our first speaker is natasha caplen, ph.d. from the university of the london, kings college. in 1996 came to the national human genome research institute at nih. and ccr thought she was really smart and hired her in 2004,she
heads the gene silencing section and she's now in the genetics branch, functional genomics by rnai. >> everyone can hear me, and the few people able to brave the storm and weather on a wet november monday, but welcome to those here, and i gather there
are lots of people online watching this in the future. my topic today isnctional genomics, concentrating on what i think of that in the concept of rna interference, i'm going to talk on how other technologies are being developed that complement rna
interference. so today's presentation, i'm going to give you a brief introduction to rnai and rnai based technology. there's not a quorum to make sure the survey is valid but i want to get a sense of the few people in the room how many of
you have done an rnai-based experiment in the lab? i've got three plus dr. moody, so 50%. were that translates into the world, i'm not sure but it gives me a sense of where i'm at. i'm going to give you an introduction and real world
cases where these are projects we've conducted in collaboration with various investigators within crr, but have been focused in my lab. i've chosen to illustrate some key features of how we apply rnai to study gene function, so i'll talk about breast cancer
cell survival, i'm going to talk about genes amplified in colorectal cancer and also breast cancer where we looked at trail-induced apoptosis. i'm going to move to just a couple brief slides talking about genome--wide rnai screening.
dr. moody, you said you're going or you've been to ncat? that will be on the work they are doing in the rnai screening area, and then as i say, briefly i'm going to talk about other functional genomic approaches. all right.
so rnai is a great fit for this set of presentations and this course because this has been a truly translational research story. something that started in plants and fungi, now in clinical trials being used for drug discovery targets with analyzing
drugs, a translational setting. so as i said, this is originally described, what we now know is the phenomena of rnai was really described in the early '90s in petunias and fungi, it was observed but when you manipulated plants and fungi you saw suppression of gene express,
but in 2006 they won the nobel prize understanding the mediator of the familiar in a was double-stranded rna. he saw it in work that i did and also tom's lab, how we could actually induce this phenomena in mammalian cells. this is really where i came into
this field, people have been taking on this in clinical trials, a very robust and well -- broadly and widely used tool for studying gene function. okay. so i'm not going to go through the mechanism of the rnai p athway in detail, just
highlights so everybody is on the same page. so rnai is not there just as a really fun tool for us. it's there as a critical pathway for regulating gene expression. the best mediators of rnai in mammalian cells are mature microrna.
so this entire pathway uses small rna molecules, of about -- end point of 20 to 27 nucleotides, some are larger but i'm going to focus on the species that are around 21, 22 nucleotides. so in microrna you have an entire pathway that is
developed, that evolved in the biogenesis of micrornas, these are expressed from genes that are within -- look like standard genes but they are noncoding, and these are involved in the post transcriptional regulation, this is a major topic, i think at
least one person that talked about some of that within this course, so i'm not going to talk about this beyond today other than saying this is the mechanism we're exploiting when we conduct rnai-based technology experiments. so over on this side we have
gene regulation. over on this side we have loss of function analysis. what is the key difference between these? in the end the key difference between the two is related to the sequences of the molecules that we're using.
so microrna traditionally has an -- interact with targets and have mismatches in the a line. of sequences of the mature microrna with the sequence it targets in the mrna. here you're seeing an effect that reduces mr rna stability, leads to degradation.
this is where you have generally a transcriptional repression as a consequence of mrna degradation and stability. we're exploiting a feature of one of the proteins and an exact match between, at a predict able fight in the sequence, degraded, you see your subsequent loss of
protein production and loss of function. i'm going to come back to this in a few moments when i discuss how we have to understand the differences between these processes if we're going to interpret data because s rnas can act as microrna, target
effects, i'll mention that briefly, you have to take that into account when designing an experiment using rnai-based technology. so why do we -- why has rnai become so important? one of the reasons is that particularly in the context of
cancer cells, our ability to profile the cancer genome on a large scale has been very much revolutionized. we know we can do large scale epidemiological studies to get gene association. genome copy number and genetic analysis on a genome-wide basis,
sequence analysis and expression analysis but systematically finding out what they mean is still a significant challenge. it's very much bottlenecking really making maximum use of all the information we're getting. so one of mine has been thinking about how we can do genome-wide,
as i'll mention later, functional analysis and genes to make use of that information. so, again, a brief couple introductory slides, when i talk about rna analysis, what is still the most widely used approach for conducting gene-specific and loss of
function studies, we're inducing a loss of function. we're comparing two populations where we have eliminated a gene, which is shown here, so what we've done is have the sirna, this is in two things, target cleavage, we get inhibit protein production, a loss of some sort
of cellular function, some sort of phenotype we can measure. we're inhibiting protein production and if a protein is stable, then you may not induce a phenotype for some time. there are different ways of inducing rnai using an sirna, a transient effect,
which means you need to see a phenotype, you have to induce something over a maximum of five to six days or use a sirna with a similar physical characteristic, a hairpin, expressed in a plasmid and be stable expressed at least in some circumstances.
