Cancer Treatment Center

>> the okay, we'll get started. this is the last lecture for traco for 2014, i would like to thank anthony thomas in building 50 for videocasts, and at frederick for helping with the videocast, jonathan weiss for supporting the traco class. first speaker today is marina

dobrovolskaia, at the nanotechnology characterization lab in frederick. immunological properties of engineered nanoparticles, and some challenges in clinical characterization. marina? >> thank you, dr. moody, for the

introduction. good afternoon, everyone. good afternoon to those who joined us in this room and also hi to everybody who is watching this presentation online. i am going to talk about properties of nanomaterials. i will start from overview and

definition, we'll talk about what nanotechnology is, what kind of medicine, we'll talk about examples of nanoparticle use in daily life, we'll see what nanoparticles can offer for cancer diagnosis and therapy because this is of special interest to this lecture series.

i will give you overview of nanomedicine, tell you about the lab, nanotechnology characterization lab. ntl, and immunological properties and case studies, how nanoparticles use chemical properties and determine interaction with components of

the immune system and review some case studies to show you the challenges with pre-clinical characterization of this material. so what is nano? there is no universal definition. the one which is most commonly

used comes from national initiative, nni, and it defines nanoparticles as objects with at least one dimension, approximately one to 100 nanometers. however, the nanoparticles are used for biomedical application, the size can extend beyond

100 nanometers, and united states food and drug administration specifically defined if material properties are determined by the size of this material, and the size is steppedded beyond 100 nanometers it will still be considered nanotechnology, the size extends

to 1000 nanometers, we're talking about objects in this size range, within the size of the molecule of glucose, to approximately the size of bacterial cells. the role in daily life according to literature, 800 manufacturer-defined products

from more than 400 companies in more than 20 countries world wide, that use nanoparticles in their products. nanoparticles are included in clothing, wound dressing, washing machine liners, used as a lens coating in sunglasses, sporting equipment, many of you

use nanoparticles throughout the summer, we apply sunscreens to our skin and skin of our children, because some of this sunscreens contain zinc oxide, carbon nanotubes are used as structural materials. nanotechnology is special for cancer therapy because as you

know a lot of drugs are insoluble, nanoparticles may act as a carrier for hydrophobic drugs to improve something your solubility, they can offer multi-functional capabilities. a review was published in 2005 where he described this concept

of the nanoparticle drug carrier, a nanoparticle with a nanosize core and it's represented here for multi-functional modalities because it contains image contrast agent, targeting molecule and some drugs incorporated into the particle.

the nanoparticles are used for drug delivery, but also for cancer diagnosis, imaging agents as a diagnostic agent, and nanotechnology detects cancer cells, most sensitive, they work with smaller volume and provide great greater sensitivity and specificity.

cancer therapy, nanoparticles efficacy occurs through two major mechanismsment one is considered as passive targeting, which relies on so-called enhanced retention, based on leaky vasculature in the blood vessels, penetrating and

entering the interstitial space in the tumor where is the drug. the second targeting mechanism is active targeting, and it is based on attachment to the particle surface of specific ligands like cytokines which recognize their receptors on the surface of cancer cells.

however, it is important to mention in order for the active targeting to work, basic tar getting still has to take place. even though we have antibodies, any ligand on the particle surface, in your for the ligand to recognize a receptor molecule expressed on the surface of

cancer cell nanoparticle has to go through the vasculature, get into the interstitial space and interact with cancer cell over there. so what is nanomedicine? nanomedicine is a terminology which is commonly used by the researchers to define

nanoparticles that are used for biomedical application. what i have on this slide is somebody demonstrating difference between nanotechnology-based medical devices and drugs, in investigational stage, and in the commercial stage.

as you see in nanomedicines that are currently under investigation, majority of the concepts are represented by medical devices and small percentages represented by the drugs. if we look at the commercialized nanomaterials, maturity of drugs

and some proportion about 33% is represented by medical devices. i prepared this graph based on etheridge, in a nanomedicine journal. if you're interested i highly encourage you to read this article. it's very interesting.

my other slides are based on data published in this article. if bee we look at nanomedicine,we see the difference between investigational and commercial nanomaterials. in the investigational stage the dominant nanomedicines, i mean representing more than 10% of

total concept liposomes, polymeric solid by solids. in commercial this is different, there are more solid nanoparticles, followed by nanocomposites, followed by other categories, actually to me this was very interesting

difference because i thought majority of the nanoformulations that approved for clinical use represented by liposomes and nanoemulsions but as you see from this data this is not the case. what are the common features of nanomedicine?

