomes? and i will come back to that molecular subgrouping later on in my talk. so how to improve the outcomeintic cancer patients if

it's highly lethal, and there is no diagnostic marker, and there is not an effective therapy that can improve the survival, even to one year or longer? so we need of course effective therapy, and there are several strategies that are being pursued.

for example identification of novel therapy targets singly or in combination with chemotherapeutic drugs, molecular subtype as i mentioned, so that a particular subtype can be targeted with this therapy leading to precision treatment, and

treatment selection based on the individual tumor type, and then one of the latest strategies that is being pursued, and i will give you a couple examples, how to improve the drug delivery, and i will show you later that when you look at the pancreatic cancer it has huge

stroma, and highly reactive, which is called dismoplastic reaction, not a lot of blood vessels. so what has been found that drug is not reaching the tumor. so how to improve that drug delivery and i will give you a couple examples.

there are many different views which are contradictory to each other, but what has been found with stroma if we make the drug reach the tumor, that can enhance the survival in the pre-clinical studies and also there are one or two clinical trials that are successful in

phase 2, and are in phase 3 clinical trials. so the second thing is of course as i mentioned we have to detect it early, and find the biomarkers that are associated with pre-cancerous lesion, because once it was developed, even in stage 1 you do

resection, people die. so we have to have some biomarker that can detect early on. so for all these things, we need to understand that tumor biology and i think understanding tumor biology to improve disease outcome.

so let me tell you a little bit about how the pancreatic cancer development progress. it has a well-defined core, and it is developed through well-defined precursor lesion, the most common precursor lesions are pancreatic intraepithelial neoplasia,

subdivided into one, two, three stages, carcinoma in situ and develops into cancer, and there are several genetic alterations commonly found, both in the pre-cancerous lesions, also in the tumor, for example k-ras mutation is present in almost 95% of pancreatic cancer, the

most common form is pancreatic ductal carcinoma, so i'm talking mostly about pancreatic ductal adenocarcinoma, and the same percentage of alterations are silencing of p-16 is found in pancreatic cancer, you can see k-ras mutation early on in the panin-1 and also p-16 alteration

found early on. in the later stage of development, you see p53 mutation, and 50 to 75% of the tumors they show p53 mutations, and smad 4 mutation is associated with distal metastasis, if the tumor s so smad4 mutation you see a lot of

distant metastasis. as i mentioned earlier, a little bit about the tumor stroma, here is the stroma which we call -- which i earlier mentioned, the huge stroma, these are the tumor glands, and these stroma consistent stromal cells and lots of extracellular matrix,

the stromal cells as tumor cells secrete number of hemokine, cytokine, a strong stromal reaction. highly heterogeneous, i mentioned heterogeneity and molecular makeup. one study rly on in 2008 by

burt vogelstein and ken kinsler's group at johns hopkins did comprehensiv analysis of 27 human pancreatic ductal adenocarcinoma, each tumor had at least 68 genetic alterations that can be grouped into 12 core signaling pathways that are associated with cell

carcinogenesis, and in each tumor they found that a different gene mutation altered some of these pathways, so two tumors, even when they had alterations in different pathways, but the genes that were mutated were different, which showed it is highly

heterogeneous. and in a study by collision et al. in nature medicine, in a cohort, based on gene expression analysis they found three subgroups, classical, quasi-mesenchymal and exocrine-like. similarly recently in "nature,"

waddell and colleagues published based on variation in chromosomal structure and the pdac in stable, scattered, and locally rearranged, unstable. in a study published by moffitt and colleagues they based analysis on stromal genes and categorize normal, activated

stromal genes, subgroup in these types, they found that the activated type and normal, they had different survival, stromal genes activated, compared to the groups with normal stromal gene expression. so i will just change gears just a little bit for a couple of

slides because i'm going to tell you how pancreatic tumors can be subdivided based on metabolic alterations also. this is a very complex slide. you don't have to understand everything, but as you know, that metabolic alteration is one of the hallmarks in tumors, many

different tumors, so in pancreatic cancer, they heavily use glucose and guy -- and glycolysis, some have mutant signaling pathway. another pathway that's unique in here, pancreatic tumors use the non-oxidated part to produce ribose phospate sugars for the

nucleotide synthesis, utilizing novel glutamine metabolism shown in orange that are used to produce nadph. they also used ldh to convert pyruvate to lactate, lactate is further used in tumors. they also are heavily used autophagy to produce building

