How to get rid of asthma that often recur

tristan sissung:okay, well, thanks for having me today. i think i saw in the -- i was billed as an m.d.ph.d., so while i appreciate the honorary degree, i'm only a ph.d. and an m.s, and i'mbasically a bench scientist who does a lot of translational research, so that's my perspective.i'm going to talk a lot about molecular pathways and that sort of thing today. so, anyway, let me launch in. i'm going totalk about pharmacogenetics today, and i think probably one of the best compelling storiesfor pharmacogenetics is this paper right here. it's a case report about a 2-year-old boywho went -- underwent a tonsillectomy. after his surgery, everything went fine. he wasin outpatient, he went home, took codeine

to manage the pain, and died a couple of dayslater of respiratory depression. so didn't really understand why this had happened.according to the paper, because he had taken the correct amount of pills, there was nothing-- no overdose due to the pills. but somebody got smart and genotyped him, and found outthat he has, instead of two copies of a gene that activates codeine into morphine, calledcyp2d6, right here, he has three copies of this gene. so everybody inherits one chromosomefrom mom and dad. in general, you have two genes. this kid had a duplication on one ofhis chromosomes that gave him three copies, so he was considered an ultra-rapid metabolizerof codeine into morphine, and while there is probably about 5 percent of the u.s. populationhas this ultra-rapid metabolizer genotype,

this probably indicates that this kid hadsome other confounding issues. it wasn't just that. nonetheless, had he been genotyped,codeine would have been avoided, and he probably would still be alive today. on the other side of the coin, people go tothe dentist, they get dental work done, they are given codeine to manage the pain whenthey get home. when they get home, sometimes they take the codeine, and it doesn't reallyhave much of an effect. this is because there's a lot of people in the population who aredeficient in cyp2d6 and cannot turn codeine into morphine. codeine has very little analgesiceffect. it's really the morphine conversion that is needed for that. so the same genecan cause inefficacy, and it can cause severe

toxicities. so i work at the nci, so a lot of my slideshave cancer drugs on them, so this is no different. there is a lot of variation in most drug therapies,especially in cancer, but, you know, you can see 3- to 50-fold variation in certain drugtherapies, and this variability is partially, oftentimes, attributed to genetics, but notalways, which leads to the next slide. i'm sure people in this room can probably thinkof more sources of variability, but i'm just going to go through them each here. so drug specific, dose schedule, the dosageform, how the drug is formulated, et cetera, can affect the variability. body size, bodycomposition, demographic variables such as

age, race, sex, can affect drug therapies.physiologic, especially disease states, hepatic and renal function, can affect how drugs arehandled in the body. environmental interactions, like drug-drug interactions, drug-food interactions,these sorts of things, can affect. and genetics is just one sort of these many variables thatwill affect drug therapy. so we see this more as a useful tool and not the "end all, beall" of determining variability in drug therapies. now sometimes the genetics is extremely important,and sometimes it's not important, barely at all. so today i'm going to be primarily talkingabout cases where the genetics really contributes a lot to the variability, and it's actuallyuseful for making clinical decisions. there are several types of pharmacogenetic endpointsthat we use at the nci. we're -- like i said,

i'm more of a wet bench kind of guy so i'vebeen, you know, handling a lot of the mining, a lot of the samples in our clinical pharmacologyprogram, primarily from cancer patients, and we will notice from time to time that thereis an association between a gene snp and some sort of clinical outcome that we can thengo and figure out why this is happening. so here we had a group of patients with prostatecancer treated with docetaxel. we found that a polymorphism in a gene, cyp1b1, was relatedto the outcome. so men carrying the wild-type *1 snp had a double overall survival comparedto people carrying the *3 snp. this gene does not metabolize docetaxel, so we had to doa little investigative work to figure out what was going on. we found out that estradiolis actually metabolized by cyp1b1. cyp1b1

is also upregulated in almost every singleprostate tumor. those carrying the *3 allele turn estradiol into a very reactive metabolitethat binds to docetaxel and adducts it. and this form of docetaxel is not very potentat all. it also interferes with microtubule polymerization, because this reactive formof estradiol will bind to practically everything in the cell, and it really likes the sulfinylgroups on tubulin. so we would have never found this interaction without the use ofpharmacogenetics, so we use it in the discovery capacity. we're also doing a lot of clinical trialsat the nih, as i'm sure you all know, and so we're often looking at variation in phenotypes.so there's a molecular pathway that feeds

into a variation of phenotypes. so here wewere studying an investigative drug that was shown to cause qt prolongation. we knew thedrug was handled by a transporter that existed in the heart, and basically, the transporterfunctioned so that when the drug got into the heart, it was pumped back out. patientswho were not able to pump the drug out as effectively, because of a genetic polymorphism,are shown here, they had qt prolongation, whereas patients who were more effectivelyable to pump the drug out had barely any, if at all, qt prolongation. so here we'relooking at a variation of phenotype. we had the molecular pathway sort of characterized. now both of these feed into clinical trialinclusion and exclusion criteria. you can

take people who are responders, non-responders,or you're going to get significant toxicities. you can take them out of your population andtreat them with other sorts of drugs, and subset your population for people where youthink the drug is going to be more effective. and all of this, of course, leads into actualtranslation of these findings into clinical practice. so today, the objectives are: to review themolecular and physiological basis for drug-drug -- or gene-drug interactions; to appreciatethe impact on drug therapy; to discuss the future of pharmacogenetics and drug developmentand treatment. so basically, i'm going to sort of give you a bird's eye view of pharmacogenesand what they do. i'm going to talk about