if you look in this graph, you see majority of nanomedicines are developed for cancer therapy, followed by infectious diseases and other applications. these concepts are designed for intravenous administration, less than 350 nanometers, most of these concepts are neutral,

hydrophylic surfaces, the most biocompatible and loss toxic, most are spherical in shape. this slide shows the funding that national nanotechnology initiative invests in the nanotechnology, even though in the last few years the budget flattened, almost triple

compared to the 2001 investment in nanotechnology and especially nanotechnology for medical applications, started in this country. so in development, there is such a concept as a valley of death. i borrowed this image from review by butler in "nature"

that demonstrates this concept very nicely. most of us who developed basic research concepts for diagnosis or treatment of cancer are on this bank, drug discovery. and in order to transition this wonderful concept into the clinical use, before patients

see the benefits, there's a huge gap, this gap is called valley of death because crossing this valley is not fast and it's not easy, it requires effort from both academia and industry and in nanotechnology there's a wonderful way for the government to help this transition, and one

such efforts is the lab established in 2004 as interagency, under interagency agreement between national cancer institute, united states food and drug administration, with drugs and diagnostics, ncl is performing pre-clinical work helping academia, health be

small biotech companies and big pharma to cross this valley of death, including physical chemical characterization. from the beginning in 2004 the lab is led by the doctor whose picture you see on this slide. we've been in operation for ten years, in fact in september of

this year we celebrated our tenth anniversary, and in this time we characterized more than 300 different nanoparticle concepts. what you see on this graph is the different kind of particles that pass through ncl. you see the dominant particles

are limb so manies followed by polymers and emulsions, followed by other formulations. later in my presentation i will show you case studies, show you our experience with some of these materials. so it is very well established

now that nanoparticle physiochemical properties such as size, charge, targeting and hydrophobeicity, the majority of case studies demonstrating this will be used from our immunological characterization. nanoparticles can be immunotoxic.

however, the immunotoxicity can be good or bad depending whether it is desirable or not. a lot of engineers of nanomaterials are designed for biomedical use, and they are designed to be immunologically radioactive. nanoparticles can be used to

improve cancer therapy, to achieve therapy from inflammatory and auto-immune diseases, they are desirable. nanoparticles may lead to undesirable side effects, hyper sensitivity reaction, anaphylaxis, coagulation disorders, and lowered defense

to pathogens and cancer. many examples come from the environmental nanomaterials. we at nco are focus on engineered nanomaterials being used for diagnosis and therapy of cancer, not necessarily intended to be radioactive, what we want to know whether the

materials have undesirable effects. why? once we understand the reactivities responsible for this immunological reactivities, remay develop the concepts to reduce toxicities. nanotechnology based

pharmaceuticals are not more intrinsically immunotoxic than other drugs in clinical use. there's increasing data demonstrating that incorporation of small molecules to the nanotechnology platform helps decrease immunotoxicity of these what i will do now, give you two

examples, from the field of small molecules and therapeutic protein. many of you are family with anticancer drug, doxorubicin, used in clinic, maybe patient develop dic originating from development of so called pro coagulant activity on the

surface of the leukocyte, what this complex is doing, it triggers coagulation, many patients have to be withdrawn from the therapy because of this coagulation side effect, incorporation into the liposome is known as as doxil, this does not have this type of toxicity.

another example is therapeutic protein, the name stems from the function, causes necrosis of the tumors but in order to achieve therapeutic efficacy one has to deliver so much protein that it is incompatible with the patient's life because tnf is an important stimulant.

there a small biotech company in rockville, maryland, what this company did, they put tnf on the surface of gold nanoparticles, it makes particle surface hydrosilic and shields from immune cell recognition, while it is delivered to the tumor site, shielding tnf from immune

cell recognition, decreasing systemic to achieve higher therapeutic efficacy from this drug, this concept is in clinical trial stage. there are a lot of changes in characterization of in 1968, it was suggested using what he called fish bone diagram

to summarize potential factors that may have overall effects. this diagram is used in the industry mostly in the products developed in management, but i use this idea to prepare this fish bone diagram to summarize factors which are critical of engineered nanomaterials.

the knowledge i used to prepare this slide is generated over many years of working side by side with two colleagues, dr. patri who for almost ten years led nci chemistry group and dr. stephen stern, head of pharmacology.

it is critical to understand transition in concept from bench to bedside. in chemistry, they include particle size, charge, shape, composition, surface coating, solubleity, architecture and ecxipient. in pharmacology and toxicology,

dr. stern summarized the biodistribution of nanoparticles, again the use of appropriate models to study toxicity, challenges and sometimes causing a problem for toxicology. this is my area of expertise. based on our experience with

hematology characterization i can say very challenging, a lot of problems come from chemical impurities and biological impurities such as endotoxin, and later in my presentation i will talk about this. i think from just looking at this slide, you can appreciate

that many of the factors are overlapping within the chemistry, toxicology and immunology. following up the three sections, three groups working together, chemistry toxicology and immunology are inseparable because i cannot evaluate

immunological compatible of nanoparticles without knowing chemical properties and i will show you case studies demonstrating this connection. this study is prepared by dr. steve stern, and the idea to show that nanoparticle stability is critical to achieve very good

drug delivery and good efficacy in terms of cancer therapy. if nanoparticles are too stable, these particles are not releasing the drug, or release the drug very slowly, as a result they are nonefficacious and accumulate in the tissues. in this example we're showing

here the concept which includes anticancer drug, and we found no toxicity, it was perfect. however, what you see from this graph, it did not release the drug, even less than 10% of the drug was released over 48 hours. as such, it was not toxic but it

was also not efficacious. another extreme point are unstable formulations, unstable formulations are those that release drugs too quickly, and that results in toxicity and not optimal efficacy. what we've seen here example of such unstable formulation which

contains docetaxel on the liposome, what steve found by studying this nanoparticle in vivo the drug is released from the liposome within five hours and it is causing off target toxicity and not efficacious. nanoparticles may change by distribution of the drug.