blocks, but what i'll try to highlight, many important enzymes, they are marked with asterisks, controlled and regulated to k-ras mutations, there are many inhibitors of enzymes in clinical trials, so i showed you this slide to show this data that is recently

published describing again the molecular subgrouping based on the metabolic alterations that they found in tumors. so they found slow proliferating and glycolytic based on those metabolites. now let's talk a little bit about what makes it so

aggressive and so therapeutically resistant, so the tumor cells and stromal cells and hematopoietic cells and inflammatory cells, they all contribute to the therapeutic resistance, and provide a very friendly environment for pancreatic cancer progression,

so let me talk a little bit about -- this is tumor epithelial cell. tumor epithelial cell secretes a number of factors, growth factor, and that activates pancreatic it stellate cell in the stroma, and this induces the deposition of hylaronic acid,

that absorbs water and produces a high interstitial fluid pressure that's suppressed already scarce vessel and this gets constricted and that drug doesn't come out of the vessel to the tumor. i will show you one or two studies how when you reduce this

interstitial fluid pressure by reducing hyaloronic acid, blood delivery goes in and enhances survival in pre-clinical models. more importantly what has been shown that from the very beginning, the immune suppression, the tumor suppressive mechanism due to the

anti-tumor immunity is heavily suppressed, so there is immune suppression because of the heavy deposition of tumor associated macrophages and myeloid derived cells, inhibit cd8 positive t cells, cells to function. so these several components in the tumor including the stroma,

tumor select cells and immune cells, they all contribute to the pancreatic cancer progression and therapeutic resistance. as i mentioned, there are several treatment strategies including enhancing the drug delivery and effectiveness of

systemic therapy, to target stroma. one of the pre-clinical models that has been heavily used in this strategy is this genetically engineered mouse model where you have mutated k-ras gene and you have active mutated p53, and they are large

so when you cross these mice, mutant k-ras is activated, p5 is activated, develop in more than 99% in mice pancreatic ductal carcinoma, median survival five months, the beauty of this model originally developed by hingorani, it faithfully recapitulates development and

progression of human pancreatic so what did they use this model, in one of the earlier studies that was published in "science," and they used the hedge hog inhibitor and found increased survival, here also decrease in liver metastasis in the mouse model, but unfortunately the

clinical trial with sonic hedge hog inhibitor failed, in fact it gave the unexpected result so the clinical trial had to be halted. and this study by hingurani publisheled in cancer cell, using the pre-clinical model that showed heavy deposition of

hyalorinic acid and combination with water provided solid mass and enhanced interstitial fluid pressure and constricted the blood vessel, enhancing the drug in these mice, so this pegph20 is in clinical trial, phase 3 trial, we'll see what happens with this trial.

but there are other studies that show stromal targeting may not always have beneficial therapeutic response. what they found is that they use the gem model, genetically engineered mouse model, deleted sonic hedge hog model, micro

metastasis is up, several pre-cancerous lesions, all there increased as compared to the pkt mice. at m.d. anderson they depleted fibroblasts from the stroma and found that depletion in fact enhanced the disease

aggressiveness in these mice, and it also reduced the survival. so tumorous stromal interaction is complex, therapeutic approaches need caution and may require new molecular taxonomy. stroma has to be engineered, we have to figure out that what

molecular makeup of tumor will have beneficial effect with this treatment of strategy. about another very promising target that is mesothelin, discovered on this campus in pastan's lab by past and colleagues, they have done a lot of studies on how it can be

targeted for treatment, and these are some of the examples which shows that mesothelin is overexpressed in a number of tumors, and they are using immunotoxin that is targeted to mesothelin in the mesothelioma, a clinical trial initiated to use this immunotoxin that is

targeted toward mesothelin in pancreatic cancer. so i showed that mesothelin background because recently in cancer cell, i think a couple weeks or a month ago what stromnes and colleagues did, engineered t cells to express mesothelin receptors, and when

they introduced the receptor expressing t cells they lysed tumor cells in the animal model, and they also enhanced survival significantly. so mesothelin is a promising target that can be -- that can be used, either in immune therapy to target pancreatic

cancer, improve disease outcome. so let me mention a little bit about early detection. as we know, there is no early detection marker that is sensitive enough for early detection of pancreatic cancer. a couple of weeks ago a paper in "nature" by the lab at md

anderson, what they found is in circulating exosomes, tumor exosomes, expression of glypican-1, they could different differentiate between benign disease and pre-cancerous lesion and pancreatic ductal carcinoma, high level of circulating exosome positive with glypican,

highly sensitive for pancreatic so they also tested if this can be used to predict prognosis in resected cases. they found that, yes, the presence of the -- the decrease of glypican 1 positive circulating exosome can predict better survival in the

pancreatic cancer patient, after resection. so these are some of the very promising pre-clinical data, some of the association studies that have the promise if validated further in different studies to come to the clinical view.