how the molecular pathway will alter phenotype,which will then alter drug therapy. and then, at the very end of the talk, the nih has instituteda pharmacogenetics program where a patient comes into the hospital, they are genotyped,and that genotype follows them around the hospital in our computer system. so if they'regiven -- if the doc wants to give them a drug, they will have to put it into the system.the system will flag if there is a genetic issue with administering that particular drug.so i'm going to talk about the drugs that we've flagged as important at the nih at theend. so just launching with the types of pharmacogenes.so probably the -- when people think of pharmacogenetics, they tend to think of these sorts of interactionswhere you have phase i metabolism, which tends

to be just redox reactions that oxidize drugs.sorry, the arrow got scooted over there. so here you just have the drug that's oxygenatedand becomes more polar. now this can have two effects. one, it can activate drugs, likewith codeine, but, in general, it deactivates drugs and makes them more soluble, readilyexcretable. phase ii metabolism is also the chemical modification of a drug. here, youtake a polar r group and add it onto the drug. so you have drug, drug r; the r is polar,it's more soluble, and easier to detoxify the drug. before i go into the cyps that i'm going totalk about, it's helpful to think about what are the major cyps that metabolize most ofthe pharmaceutical armamentarium. in general,

it's cyp3a4. 3a family probably metabolizes40 to 60 percent of the drugs that are available right now. this is an old slide, but littlehas changed in the last 13, 14 years. this gene really does not have very many geneticpolymorphisms that are very predictive, so i'm not going to really talk about cyp3astoday; however, the next two most frequent metabolizers of drugs, cyp2c9 and cyp2d6,do have some very important genetic variants that will alter their activity. so i'm goingto talk about those today. phase ii metabolizing enzymes tend to be theugts. you have -- you have these ugts in the liver, the glucuronidate drugs, and make themmore readily excretable in the bile and urine. then your sulfotransferases, and then a hostof others that are more or less important

in the major metabolism of multiple drugs. i'm going to talk about tpmt today. even thoughthis is a very small sliver, this particular gene is quite important in pharmacology. soi'm going to give you the first example here, cyp2d6 and tamoxifen. it's already mentionedcodeine will activate -- or, i'm sorry, cyp2d6 will activate codeine. cyp2d6 actually alsoactivates tamoxifen. when tamoxifen was developed, people were thinking, "i believe that thendm, or the 4-hydroxy, were the major metabolites that were actually active." relatively recently,some studies at georgetown proved that it was endoxifen that's really the active compoundof tamoxifen. endoxifen is formed through n-desmethyl tamoxifen, which i'm going tocall ndm, and it forms this compound which

is 3- to 100-fold more active than tamoxifenor ndm alone. also, when tamoxifen was being used, peoplenoticed that ssris actually inhibited the hot flashes that people would experience whenthey were, you know, undergoing tamoxifen therapy, and i don't think people really understoodwhy until recently, when they found that really, what they were doing, was inhibiting the enzymethat formed the active metabolite. so you had less active metabolite and less hot flashesdue to that. so it's kind of useful to think about, "how does the population break downin terms of cyp2d6 genetics?" we would expect, just to back up, that people who were deficientin this would have more n-desmethyl tamoxifen to endoxifen ratio. people who were very wererapid would have more endoxifen to ndm.

the poor metabolizers who do not form as muchof the active metabolize comprise probably about 10 percent of the population, roughly,and they're at the top right here. on the bottom right, you'll see about maybe another5 to 10 percent who are ultra-rapid metabolizers; they form a lot of endoxifen and the drugis actually, probably, more effective in these people, especially. when the drug was developed,though, these two extreme ends of the genetic spectrum here were not the general population.the drug was really developed for people sort of in the middle. and the people at the ends,unfortunately, don't benefit as well from the drug, or they have more hot flashes, moretoxicity to deal with. so, you know, this is how the population breaks down. go aheadand talk about the plasma concentrations of

the drugs now. so if you look at the endoxifen to ndm ratio,and you take the population, look at their plasma concentrations, you get -- they putit on a normit plot, which just is sort of a statistical method to figure out what groupsof people comprise this population. you'll find four bell-shaped curves that are verydistinct of endoxifen-to-ndm ratio. these people on the left end here have little endoxifento ndm, these have high endoxifen to ndm, so we would expect then, that if cyp2d6 wasreally an important genetic predictor of endoxifen concentration, that you would see this curveenriched for poor metabolizers and this one enriched for rapid metabolizers. and that'sexactly what you see. draw your attention

to the right-hand side of the table here.the poor metabolizers over here are the major constituents of group 1, which has low endoxifen.the ultra-rapid, or extensive metabolizers, are those that comprise group 4, which havehigh endoxifen. here's another way to look at the data, andi want to point something out here. the poor metabolizers tend to cluster low on the endoxifen-to-ndmratio, whereas the extensive metabolizers are high on it. however, you'll notice howmuch the data really spread here. there's several extensive metabolizers that look likepoor metabolizers. this is because this gene is not a perfect predictor of anything. however,it is still a very useful predictor. so if you look at patients with extensive metabolizingversus poor metabolizing, how long it takes