i'm using example of doxorubicin. this drug accumulates in bone marrow, also it didn'ts to the heart. doxorubicin common toxicity is cardiac toxicity. however, this drug is incorporated into the different

nanoparticle platform. these particles may change distribution of the drug, in case of the doxorubicin from bone marrow and heart it moves into the skin and so as a result doxo has new myelo toxicity, now it has tpe, palomar plantar erythrodysthesia.

and same drug, instead of the bone marrow in heart, this nanoparticle takes doxorubicin into the kidney, there's no cardio toxicity but there is nephrotoxicity. drug carriers change, it may result in altered distribution, which may change toxicity

profile of the drug. i mentioned i was showing you the diagram that very often excipients are toxic. we looked at compatibility and found it is hemolytic. in this case we didn't even need the test, you can see there was no dent row site left

-- no dendrocyte left. 165 nanometers were expected for this particle, but then the same formulation was analyzed by transition microscopy, in addition to the nanocapsules, they also detected my cells. we found this the

brij 78 is present at the concentrated above critical cmc, so this could be reproduced and they look identical to what we observed with formulation, but concentration of surfactant, and they may interact and cause toxicity, so the toxicity

is caused by excipients and not nanoparticles. the similar relationship exists for other types of the particles, but all particles are different and i'm just using one example. we study here interaction of the

particles with platelets and we're using platelet degradation as endpoint, we transition to the larger particle, the reactivity with plasmaic membranes increases. again particle size is important. however size in biological

context may be different due to the interaction with plasma proteins. many of you may have heard this definition protein corona which refers do plasma proteins bound to the particle surface after the introduction of particles into the bloodstream from our

earliest studies with gold nanoparticles, particles have theoretical size of 30 nanometers and these particles are studied under pristine conditions, it doesn't matter whether at the electron microscopy or dynamic to measure their size, we measure the size

which is very close to nominal size of these particles. however after these particles are incubated with human plasma, what we see is that size measures by tem and afm does not change because they look at the core of the particles, but as measured by dynamic light scan

almost doubles. what is important here is our body sees this 13-nanometer gold particle as 18 nanometer particles. because of the protein corona formed on the particle surface, this is very important to recognize.

the surface charge is another property which is commonly used to evaluate the activity with biological systems. again, in this case, i'm using example of a cat ionic dendrimers. what our chemists did, they masked 25% and we used particles

to look at the degradation and another thing to factor in, we see a decrease in plate late aggregation and keep masking them and neutralize the surface, we neutralize reactivity of particles with biological membranes. these case studies demonstrated

zeta potential is important, less surface amines, less area with platelet. in this case i'm comparing two different dendrimers. look at this blue graph. you see the same trend, with the red bars, we transition from the smaller particle generation

three to the larger particle generation seven, we see increased reactivity with platelets. if we compare same generation, we see the triazine are less biologically active than their counterpart. why is that?

this is pamam, architecture is different. they have less number of surface groups on the particles surface and we know from the previous studies the number of surface amines are important determinant of particle reactivity with biological membrane.

i hope by now i convinced you particle size and zeta potential are important determinants of nanoparticle toxicity. however, not always they allow to discriminate between i would like to use doxils including lipodox, and i will talk about these three

formulations because those formulations are available in the united states. our chemistry group did an analysis. they did not detect any difference between what we call old doxil, the doxorubicin available in the united states

before 2012. the company experienced problems in their facilities resulting in worldwide shortage. then this problem was addressed, the same manufacturer produced new doxil, this is available after the shortage and we compare it to the lipodox.

according to the physical chemical characterization, there is no difference in this formulation. however, we look at the complement activation, we do see the difference, old doxil is reactive with complement. we used this material as a

positive control in our complement activation studies. but the new doxil and lipodox does not have this toxicity, and this data are in the agreement with in vivo clinical data, data in animal model of of carpa. this is very good news for the patient in that whatever the

difference in the new formulation, the toxicity was decreased, but this relationship could be the other way around. this is why it is very important to understand what processes are responsible for the discrepancies in biological properties of the material.

now i will switch the subject a little bit and talk about endotoxin, for those of you who are not familiar with endotoxin i'll give a brief introduction. endotoxin, lip oh poly saccharide, is a component of the membrane of gram-negative bacteria.