so this is the last part of my talk, and i will take about ten more minutes to tell you about some of the inflammatory mediators because many of the inflammatory mediators are found to affect the development and progression of pancreatic cancer in many of the animal models,

and also its association has been established, with the human pancreatic cancers. so some of the examples are chronic pancreatitis, increased risk of developing pancreatic saner is. nf-kappa b signaling enhances some enzymes that plays

important role in tumorigenesis, cox2 and nos2, and many inflammatory cytokines, you see a desmoplastic stroma, highly inflammatory. you see inflammatory changes during development of pancreatic cancer, panins in this model. also although mutant k-ras is

important for the development of pancreatic cancer, it has to be maintained just the mutant k-ras is not sufficient to induce it has to be maintained at an oncogenic functional level, at the pathologic level, maintained through several inflammatory mediators, for example pge 2,

that's what he described in his elegant study that high sustained ras activity initiates nf-kappa b, several inflammatory mediatos, one is pge2, that maintains the enhanced k-ras activity. so one of the interests in my lab, i will show you three or

four slides some of the data that we have generated in our lab, and one of the interests is macrophage migration inhibiter to factor, expressed in many cells, primarily it has been described as a regulators of immune and inflammatory responses.

it is expressed in a number of different tumors, including pancreatic cancer, it activates a number of different oncogenic signaling pathways, for example akt, nf-kappa b, erk 1/2, catalyzes reactions that produces inflammatory mediators, for example cox2 and nos2 and

inhibits some of the p53 function and interferes with rb/e2f pathways. based on these and other function mif is described as a link. we tested a hypothesis that mif contributes to pancreatic cancer and predicts disease outcome.

first of all, we screened pancreatic cancer patient samples, and we found and increased expression of mif in tumors, as compared to normal adjacent duct in non-tumor tissue and we also found that a higher expression of mif in tumors was associated with

poorer prognosis, this is the kaplan-meier analysis, also the univariable and multi-variable analysis showing mif an independent predictor of prognosis, and then these studies have to be validated, so we validated in several different cohorts, two cohorts

are shown here that increased mif expression in tumor predicted poorer survival in pan pancreatic cancer patients following resection. these findings show an association, you have high mif in tumors, predict poor what does mif do actually?

we did an in vivo orthotopic model, we found cells developed accelerated tumor growth and increased metastasis here as compared to controls, and interestingly if you look at these two tumors, histologically, 100% of the mif-expressing tumor are all

poorly differentiated, solid growth pattern, as compared to the control tumor which was moderately differentiated, and when you do gene expression analysis on these tumors you see a drastic change in global gene expression, and some of these genes, they are associated with

emt as you can see. so what does it mean? recently, there was a paper, and i was very happy about it, that david and ben published that exosomal mif, if the exosome have increased expression of mif, that goes and prepares the metastatic niche in liver, stage

1 patients who are resected coming back to my original point that why some of these stage 1 patients, they die faster than others, so here is some answer that they are -- they have enhanced levels exosomal mif, they also found stage 1 patients who have increased exosomal mif,

they show metastasis later on as compared to those stage 1 patients after resection who do not have enhanced mif, that's good, consistent with what we have earlier described. so what we did, we took the human pancreatic cancer, divided into two groups, one with

increased mif, one with lower mif, you see the h/e, tumors with lower mif had well differentiated tumor, high mif, poorly differentiated tumor, so the questions here you're asking, are there molecular distinction between the two tumors, and what is the

mechanistic and functional role of mif in tumor progression? so very talented postdoc in my lab used a strategy by doing transcriptomic profile to answer some of these questions, and he tested hypothesis mif regulates microrna associated with tumor progression and disease

aggressiveness in patients with pancreatic cancer, he found there are several differently expressed micrornas when you compared mif high to mif low tumors, and some of these micrornas that are differentially expressed between these two groups of tumors, they