them to have recurrent breast cancer, you'llsee this, where patients with extensive metabolism are benefitting much more from tamoxifen thanpatients who are poor metabolizers. so, basically, we think that the poor metabolism group herereally is not benefitting as much from tamoxifen. they should probably be given another drug,such as an aromatase inhibitor or something else, whereas people who are extensive metabolizersprobably benefit more from tamoxifen than they do from other drugs. so when you think about, you know, this issuein terms of, "how does tamoxifen stack up with one of these aromatase inhibitors," forexample. tamoxifen is causing a little bit more recurrence, however, this part of thekaplan-meier analysis here is composed of

a lot of poor metabolizers who are sort ofdragging down the efficacy of tamoxifen. and right now, studies are really trying to comparethese two curves to see if taking poor metabolizers out of here and moving them to here will actuallyimprove this curve. and some early data from one of these trials is indicating that poormetabolizers that are switched to anastrozole after two years of tamoxifen experience noincrease in breast cancer recurrence. so the poor metabolizers who are switched are actuallydoing better than they would have done on tamoxifen, is really the idea. so i talked about a phase i metabolizing enzyme,cyp2d6; now i'm going to switch gears and talk about phase ii metabolizing enzymes.we'll talk about thiopurine methyltransferase

and 6-mercaptopurine and its analogs. so thethiopurine methyltransferase just simply methylates drugs and deactivates them through methylation.6-mercaptopurine and its analogs are used to treat all, inflammatory bowel disease,and autoimmune disorders. they're fairly heavily used in the transplant community as well,especially azathioprine and the transplant community, i'll mention that in a minute.these drugs basically just incorporate cytotoxic thioguanine nucleotides into the dna, whichcauses the cell to die. however, they also do a second thing. they inhibit de novo purinesynthesis, so the cell is not as able to synthesize dna and divide it as it otherwise would be.so they're very good drugs. 6-mercaptopurine was heavily used in childhood all, and someof the initial pharmacogenetics studies actually

were very concerned with this drug becausethis drug can cause severe hemotoxicity, in childhood patients can cause death, so st.jude was very interested in it, and it was heavily developed at st. jude. so the tpmt, which basically functions totake azathioprine, which is converted into 6-mp, right, and then it goes into one oftwo fates, inhibiting de novo purine synthesis or incorporating it into dna and leading tocytotoxicity. but before it can do that, it will see a lot of tpmt in the blood and othertissues, where it just gets methylated and inactivated. so when the drug was developed,the dosing was based off of people who were very able to metabolize mercaptopurine throughtpmt and inactivate it.

so the metabolism of these mercaptopurinedrugs is decreased with polymorphic tpmt variation by up to 200-fold. so 200-fold is a very largenumber in any therapy, and it has a lot of cytotoxicity in patients who are not ableto methylate it and get rid of it, and these are the kids that are really experiencingvery severe toxicity from 6-mp, so i'll talk about the snps in a second. the rapid metabolizersare resistant to the drug, the slow metabolizers are at risk. so the rapid metabolizers arethese wild-type individuals who have functional tpmt. they're about 80 to 98 percent of thepopulation, depending on which population you're looking at. the intermediate metabolizersare -- they carry one wild-type allele, and one allele that's not functional. and they'reabout 65 -- they need about 65 percent of

the dose, but they're -- they have some toxicitybut it's not nearly as severe as this group down here of slow metabolizers, who carrytwo copies of these two tpmt-deficiency alleles. and they carry about 10 to 15 percent of theoriginal dose. and if you're talking about kids, these people are also at risk for secondarymalignancy; so you give them these drugs in childhood, they can develop cancers lateron because they were just administered too much for what they needed. i'm -- just got some results back from thelargest pediatric cohort treated with azathioprine, and the results are very positive. the exactsame thing is going on with azathioprine as it is with 6-mp, and the results should bepublished within the next year. so it's not

only 6-mp that's affected, it's these otherdrugs as well, and it's not just pediatric patients, it's also adult patients. oh, by the way, i wanted to mention one otherthing: the genetic variation in tpmt explains 95 percent of these hemotoxicity issues with6-mp. so all of this information is high level ofevidence. we'll talk about high levels of evidence in a minute, but it's made it intothe package insert of 6-mp, at least, and the package insert says that "substantialdosage reductions may be required to avoid the development of life-threatening bone marrowsuppression in these patients." now i'm not a clinician, but i've heard that there isnot a lot of genotyping in these patients

going on, and this is something that probablyneeds to be translated clinically to avoid some of these severe toxicities, especiallyin children. so i'm going to switch gears again, to talkabout ugt1a1. this is also a phase ii metabolizing enzyme, very important. it is involved -- first,let me talk about the snps. so you have these ta repeats in the promoter of ugt1a1. normal,functioning ugt1a1 has six ta repeats. a gene that carries seven ta repeats is expressedmuch less effectively in the liver. and if people carry two copies of this allele, calledugt1a1*28, they have a decreased expression and function of ugt1a1. ugt1a1 is the primaryglucuronidator of bilirubin, so these patients have a slight jaundice phenotype, known asgilbert's syndrome, and this is about 10 percent

of the u.s. population has this deficiency.there are some other snps that also that are predictive, i'm not going to go through them,though. these snps explain about 40 percent of the variability in glucuronidation reactionsas a whole. glucuronidation is absolutely key in irinotecantoxicity. so irinotecan is administered iv, goes into the blood. these carboxylesterasescleave certain groups off of irinotecan that turn it into its active metabolite, calledsn38. sn38 is rapidly glucuronidated by ugt1a1, and is completely detoxified when that happens.if a patient is unable to glucuronidate their sn38, the drug becomes very toxic, and youcan see some severe adrs again. however, this is very dependent on the irinotecan dose.this is really what i wanted to bring up.