it may lead to septic shock, resulting in organ failure. nanoparticles can be contaminated, very common in pre-clinical stage. based on our experience at nco, 30% of pre-clinical nanoformulations fail due to the

endotoxin contamination, endotoxin in nanoearly pas is bad because it results in wrong conclusion, may confound results of toxicity studies and responsible for undesirable toxicity, some nanoparticles, not all, some may exaggerate inflammatory processes of

endotoxin, also because it may create problems with immunogen isty of protein based active pharmaceutical ingredients or target ingredients of induction of activity by concentrations of lts alone and in the presence of cationic

dendrimer. one of the biggest challenges, the grand challenges in my opinion, is accurate detection of endotoxin. i summarized some examples, looking at the nanoformulations, and amount of endotoxin detected by three forms of lal assay,

used in pharmaceutical industry to quantify contaminated endotoxin. what we found is majority of the nanoparticles interfere with the gel-clot lal. for some it doesn't matter, the results are consistent, but for some materials like in the case

of this nanocrystal formulation, relying on gel-clot may lead to overdose of endotoxin. the reason why this is important is because gel-clot lol is fda preferred method. so what we did, we conducted collaboration with fda looking at nanoparticles, and the

ability to interfere with gel-clot and other tests. we proposed this decision tree which informs us in choosing what is i appropriate. the decision tree is based on physical chemical properties of at a wavelength of the allele assay we use turbidity.

if it is turbid we don't use turbidity assay, then we use gel-clot, but the main message from this is that we use more than one lal and at the end compare the data. if the results from two lal are in agreement we report results. if they are in disagreement we

use biological assay such as macrophage test to verify lal findings, this approach is useful in many cases, this is one of the examples. we tested this by chromogenic, the same tested by turbidity gave us 21.4. how do we know which data is

correct? at the dose at this which material is used, if this value is through, material should not be pyrogenic. we found this material is pyrogenic, at this point we worked with them on purifying this formulation from endotoxin

contamination, and purified formulation was tested. you see the result of lals are in agreement, detecting lower quantity and the material was nonpyrogenic. so this case shows us the utility and usefulness of the supplemental lal test with

biological assay, not all this approach is working. biological tests are not always helpful. this is example. this is commercial formulation, nanoformulation, these were studied by turbidity. there was no problem for

different lals, for this formulation we do see discrepancy between turbidity, chromogenic and gel-clot. then we use particles to verify lal finding with macrophage activation test. one of the important steps is to see this.

we take the particles and deliberately spike them with endotoxin at known concentration which tells us whether -- this provides valid data. we see it, the data is reliable because we can recover endotoxin which was spiked into the samples, but this approach is

useless with here because the materials contain cyto toxin there are some approaches to find out endotoxin contamination but it is not straightforward and it requires the use of combinational methods. another very important parameter in studying endotoxin

contamination in nanomaterials is the use of various reagents. in immunology, we have a lot of agents known to neutralize. one is a peptide antibiotic used to treat bacterial infection, and what immunologists commonly do, they have a doubt whether the immunostimulation is caused

by endotoxin or test material, in our case nanoparticles, euse pmb to add into the reaction mixture and see whether we inhibit biological effect or not. for example, here we look at the cytokine induction, we inhibit cytokine induction, same through

this case. we studied metallic nanoparticles, inducing cytokines in both cases. this is tested in the presence of pmb, in this case we do see inhibition. trained immunologists are term this data, cytokine induction

caused by nanoparticle, in this case cytokine induction is caused by endotoxin contamination nanoparticle. but this is not necessarily the and i would like to share with you some data showing that pmb, because it is cationic material, may interaction with

nanoparticle, pmb interacts with lps electro statically, so material can be neutralized by pmb, in this case the metallic nanoparticles have size of 26 nanometers, and approximately minus 47. you can see by transmission electron microscopy, the zeta

potential, the material is neutral for minus 47, it becomes 9. pmb is a useful reagent sometimes but it has to be used with caution in that it may change physical chemical properties of the nanoparticles as well.

this is one of the challenges in pre-clinical characterization, in addition to characterization under pristine condition and relevant condition we also haves to understand whether additional reagents that we use in nanobiological tests can influence particle physical

chemical properties, the result of the tests may not be valid. nanoparticle sterilization stability is another grant challenge. i use the example of citrates. gamma radiation is a common method for sterilization and common method for deeper

irradiation. the properties do not change but silver particles disappear, and what we see, we see appearance of beautiful structures that look like flowers and swans, but anything but the nanoparticles. in fact, the gold material which is available from yeast is

sterilized by gamma radiation but in silver biologic properties also change. this is unsterilized silver colloids, as we increase the dose of gamma radiation they become more toxic and interact with membrane resulting in degradation.