are also associated with survival, some of these are mentioned here. so these data were validated in multiple cohorts, patient cohorts of pancreatic ductal adenocarcinoma, and i am just cutting short the experimental part, and what we found that

there is a novel mif induced signaling pathway, which increased mir-301b through pi3 kinase pathway, and mir-301b are targets in nr 3c2, the receptor gene, and its function is not described in pancreatic cancer, we found it inhibits ent, this inhibition of nr3c 2 enhanced

emf and enhanced pancreatic cancer progression and we validated this in the clinical centers, and this study is under review for publication. so what we are going to do with this data? so we are testing the hypothesis that mif 301b/nr3c 2 signal is a

potential therapeutic target, we used the same genetically engineered mouse models i earlier described, the tumor expresses a high level of mif, so we genetically deleted mif and generated mif-deleted kpc mice, and what we found is that mif deficient kpc mice survived

significantly longer as compared to the kpc mice with wildtype mif and showed a lower metastasis as shown here and specifically in liver. and we isolated the tumor cells from these mice, and you do find that mif deficient mice, they have (indiscernible) this is the

western blot analysis, just consistent with data, and what we also found that it has decreased mir 301b, mif enhanced, when you delete mif it down and nr3c 2 comes up. here are the seq analysis for localization just to show you what it is localized, these are

are the tumors, this is the mif-deficient kpc mice. so now we have the genetic in the pre-clinical model, when you inhibit mif, it enhances survival, reduces metastasis, so now there are several ways you can inhibit mif. there is anti-mif antibody.

there are small molecule mif antagonists, anti-mif receptive antibodies, also mif receptor antagonists. so anti-mif antibody is in phase 1 clinical trial for different solid tumors, i just found out that it's in phase 2 clinical trial for colorectal cancer and

ovarian cancer, and we're talking to the company, which has humanised mif antibody, and if they would like to collaborate with us and give us the drug to start our clinical trial, and there is also in collaboration with richard bucala, who has actually

also small molecule inhibitor, we will be using these inhibitors in the pre-clinical model and ultimately in the clinical trial. so summary, higher mif expression is associated with poorer outcome in pdac patiens,

mif enhances growth and metastasis, and nr3c 2 is negative regulator, mif-deficiency increased survival and reduced metastasis, and i would like to end with this slide which i showed in the beginning that to increase the outcome or to improve the

outcome in the pancreatic cancer patient, we need to work not only on the early detection but also on the effective therapy and i think understanding the pancreatic tumor biology and utilizing the biology in developing treatment strategy is a key and the molecular

subgrouping that can lead to the precision treatment strategy for pancreatic cancer will be extremely helpful. i will stop here and answer your questions, if you have any, and exactly 45 minutes so thank you very much for staying. thanks.

[applause] >> (inaudible) >> say that again. yes, in the resected patients, you can do easily, right? you have the biopsy, you can do it. in the advanced stage patient, that's why it's important for

example looking at the circulating tumor cell, and there's the recent paper that you can take out from the portal without doing so much damage to the patient, circulating tumor cells, and they have, and i did not mention that study, just published, and you can in fact

identify some of the genetic alteration and some mutations, so these are the initial studies which i described to you. so now we can do the endoscopic ultrasound and take out the biopsies in those patients, and if possible and with further advancement, it may be possible

to find out in serum or blood or in the circulating tumor cells whether the tumors can be distinguished. >> sure. she's these have important and good questions. we have to deal with the

intratumor heterogeneity, so those are the questionses questionses that we don't have answer, how much of intratumor heterogeneity is important to achieve the treatment outcome. we don't know yet, but, yeah, all those are important yes, please.

>> yes, so we don't know that why they are hyper vascular, as opposed to the other tumors they have increased angiogenesis, and they are heavily vascularized, but one of the hypotheses that stromal -- the huge stroma and they suppress the formation or expansion of the new blood

vessels but there is no clear-cut answer why they are hyper vascular, yeah. you know, enhancing vascularization and then providing drugs is one of the strategies that is being but we don't know why it is hyper vascular other than the

fact it has compact and huge -- when you touch the tumor it's like a rock. like i was fortunate, i'm not a clinician but with some of the collaborators i attended some of the -- scrubbed in in some of the surgery, and i could see first hand, and they showed,

look at this tumor, you will not see this kind of tumor. it's like a stone. and so i don't know. i don't know the answer of the hyper vascularization. thank you very much.

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