at high dose, almost 100 percent of the patientswho carry this snp get a severe hemotoxicity, whereas, you know, a moderate amount of patientswith wild-type alleles get the hemotoxicity. however, if you go down to 125 mgs/meter squared,the -- this snp no longer really matters at all. so this is a very dose-dependent situation,and so sometimes when we think of pharmacogenetics association, we have to consider other issuesother than just the gene. yeah, let me go on. so irinotecan toxicity through glucuronidationreactions has made its way to the package insert of the drug. the package insert -- thisone says that the glucuronidation of bilirubin, such as those with gilbert's syndrome, peoplewith that will be at a greater risk of myelosuppression.

i think the updated one actually does listugt1a1*28 now. switch gears from phase ii metabolizing enzymesto transporters. going to talk about one transporter in particular that's been very highly studiedin the past five years, and i think is on its way to making it into pharmacogenetics-directedtherapy. it's this oatp1b1 here. so a patient receives a statin, it goes into the gut, goesthrough the gut wall into the portal blood. it can be metabolized in the gut wall by cyp3a4,or pumped back into the gut wall by mdr1 and mrp2. once in the portal blood, it basicallyneeds to see an oatp. oatp1b1 is the primary transporter of simvastatin. there are someother oatps that are very important, but unless this statin sees an oatp, it does not veryeffectively get into the liver cell. once

in the liver cell, it's metabolized and eliminated.some of it makes it into the bloodstream, and, you know, you have varying levels ofauc exposure in these patients. what happened there? here's a slightly more complex version ofwhat's going on in the liver cell. there is a snp in this gene, a single nucleotide polymorphism,snp, in this gene that affects how much statin actually gets into the liver cell. the snpis what's called a non-synonymous transition. you have n in most people, those are the wild-typeallele, at position 130 gets changed to a d, and this actually has a great effect onauc exposure of statins. we knew this back in 2006; a very good paper was published showingthat thing is heavily linked to the auc of

statins. now, greater exposure to statinscan lead to statin-induced myopathies. so in patients carrying the snp that can't gettheir statins into the liver cell as well, you worry that they're overexposed and they'regoing to get a myopathy. another study was published more recently,looking at 500,000 alleles in the genome. i love this study. it shows that only onepolymorphism was associated with statin-induced myopathy, and not only was it associated,it was several orders of magnitude over the association threshold, which was just denotedby that brownish line there. this snp is almost in 100 percent complete linkage, meaning it'sco-inherited with that ni30d snp. so this snp is probably just a passenger that's ridingalong with the n130d snp, causing overexposure

to statins and statin-induced myopathies.this group also took these data into a validation cohort, where they had cumulative percentagesof myopathy, and they found that, again, they see the same snp is -- about 20 percent ofthe patients are getting statin-induced myopathy, and about 60 percent of statin-induced myopathycases could be attributed to this snp. so this is a very predictive allele, and thepresent snp has a 15 percent representation in the u.s. population. so this is a veryfrequent snp. there's a lot of people getting statins that are probably at risk for myopathy,just due to this issue alone. at this point, the fda has not really weighed in on whetheror not we should genotype for this one yet, but i think it's coming soon, and at the nih,we are genotyping for this.

i'm going to talk about targets today as well.so, you know, drugs are designed to bind to something in the body, and, you know, so theseare drug targets. most people, when they think of drug targets, think of, you know, yourimatinibs of the world where they -- it's targeted to a somatic mutation in somethinglike bcr-abl. i'm not really going to talk about that today because i'm really concernedmore with the germ-line variation, the dna that mom and dad gave us, not mutations intumors. there are other types of targets that are subject to germ-line variation, and i'mgoing to talk about that instead. so before i get to the targets, here are twocytochromes, p450, that take warfarin and convert it into an inactive form of warfarin.so these -- more hydroxylation through 2c9

and cyp4f2 leads to less active warfarin inthe bloodstream. but i'm not really going to focus on the cyp story, i'm going to focusover here. warfarin is designed to bind to the vitamin k oxidoreductase c1. by doingso, it reduces the amount of reduced vitamin k, which reduced vitamin k is pro-clotting-- has a pro-clotting function. so warfarin binds to this target. there is a snp in thistarget gene, vkorc1, that has the -- causes the expression of the gene to go down by many-fold.so if a patient lacks sufficient expression of vkorc1, warfarin will bind it all up andcause bleeding events. brief aside on cyp4f2, it was fairly recentlydiscovered, using a platform i'm going to talk about in a minute, called the dmet platform.here's the association, it's very strong.

the fda has, again, not weighed in on thisone, but i think it's going to be up and coming. so here is the incidence of warfarin sensitivity-- i like this paper a lot -- showing basically what causes warfarin sensitivity in the generalpopulation. and you can see this sort of red/pink piece of the pie chart and this yellow pieceof the pie chart, correspond to cyp2c9 and vkorc. so about 40 percent of warfarin sensitivityin the general population can be attributed to these polymorphisms alone. incidentally,this cyp2c9 polymorphism, which metabolizes warfarin, is about 1 to 15 percent of theu.s. population. vkorc variants are more frequent, especially in caucasians; about 40 percentof us carry these snps that lower vkorc1, and it's about 12 percent in african americans.