i would like to finish reviewing my presentation reviewing another grant challenge which is common for other areas of drug development, this challenge is related to in vitro and in vivo correlation. the likelihood of identifying immunotoxicity or any other type

of toxicity increases as we progress into clinical trials, but in vivo testses are more expensive and they have less throughput, the application in pre-clinical studies is limited. therefore there is a growing recognition of the need for the screening methods and there is a

requirement for those methods to be predictable and valid and give us informative and reliable data. there's the effort worldwide in understanding predictability of in vitro tests. i would like to step back and give you examples of the lessons

which was learned from biotherapeutics from demonstrate why in vitro methods are i will talk about tgn 1412 compound. what happened here? there was a company in europe tegenero that developed antibodies cd28, what it was

expected to do, for the t-cells to interact with the antigen presenting cells, two events are necessary. one is interaction within t-cell receptor on the surface of t-cell. this interaction gives information to t-cells that

there's an antigen, t-cell has to react to. but this interaction is not enough to stimulate activation of the t-cells. the second interaction is necessary for cd28 and 80 and 86 on the surface, the second interaction takes place t-cells

now have okay or green light to go with proliferation, production of cytokine in response to antigen but in cancer, for example, there is a mechanism, cancer cells develop to inhibit this interaction which inhibits t-cells, the idea was to stimulate, to promote the

cell activation. studies were done in nonhuman primates and rodents. they transitioned into the clinical studies, and injected this material into human volunteers, six out of six patients were delivered into the intensive care unit due to the

cytokine storm, they almost lost their life. they had to stay in icu for months, and some of them lost their fingers and toes result of the cytokine storm, necrosis of tissue, very devastating example, the research community especially in biotechnology

proteins was very affected by this. later then studies were done to understand the reason for this toxicity, we found in vitro experiments, exposed to the compound, high levels of tnf, if the test was used to supplement pre-clinical studies in monkeys

and rats, then the life of the patients may be saved and maybe not placed in such a danger like in this case. so this is example demonstrated the necessity and importance of in vitro test. nothing happened like this in nanomedicine but we

used cytokine tests. we look at compatibility. we know from the literature and our work the common acute toxicity include hemolysis, complement activation, thrombogenicity, phagccytosis, pyrogenicity and cytokine

reduction. in vitro methods are predictive and helpful. we look at the nanoparticles that have identical core, identical hydrodynamic size and potential, the surface contains different ligands, i just for the purpose of this presentation

named them formulation 1 and formulation 2. these particles were studied in pre-clinical studies in vivo in rodents, and in rats and rabbits. the toxin was undetectable in both formulations by gel-clot lal and formulation 2, but

formulation 1 was toxic. our pathologist analyzed the tissue from the animals and found congestion which was very similar to that seen in septic shock, so even at some point we thought the particles contaminated with endotoxin but again they did endotoxin and did

not find any. analysis of blood samples from animals the show high cytokine levels. this particle were tested in vitro, the toxicity was reproduced for the formulation 2, formulation 2 was induced, formulation 1 was not toxic in

vivo did not induce cytokines. this study shows the activity of the test. in summary i would like to say each nanoparticle is unique, characterization is important, not just dls and zeta potential. and the key factors that are critical in pre-clinical

development include as size, charge, competition. their functional properties, it is important to understand structure-activity relationship, because the beauty of nanotechnology it can be engineered and bioengineering these properties we may deliver

interaction with certain components of the immune system or certain components of the biological system or avoid this interaction. endotoxins and sterility and sterilization are common challenges viewed in pre-clinical development and the

need for predictive in vitro methods like cytokine induction test. i would like to say thank you to my colleagues at nanotechnology i'm proud for being a member of this group. we have very talented scientists can expertise in drug delivery,

chemistry, immunology, oncology, cell biology and we have wonderful technical support team, majority of the data that i shared with you today in immunology is generated by tim porter, barry noone and i would like to say thank you to the collaborators.

dr. jan simak from texas christian university, so we could understand the importance of the particle competition, and how particle composition influences biological reactivity of this material. if you have any questions, after this presentation, please feel

free to send e-mail, and visit the website to look at our publications, to look at how you can collaborate if you have a nanoparticle concept and i would like help transitioning the concept from clinic to the bedside. with this, thank you very much

for your attention. and we have a few minutes to answer questions. [applause] [low audio] >> the question was, how many nanomedicines are approved and being used in the clinic. i cannot tell you like a number.

i don't have a magic number. i would like to say that there are a number of nanomedicines currently in the clinical use, and for cancer therapy two very common examples -- as far as i know nanoparticles are developed to target

different types of cancers, but one used for breast cancer, and the advantage of this medicine is not specifically to target the cancer cells but reduce toxicity common to formulation. for cancer therapy, the current clinical examples demonstrate the utility of nanomedicines to

reduce toxicity and still provide therapeutic efficacy but in pre-clinical pipeline there are a lot of concepts that contain targeting, like targeting to her2 receptor on the surface of cancer cells, folic acid is another common small molecule used to target

for colon cancer, including ligands overexpressed on the cancer cells missing on the normal tissue. >> that will do it. >> thank you. >> and we have another announcement, being that -- for those of you who are want to get

a certificate of completion of the traco course you can take the final examination, we're in the process of loading that onto the website, and it should be ready by the end of the week. basically we have one question from each of the lectures, multiple guess exam, you newed

to get 70% of the questions right to pass the examination. okay. so initially we had perwez hussain scheduled to close the course on pancreatic cancer but he had a schedule conflict so he spoke earlier. so i'm going to close the course

talking about small cell lung cancer. here we go. so small cell lung cancer, it's a type of lung cancer that's very deadly. it kills approximately 25,000 people in the u.s. annually. and it's a neuroendocrine