if you look at the package insert, you'llfind this little table which gives you a warfarin starting dose based on these two snps, or,actually, it's three snps, in vkorc1 and cyp2c9. there's even a neat little iphone app thatallows you to put this information in and get a warfarin starting dose. it's prettyneat. in this case, if the warfarin was already -- dose was already decided upon based oninrs, then, obviously, you don't need this information, but it still is useful as a startingdose -- to decide on a starting dose. okay, i'm going to switch gears again. soi've talked about targets, now i'm going to talk about genes that have effects that arenot necessarily related to the target but are sort of ancillary, you know, targets themselves.okay, so, you know, i'll show you what i'm

talking about in a second if that doesn'tmake sense. so you have a tumor lysis syndrome. you have cellular breakdown, which spillsout a lot of dna. this dna is catabolized into a lot of purines. these purines can causehypouricemia. this uric acid can precipitate in renal tubules and cause renal failure,so this is known as tumor lysis syndrome. a drug is given to avoid this -- actuallytwo drugs, allopurinol and rasburicase can be used. rasburicase, here, takes uric acidand converts it into a readily excretable form of uric acid called allantoin. here isthe actual reaction up here. when urate is converted into allantoin it produces a lotof hydrogen peroxide. this hydrogen peroxide is clear by glucose-6-phosphate dehydrogenate.there is a group of people that do not have

functional g6pd. they tend to be mediterraneanin origin, and it's the same group that cannot eat fava beans, which is why i have the broadbean up here, because the toxin in fava beans will actually cause the exact same thing tohappen. they'll get severe hemolysis due to too much hydrogen peroxide. just an interestingaside, it's thought that this population has this deficiency because they want to producea lot of peroxide in the bloodstream because they want to combat malaria. it's a kind ofinteresting idea. so, anyway, genotyping for g6pd is a very, very good predictor of g6pdfunction and so this is a genetic test as well. and the last type of gene-drug interactioni'm going to talk about are these hypersensitivity

reactions which are becoming increasinglyimportant, i think, in pharmacotherapy. so a drug like abacavir goes into an antigen-presentingcell where it sees one of these major histocompatability complexes. these image c proteins are encodedby human leukocyte antigen, which is called hla. these are the genes in the genome, soi'm going to say hla referring to these proteins here, the genes for these proteins, anyway.these proteins will bind to your drug, go out and start to amount an immune responseto the drug itself which causes hypersensitivity. and it's really -- it's a stevens-johnsonsyndrome in general. and here's a kid with stevens-johnson. this is really considered-- it's starting to be considered malpractice to not genotype for this before you give somecertain drugs, especially abacavir. there

are similar results with carbamazepine andallopurinol, still only recommended by the fda, but it's still extremely predictive ofhypersensitivity reactions. just a simple genotype test can really tell you who's goingto get it and who will not. about 5 percent of patients get abacavir hypersensitivity.if they have one of these hla loci, you can have up to 103-fold odds ratio of risk ofgetting hypersensitivity reactions. it's 100 percent positive predictive value; if thepatient has this genetic background, they are almost certain to get a hypersensitivity.it also has a 97 percent negative predictive value; if they don't have the snp, you canbe 97 percent sure that they're not going to get hypersensitivity. and here is one ofthe -- the conclusion of one of the sentinel

papers investigating this, i'm just goingto read it. "in our population" -- australians -- "withholding abacavir and those with hla-b*5701or these other hlas should reduce the prevalence of hypersensitivity from 9 to 2.5 percentwithout inappropriately denying abacavir to any patient." and i think that's really avery good summation of the power of these hla genotypes. so i have sort of given you the bird's eyeview of all of the pharmacogenes that are currently out there and are probably movingtowards the translation side. now, i'm going to just briefly mention one of the platformsthat we use to actually get the genotypes in these patients, just talk to you a littlebit about it. this chip, it's an array-based

technology called dmet, which stands for drugmetabolizing enzymes and transporters. it has 2,000 variants and 235 pk/pd genes, soyou can see all of these phase i enzymes, you'll see the ones i mentioned in there;the phase ii enzymes, you'll see the ones i mentioned in there; transporters you'llsee the ones, again, the slco1b1 is in here. and then these other genes that can have effectson pk/pd, so here's g6pd, for example, cytidine deaminase, which is important for certainother drugs, et cetera. this chip is actually -- it only costs about$500 to do the chip, and if you batch a lot of samples, as we've learned, it actuallycosts only about $50 a patient. so it's not some outrageously costly thing to do. however,it does have one major deficiency that we've

identified, and that is that it takes threedays to actually get data out of this, and that's a fast turnaround time. so for a lotof these drugs, if you need the information right away, you cannot get it, it's just notpossible. this isn't csi miami; we can't just genotype something in 15 minutes. so, basically, what we've done at the nihto combat this issue is we have made a policy where a patient gets admitted, and then theyget this genotyping test done; the information follows them around so that if a clinicaldecision has to be made rapidly, then this information is there and available, and willbe flagged to the clinician who is going to give them the drug.