cancer. most cancers are epithelial in nature. but this cancer is neuroendocrine, so it makes biologically active chemicals then that it can use as growth factors. it's treated with chemo and

radiation therapy, but then relapse frequently occurs. we mentioned before that in cancer there's clones of cancer cells, and initially the first clone may be responsive to chemotherapy but as that clone is killed, the second clone may grow out and then it may be

resistant to chemotherapy and that can lead to death of the patient. in any event, the small cell lung cancer is deadly, median survival time is less than a year. and so when you do the pathology analysis, you see that there's

lots of very small cells, very prominent nuclei, and the tumor. so it's very easy for the pathologist to distinguish small cell lung cancer from other types of cancer. and using the electron microscope then you see that there's these granules in the

small cell lung cancer cells, and these contain biologically active growth factors. so with small cell lung cancer, like most lung cancer, initial symptom is cough, you get pain in your chest, what happens is the cilia in the normal lung cells disappear as the cancer

progresses, so then you can't exchange the carbon dioxide readily for oxygen and you have shortness of breath and that can lead to pneumonia, and a tell-tale sign is you cough up bloody sputum. it's traditional diagnosed with the chest x-ray but there's

other ways of detecting it. a popular way now is spiral ct for early diagnosis of the lung here is a chest x-ray, you see a mass here, and this is a cancer then in the lung, you see the ribs. and using a ct scan, you see abnormalities of growth.

these are tumors. using a bronchoscopy you can see a protuberance in the throat. so in terms of staging you can use various techniques such as the ct scan, mri, pet scan, radionucleoide, bone scan. if the cancer is detected early

they try to do surgery but the survival time is six months with small cell lung cancer, using radiotherapy the survival time slightly increases to ten months, but it all goes fast, death within a year. so small cell lung cancer is very responsive to

chemotherapeutics, we see wide use of platins, and various entities such as irinotecan. you can use combination chemotherapy. there's other combinations as well. so the best thing though is combine radiotherapy with

chemotherapy, and the survival then increases from 10 up to 34 months. but then there's often relapse, as the second clone grows out, and after relapse the chemotherapy is often ineffective. and the small cell lung cancer

undergoes metastasis to various organs such as liver, bone, lymph nodes, and when the metastasis goes to the brain the patient has a few weeks to live. so we mentioned before that most lung cancer is associated with tobacco smoke, and you inhale various carcinogens, they get

activated and mutate the dna. really very little is known about the origin of small cell lung cancer, is it derived from initially endocrine kulcitsky cells or possibly stem cells? we don't know. one thing in tobacco smoke is nicotine.

nicotine and carcinogens such as nnk can activate akt leading to survival of the cancer cells. so the nicotine can bind to choline receptors. nnk forms dna aducts, this can lead to mutations in the lung cells. nnk also causes akt

phosphorylation. nnal can be measured in the urine of patients by gas chromatography. the only way to get nnll in your body is if the cigarette smoke is then breathed in and then this results in an indication that you've been exposed to

cigarette smoke. so nnal is increased in nonsmokers who breathe in cigarette smoke so secondhand smoke can increase nnal in your body. the unique thing of lung cancer, in the 198 0s, nci made cell lines out of biopsy specimens,

so with lung cancer we have hundreds of cell lines to be studied, in contrast other tumors you're lucky to have a dozen cell lines that you can work with. we have hundreds of cell lines then for lung cancer study, and basically the bone marrow

aspirates were collected from patients, mono nuclear cells were isolated, the lymph node aspirates were disassociate and self suspension obtained, and the cells were ten put in a serum-free medium and all the normal cells died, but the cancer cells can make their own

growth factors and hence they grew. for small cell lung cancers surprisingly they grew as floating cells. they do not adhere to traditional petri dishes or flasks. and so for the small cell lung

cancer then we have neuroendocrine tumors, and these vesicles in the tumors secrete growth factors, that can then bind to receptors on the cell surface, stimulating growth. and we'll be looking at igf 1, and the bombbasin, both important for the growth of

small cancer cells. so between 1982 and 1984, 31 lines were established, and these cell lines when you inject them into nude mice, tumors then rapidly formed. and these small cells, they have high levels of bombesin pep sides and an enzyme that's

traditional found in neurons, and they have high levels of neuron specific enolasi, so hence these were then classified as neuroendocrine tumors, and over a 20-year period, over 100 small cell lung cancer cell lines were developed as well as 100 nonsmall cell lung cancer

cell lines. so looking at animal models, we talked about the a/j mouse where you inject it with carcinogens such as urethane and adenomas form on the surface of the lung and you count the number of adenomas on the lung, as an index of how many tumors you

got. so for the various lipids in the cell lines, one thing you can study is production of prostaglandins and leukotrienes. so the nonsteroid all antiinflammatory drugs, such as aspirin inhibited growth. you add prostaglandin e2, it was

reversed, suggesting it is a mediator of the lung cancer growth. in this case animal studies were done, and in the presence of endomethcy, in the number was reduced relative to animals not given any treatment. and if you looked at specimens

then of the lung in the mice, and you looked for cox-2, you found large amount of enzyme in the bronchus, in this case the color of the immunoreactivity is brown, purple is just a counterstain of the tissue. and you see lots of immunoactivity in the broncus,

the bronco broncheoli and alveoli. we see intense staining in the epithelial, moderate in muscle but not cartilage. in the adenoma, there's also scattered cellular staining. so there's two types of enzymes, and here in this experiment we

added epidermal. it's thought in lung cancer, the 2 enzyme is important for proliferation, and if you have cell lines and add prostaglandin you get phosphorylation or activation of egf receptor, and the antagonist blocks the type ii receptor on the lung cancer

cells. it phosphorylates egf receptor and caused erk phosphorylation in a dose dependent manner and the erk can go in the nucleus and turn on various oncogenes. when various genes are activated at f sites, we get a doubling of vegf, and increase the vegf

mrna. and here is a little cartoon summarizing what we think is going on. that being that the egf receptor will activate cox2, increase expression, leading to increased pge2 production, which binds, the ep 2 receptor can cause

transactivation after egf receptor, phosphorylation, so this is basically an autocrine growth cycle, ultimately transforming growth factor alpha gets released, which further activates the egf receptor. and once the ep 2 receptor is activated it can cause an

increase in vegf expression. so it's not only a growth factor, stimulating the egf receptor, it's also an angiogenic factor increasing vegf expression. so in terms of cox inhibitors, the nonsteroid all antiinflammatory drugs can

inhibit cox1 and cox2. when you take aspirin side effects can occur such as stomach ulcers. a great effort was made into making selective cox2 inhibitors and the one that's traditionally used is celecoxib, and the goal then is to block have minimal

side effects in patients. so there's lots of clinical trials going on, using celecoxib, and there's phase 2 studies, studying effects in heavy smokers, there's also phase 2 studying in the face of paclitaxel and carboplatin.

we have in small cancer lung cell several things going on such as rb inactivation in 90% of patients. we mentioned p53, it's a tumor suppressor gene, it gets inactivated in 90% of small cell lung cancer patients. another thing that's inactivated

is fhit, in 3/4 of the patients, and then bcl2 is overexpressed in 85% of the small cell lung cancer patients. so again in lung cancer we mentioned there's no one thing that causes it. but since it takes decades for lung cancer to form, several

things lead to the lung cancer proliferation. so p53, we mentioned previously it causes g 1 to s-phase check point inhibition in most cells driving program cell death or apoptosis after dna gets damaged. if the p53 gets mutated, then

the cancer cells will readily proliferate. for the retinoblastoma genes there are mutations, wild-type is frequently lost and subsequently the rb protein is pretty much inactivated, in 90% of small cell lung cancer patients.

the fhit gene present on chromosome 3p14, loss of fhit protein is associated with smoking, and there's a lot of investigation going on to see if it is a tumor suppressor gene associated with apoptosis. so for the bcl2s overexpressed in about 85% of the small cell

lung cancer patients, the bcl2 suppresses apoptosis, and responses to chemotherapy, and radiation therapy. trials being done basically trying to stop the bcl2. so in terms of molecular abnormalities, there's loss of chromosomes in 3p, 4p, 4q, 5q,

8q, 9p, 10q, 13q, 17p and 22q. we'll look at myc, an oncogene. tyrosine, and we'll close looking at bombasin receptor. so 3p deletion is dramatic in the chromosome of small cell lung cancer patients, and 3p is a very early event, and subsequently 5q, 13q and 17p

deletions will occur, p53 is on 17p. microsatellite alterations in lung cancer there's a laddering of short-term dna at multiple loci, it may be useful for early diagnosis of lung cancer using sputum, bronchial washing or blood.

myc can heterodimerize and facilitate cell cycle progression. another thing that happens is lkb 1 is inactivated in half of the small cell lung cancer lkb 1 causes phosphorylation of amp activated protein kinase, resulting in tumor growth.

and tgf-1 binds with high affinity. and it's actually am dimer where the igf 1 binds to the alpha subunit, tyrosine kinase activity is on the beta subunit, so when the alpha subunit finds igf 1, it causes a confirmation change to the beta subunit

causing protein phosphorylation. and downstream events include activation of map kinase, growth of c-phos and growth of lung cancer cells. so here is the igf1 receptor, we got a monoclonal antibody, which binds to the igf receptor and recognizes 90 kdal subunit.