we've based the -- talking about our experiencewith pg testing at nih, we've sort of used this website called pharmgkb, which is runby a lot of the pharmacogenetic experts in this country. they are all a part of a networkcalled pgrn, and they have really curated the pharmacogenetics literature very well.so if you're interested in this, pharmgkb is an excellent resource for learning more.they've published levels of evidence, so we have only selected those that have the highestlevels of evidence that are available; published control studies of good quality relating tophenotype and/or genotype patients; healthy volunteers having relevant pharmacokineticand clinical influence. pretty much everything i'm going to discuss today has that high ofa level of evidence. it also has a very high

level of clinical relevance, so even thoughmaybe you have a high level of evidence that a snp is associated with some outcome, thatoutcome may not be that clinically important. so they've also curated the clinical importanceof this, and all of these genes i'm about to talk about have a high level of clinicalimportance as well. so i'm just going to go through the list,because i think, you know, you may see some of your favorite drugs on this list, and i'mgoing to keep it short so that i don't keep you here for too long, but here we go. abacavir,i already mentioned this one, hla, b57o1; this one is recommended, so if an investigatorwill get flagged, this says you really should -- you really need to get this genotype beforeyou can administer abacavir. and even though

it says -- the test says tbd, our laboratorymedicine branch actually runs this test all of the time so we're currently processingthis snp through that branch, anybody treated with abacavir. allopurinol, another drug withhypersensitivity reactions. same story, it's recommended and can be run through the labright now. azathioprine or any of these mercaptopurine drugs, i already mentioned these so i won'tgo through the mechanism; this is also a very, very highly, strongly-recommended snp to testbefore administering any of these drugs, and we can actually use the dmet platform to doso. carbamazepine is another hla. the fda recommends testing this in asian populations. now, this is an issue here. so i have a friendin -- i'm from california -- i have a friend

who's grandfather is -- was one of the originaljapanese immigrants to the united states, and he doesn't look at all asian, but he hasa significant part of his genome that is asian. he wouldn't identify himself as asian, hewould identify himself as a caucasian. if he was treated with this drug, because hewasn't asian and we decided not to genotype him, then he could potentially experiencesome severe reaction here. so we've decided that really looking at a person's self-identifiedrace is not the way to go about this. we really need to actually genotype every patient tofind out if they have this snp or not. so this one is actually very recommended; testis, again, through the laboratory branch. clopidogrel, plavix, the poor metabolizershave non-responsiveness to clopidogrel. higher

doses may be needed in these patients, orthere's new anti-platelet agents out that can be used instead of clopidogrel. this onewe consider optional or available, but we assume that since the information's alreadyavailable to the clinician, that they will just opt for one of those other anti-plateletagents. codeine, i already mentioned it; we don't use a lot of codeine at the nih. thisone's still is optional or available; the dmet will give you the information. fluoropyrimidine'smetabolized by dpyd. patients with deficiencies of dpyd will have some potentially fatal toxicities,so this test is recommended, and it's already available to the clinician by the dmet chip.interferon alpha has an association with il28-î² snp. this is -- one snp is very predictiveto who is going to respond well to this drug,

and then another is predictive of who willnot respond well to the drug. we consider this optional or available. we have to gooutside of nih to labcorp to really do this one. irinotecan, i already mentioned it. we-- dmet chip already tests ugt1a1 so this one's already being used. isoniazid with nat2;nat2 is a phase two conjugating enzyme that acetylates isoniazid and gets rids of a veryreactive intermediate metabolite. if people are slow acetylators, they're have a threefoldincrease in drug-induced liver injuries. this one is considered optional or available; thedmet tests it. cyp2d1, similar story, go through it optional or available. phenytoin: difficultdrug to dose. there is some variance in cyp2c9, which affect the toxicity and efficacy. thisinformation will be available for dosing phenytoin.

phenytoin also causes some hypersensitivityreactions, and there's an hla that's predictive, so this one's strongly recommended, and thetest is done through the laboratory branch. rasburicase, which i already mentioned; g6pdgenotyping is already available through dmet. statins and oatp1b1, mentioned it; test isavailable through dmet. tamoxifen 2d6; test is available through dmet. warfarin: samesnps, dmet test. and then we have the molecular pathology laboratory who is already doingall of the somatic mutations for these targeted agents, so i'll just run through the targetedagents and not mention much about them. trastuzumab, lapatinib, imatinib, dasatinib, and nilotinib.and imatinib also affects kit, so we have the molecular pathologies test kit for us.gefitinib, renotalib [spelled phonetically],

and these others. braf inhibitors, egfr inhibitors,ret inhibitors; alkylating agents, and that's it. so those are all the drugs that we haveimplemented at this point at the nih in the pg testing arena. so just a couple of final thoughts. how manydrugs have pharmacogenetic markers in the label? well, at this point, there are 114of these drugs, and if you go on to this website at the fda, you can look at all of these drugs.how many drugs had fda recommendations that are actually actionable? seven have boxedwarnings that -- where the testing is very important; 29 have indications and usage information;and 24 will give you information about the dosage. so a subset of those are actionable.and the last slide here, just considering