you see tumors grow in nude mice, in the presence of alpha ir 3 tumor growth was greatly slowed. so the igf 1 enhances survival of the small cell lung cancer and it phosphorylates, leading to increased survival. pi 3 kinase inhibitor inhibits

phosphorylation of akt causedded by igf-1. here we see phosphorylation, causing increased expression of bcl2 inhibiting apoptosis of the lung cancer cells. so another tyrosine kinase receptor that's present on the small cell lung cancer cells is

c-kit, and scatter factor binds to c-kit, and the c-kit is often overexpressed in the igf receptor in the small cell lung cancer cells and c-kit can be inhibited by gleevec. the c-kit is a 976 amino acid membrane, 520 amino acid

extracellular domain. atp binds to kinase domain, and various proteins then are phosphorylated on tyrosine amino acid residues. so finally we get to the grp receptor, and i published in 1980 that the small cell lung cancer cells had very high

levels of bombesin-like tides. p e t ides. we elicited the an antibody, but the tumor size was greatly reduced given the antibody. so subsequently, clinical trials

were tried with this antibody, but it only worked in one out of 13 patients, so subsequent efforts were discontinued, but then the field sort of switched and antagonists were developed against the grp receptor, so you see the grp receptor has 384 amino acid residues, crosses the

plasma membrane seven times, the grp binds on the extracellular side, and what happens is this third loop will then cause turnover, so when it binds to the receptor it gets metabolized. a curious thing is with men now, the cancer incidence in the lung

is rapidly decreasing, with women it's sort of leveling off, and one interesting thing about the grp receptor, it's on the x chromosome, so subsequently the females have a double dose of grp receptor relative to males and it remains to be determined if this is why women are more

susceptible to getting lung cancer than men. so in this case, we're adding grp to small cells loaded with dye and we're watching the calcium go up as a function of time and when it gets metabolized ip 3 is one of the resulting products and then this

will cause release of calcium from intracellular organelles, it peaks and declines with time. the increase in cytosolic calcium is transient. this is a different way of looking at it graphically. add grp and calcium increases, it's a transient increase, and

then we identify an antagonist that blocks the receptor and they block the increase in calcium. so these antagonists are small molecules. and the one that blocks the grp receptors is pd 176252. and another thing that the grp

receptor can do is cause transactivation of the egf receptor. we saw prostaglandin e 2 will i do this, the increase in tyrosine phosphorylation is quick, it occurs within a minute, and then the antagonist, the pd 176252, will block the

increase. and when the egf receptor gets tyrosine phosphorylated, events downstream include erk activation, we see the bombbasin causes erk activation, antagonist will block that as well. and in terms of growth, one of

the things that we mentioned previously is the gefitinib used in patients with mutation the egf receptor, one goal is then to increase the potency in patients with wild-type receptor and when we add this antagonist the pd 176252 the gefitinib response curves shift to the

left and pd 17 6252 increases the magnitude, it's possible the antagonist in combination with gefitinib may be used to inhibit the lung cancer growth. so in summary it's a neuroendocrine tumor which initially responds to chemotherapy but subsequently

relapse occurs. there's multiple clinical trials in progress to improve treatment. we want to close by focusing on smoking. so 85% of the lung cancer cases, the patients are smokers. and so nicotine is what comes

out of the cigarette smoke that makes it very addictive to various people. so in terms of getting people to stop smoking, one of the things that they do is there's lots of nicotine replacement therapy now. so this includes the gum such as

nicorette. you can use a patch which is the nicoderm, there's a nasal spray, nicotrol, and now there's even a nicotine lozenge, and each of these delivers on the order of 10 mgs of nicotine per day to and there's also some pills that are used such as bupropion, an

antidepressant, lately in vogue is chantix. there's lots of things to take to get over the need to have nicotine, and then the question is will that help you stop smoking? and in many cases it does, but this is a very difficult thing

to get people to stop smoking, basically if you take -- start smoking when you're a teenager it's hard to give up. and people will often smoke for 10, 20, even 30 years. but if adults start smoking later, such as when you go to college, it's fairly easy to

give up. and these people, they tend to smoke for just a few years, and then they stop. so what the u.s. has done is they tax the heck out of tobacco, and as a result teenagers can't afford it, and the number of teenagers smoking

has gone down dramatically. but for people who are still smoking, cessation can be achieved with medication and health care professionals, and early failure is a normal part of trying to stop smoking. so people when they try to stop smoking oftentimes it will work

for a short while, and then they will go back. but then a year later they may try again to stop smoking, and again it doesn't work. but eventually, it does work, and people will stop smoking. and smoking cigarettes certainly leads to nicotine addiction, but

ultimately the way most smokers quit is they just quit cold turkey, after a gradual reduction in the number of cigarettes that they utilize each day. so the health benefits of stopping to smoke within 20 minutes blood pressure starts to

decrease, heart rate decreases, within half a day carbon monoxide levels in the blood return to normal. within two days, the sense of smell and taste returns, within nine months there's a decrease in cough and shortness of bret, within ten years the risk of

stroke is normal and the risk of dying from lung cancer is reduced by 50%. so now in this country we still have 45 million smokers. the good news is only 1 in 6 will actually die from lung we don't know why, all the smokers won't die from lung

many die from other things such as heart disease, but now in the u.s. we have 45 million ex-smokers. and that's a very, very good thing, because after ten years, the risk of getting lung cancer is dramatically reduced. and here is some references.

and that's the end of the traco course. are there any questions? well, thank you for attending. i wish you all a happy holiday season, and we'll have the exam up soon if you want to get a certificate of completion from the course.

thank you.