the prevalence of use of pharmacogenetically-affecteddrugs. there's about 24 million people -- this was in 2008 -- using drugs that are -- thathave pharmacogenetic information that's available that you can just genotype them and know what-- know more information, anyway, about what to do to make clinical decisions. there'sa lot of people using these drugs. this number is just ever increasing, and eventually, theythink this stuff is really going to be important in clinical medicine. and doug figg, my boss, always ends his talkby saying, one day, he envisions a child is born, the child gets a dmet chip-like genetictest, and that test can be carried with them through life on a thumb drive, and they cango hand it to their doctor one day, doc put

it into a database, it'll tell them, "don'tgive this drug, do give this drug." so, that seems to be the way that things are going.and so that's all i have to say, and thank you very much. [applause] male speaker:comments or questions? yes. male speaker:if i want to start a patient on clopidogrel -- tristan sissung:[affirmative] male speaker:-- how do i find out if it's going to be effective,

what do i actually do? male speaker:could you -- could you paraphrase the question [inaudible]? tristan sissung:yeah. so the question was how do you find out if a patient is at risk for clopidogrelinefficacy? and you can use a few options. first option is you can send it off to haveit genotyped by a private company. there are several private companies out there rightnow doing this. the test really needs to have, i think, three different alleles, and eachone of those alleles can cost a certain amount of money. we've found that it's actually cheapestto just have the dmet chip run on people.

you can take the blood sample, you can sendit to the coriell institute, they will give you the information back. a guy named normangerry there is the guy we run through; he's doing all of the nih studies. you can getthis information back, and then make the decision based on that. male speaker:yes? male speaker:thank you for a great talk. you've raised a lot of important issues. i'm sure i seeat least one patient a week that's either slow or rapid metabolizer that's not doingwell clinically. male speaker:there was a dr. flockhart [spelled phonetically]

in prior practice that used to do consults,so how can we get consult in terms of private practice to help us, because these two issues,one is a specific drug, one is -- metabolizers slower that might affect many, many drugs,and that might be beyond the expertise of the private practice doctor. tristan sissung:that's absolutely right. i know there is some agencies that are -- that are springing upthat offer pharmacogenetic consulting to clinicians. it's a very new thing, you can google searchit, i know that doug figg was approached by one of these agencies, i forget the name ofit, but we're also at the nih, and i'm sure we can -- we can direct you in the right direction.i think my email is up here. and if we can't

help you, i'm sure we can put you in touchwith somebody who can at this point. male speaker:those of you who are entrepreneurs, it sounds like that's an opportunity. tristan sissung:it is definitely. [laughter] male speaker:i want to reiterate the excellent nature of this program, and quite timely and relevantto private practice. interestingly enough, just from an historical point of view, the6-mp discoverers won the nobel prize, you may be aware of that, [inaudible] in the 1980's,but to take that a step further, there have

been some recent guidelines that have beenpublished by a national -- our national organization suggesting that hla-b5801 profiles be obtained,and that certain groups of patients who are going to be admitted allopurinol, and happento be the kahn [spelled phonetically] chinese and certain thai subgroups. but getting back to your california story,you wonder how many of these particular groups may be here and vulnerable because this isso important for the allopurinol hypersensitivity syndrome. so from bench to bedside, this isrecommended, we're looking at the economics of this as we speak, and to the practicalityand bench to bedside we are told that this hla-b5801 is now available commercially. isthis in the area that you are -- have studied

more than your slides? tristan sissung:i'm not an expert on hlas by any stretch of the imagination, but i do know the allopurinolstory, and i agree with the sentiment that we really need to genotype everyone. so i'mnot sure exactly -- can -- is there -- did that answer your question, or? male speaker:well, a statement and a question, just to point out the relevancy of this discussionrelevant to clinical practice. tristan sissung:yeah. so, yeah, i think that this needs to be genotyped in clinical practice; it absolutelyneeds to be done because it's so predictive

of who's going to get these toxicities, it'svery important. male speaker:[inaudible] the national organizations who are suggesting it. this may entertain anotherlow culpability by not doing it. tristan sissung:that's true. i actually -- i looked up before i came here -- i always look to see if therehas been yet a lawsuit for malpractice about one of these things popping up. nobody hasyet sued anybody and won, as far as i can tell from google, for not doing one of thesehla tests. however, i have found -- you mentioned allopurinol -- a woman was misdiagnosed withgout, was given allopurinol, got stevens-johnson, sued, and won $6 million. so, clearly, itis, it is something that needs to be addressed

clinically. male speaker:well, you'll see [unintelligible] on television very quickly on this matter, i think. tristan sissung:lawyers are entrepreneurs, too. [laughs] male speaker:i'm reminded of norman shumway in response to a congressional question at a hearing madethe observation that none of us are purebreds. tristan sissung:that's definitely true, especially in america. we are very admixed. female speaker:hi. thank you --

tristan sissung:hi. female speaker:-- for a wonderful talk. tristan sissung:thank you. female speaker:i'm curious -- i remember what you mentioned about package inserts having warnings aboutgenomics, and you also talked about [unintelligible] and how that's not really helpful, how youhaven't actually genotype everyone. so i wanted to know if you have an opinion or if you'doffer your perspective, considering translation, what role or lack of role do you think thesepackage inserts are playing right now in the translation of this pharmacogenomic informationas to actual use in practice.

tristan sissung:yeah, thank you. so there was a paper published by the people at st. jude who came up withthe tpmt observation, and they talked about genetic excellence, that the genetic testsare held to a higher standard than your standard clinical assays just because they're -- peoplewant them to be so predictive of everything, although they never really will meet thatbenchmark. so i think that there is a lot of resistance out there right now to implementinga lot of this stuff because of that issue. secondarily, the cyp2d6 tamoxifen story hasbeen recently stalled by two published studies that came out at the san antonio breast cancersymposium showing no relationship between cyp2d6 and tamoxifen outcome. now, these twostudies were fundamentally flawed. there's

a editorial by mark ratain in cancer letterstalking about how these two studies both violate a fundamental law of nature: the random sortingof alleles amongst populations. and the reason for this is that these folks genotyped tumorsand did not genotype the germ-line dna. the tumors get mutated, and it's not an accuratereflection of what's going on in the liver, how much endoxifen is actually being formed.so these studies have a lot of impediments to them that are outside the control of alot of us who are doing the science, so... male speaker:thank you. male speaker:yes, again, i'd like to thank you for an outstanding talk. [inaudible] ph.d. initiative of thehospital, so when i hear something like this,

you know, my mouth waters a bit. and i wonderedis the institute interested or thinking about perhaps doing some test drives in communityhospitals in terms of typing individuals coming in and seeing its impact since you all put[spelled phonetically], certainly at nci. where are you with that? tristan sissung:i think that, you know, our group would be partially interested in -- doug price herehas come for some moral support, he's a fellow staff scientist in our lab, so, i mean, ithink we could probably talk to doug figg about that, maybe doing some of those studies.juan lertora is the guy that runs the pg program right now at nih, and i think you could definitelyapproach him and ask. he would be -- he's

always interested to talk about this sortof information. male speaker:would you comment on the role of -- the traditional role of pharmacists in protecting patientsand how you see that evolve? tristan sissung:well, i mean, for this, i think -- pharmacists are not geneticists, and i know that verywell because i am a geneticist and i have to deal with pharmacists all of the time.i think that what needs to really happen here on the pharmacy side is that we need to havesome very good curated databases where you can just put in genotype information, andthe people who are experts in genetics and all of the other fields that are needed toreally understand this information, that this

database just spits out a clinical decisionthat should be made, rather than having the pharmacist do it all. male speaker:so, in fact, at the end of the day, one could conceive of a system that doesn't lead toalarm fatigue, which happens now a lot in pharmacies, i think. get a bunch of interactionmessages, and eventually a pharmacist ignore them. it's going to take a lot of work itseems. tristan sissung:yeah. male speaker:there is a small, but significant incidence of -- sorry -- small but significant incidenceof fatal malignancies, lymphomas, i believe,

in inflammatory bowel patients, and mayberheumatoid arthritis patients [inaudible]. any data on genotyping those? tristan sissung:i don't know of any, but i'm more of a cancer researcher so i can't say that there is not.i was actually recently diagnosed with psoriatic arthritis, and my doc actually mentioned thatto me when i went to him. so -- male speaker:could you repeat the question? could you repeat tristan sissung:oh, i'm sorry, the question was basically there's secondary malignancies in certaindiseases like arthritis, inflammatory bowel disease, and the question was, do you seesecondary malignancies that are related to

those diseases, i think is basically whatyou're saying, right? male speaker:or is there a genotype that would be predisposed? tristan sissung:or a genotype that's predisposed. so that's more of a risk allele, less of a pharmacogeneticallele. i could see maybe that if you were treated with azathioprine for inflammatorybowel disease, that you might see secondary malignancies in patients with certain variants,but the disease alleles, i just don't know much about. male speaker:you raised an important issue in terms of clinical trials, and that is, you know, maybewe should lower the patient population to

people most likely to benefit. one examplethat i see every day is glucosamine chondroitin works in a subset of the population, but it'ssaid ineffective when you look at the whole population. tristan sissung:interesting. male speaker:are we any closer to using genetics in clinical trials to make drugs more effective? tristan sissung:there are several out there in the literature right now. they're finally doing this, whichis exciting. i mean we really needed the prospective side of this. now, i know that there is someresistance to drug companies to do -- from

drug companies to do these sorts of studiesbecause they want their drug to work in the whole population and in any one subset. sooftentimes you'll see these prospective studies already being done on approved drugs. i'mnot aware of any drugs that are being developed at this point with pharmacogenetics in mind,but i also don't work for drug companies, so i don't really know for sure. [laughs] male speaker:other comments or questions? yes, sir? male speaker:in the world of saving a few bucks, have you ever noticed any difference between a genericdrug and a -- from a genetic point of view -- the same drug produced generically versusthe standard drug?

tristan sissung:i don't think anybody has ever done a study like that. i think we primarily assume thata generic and an on-label, or, i'm sorry, i forget the name, you know, a drug that'sproduced by a drug company are the same compound. so i don't think we ever look at genericsversus the drug companies' drugs. male speaker:so the american college of physicians did a survey on something like 500 of their fellowsand members, and asked a bunch of questions about this sort of thing, and found that,a) each of us believe that this is a really important field for future practice of medicine;and b) felt very incompetent in being able to use it. and it seems to me that revolvesaround competency rather than knowledge. and

one of the reasons we were very interestedin having pharmacogenetics talk here is that this one is very, very close to the clinicon the bedside. and it seems like maybe we ought to do some more of this. what do youthink? i see heads nodding, maybe we should do a bit more of it. i want to thank you verymuch, dr. sissung. tristan sissung:yes, thank you very much.