what are people using rnai-basing at the nothing for? gene function, pathway and network function, molecular target identification that i'll talk about today, how a compound is working or trying to find ways of combining two drugs
together to improve the efficacy of both of them. also we've used it in the context of biomarker validation so i won't talk about that today. so in the lab, when we're developing an rnai-based experiment, there's a number of
things we need to consider. probably the most key is the model system that you're going to ask the question. as you're going to see going forward, what we've done is a number of studies where we have conducted rna experiments, but the only way we could interpret
what the function of a particular protein was, was by remembering what the context is of the question we were asking. if we're studying in breast cancer we need to be careful we're looking at a full range of breast cancer cell lines. if we're looking at colorectal,
we need to look at this in the context of a range of colorectal cancer, the only way to truly interpret the data that may be coming from tumors by definition in their genetic background. we're making sure we choose the right model system and optimize inducing rnai.
we can use sirna which is what my lab does, we can also use shrna, and we need very good assays. i've worked with a great maybe postdocs who say my sirna doesn't work. the answer is no, it's usually your antibody that doesn't work.
this step is just as important as the model system and obviously having a phenotypic readout is critical. so i'm throwing in a couple not quite old slides but to give you a sense of what a successful rnai experiment looks like in my lab.
this is looking at mrna express for a well-known gene involved in colorectal cancers and others, these have been defined that they don't have sequences matching anything in the mammalian genome for protein encoding genes.
tease are not scrambled. they are well documented controls and i can strongly urge that you look at those. we would also run in something like this an untransected control, so we're making sure the untransected and negatives will look at that.
and we do a minimum of at least two s-rnas. as we move down through a complicated study we might end up with just looking at one s-rna but the study at the moment we studied with six against multiple genes, as we go through the paper we go down and
get more complex phenotypes but we're measuring down to one or two. we always start with a number to be rebust. a paper with one rna goes across my desk it gets immediately rejected. so i would not advise me to
review under those circumstances. this is looking at a protein level. we've looked at the knockout again with two over time, and this comes back to the issue what we're looking at is the blockage of production of
protein. your phenotype will be dependent on whether the protein stability becomes degraded enough that you now no longer see that protein. so we often have to do -- i make people do complicated time courses so we know at which point we should be looking to
start to see a phenotypic effect. some proteins degrade quickly and you'll see an effect in 24 hours. some do not. some you may have to go out for several days before you see an this is very important to
consider when looking at any particular protein of interest. it astonishes me still, one of the critiques when we talked with postdocs that talk to us about it, i want to do an experiment, what's the half-life of the protein you're trying to silence?
and that's just information that they either don't know or it's not available. i say you have to figure that out because you have to know when to look. it's very important. now back to a point i made earlier, all the different
features you need to consider when you're thinking about how to design rna experiments. this comes back to this question of off-target effects. i said earlier in sirna it can act as a microrna, because the microrna is a key portion that interactings to find a
particular target within the three prime utr, it's actually just the first seven nucleotides on this end of the microrna, known as the seed sequence. if there's a match with another protein encoding gene in the three prime utr it can have an effect on those targets.
that's why we often use multiple s-rnas. if you see a consistency of phenotype you'll have more confidence you're seeing an i will mention other ways to look at this in a moment. one of the key things is also -- some of these features of how to
design experiments of sequence independent, i described here some that is sequence dependent, silencing of unintended target, you want to use to control for this your lowest possible dose, negative controls and multiple factors, the easiest way to address this.
there are some other features that have been described in the literature but you just need to be aware of it. you can saturate it and in some cell types and circumstances you can induce immune response. this is relatively well described in the context of
people trying to develop rnai therapeutics about for most people it's not going to be an issue in the lab. i'm going to briefly talk about one way a former trainee in my lab who actually runs the trans-nih screening facility is try to think about a clever way
how we can control further for these s-rna microrna-like interactions. this is a busy slide. i pulled it from a complicated paper. i wanted to have this so you have this as a handout for future reference, is what we're
looking at here is this key figure here, what they have learned is that if we mutate just three nucleotides in the center part of the s-rna, positions 9, 10 and 11, what you can do is make a sequence-specific control for each of the s-rnas you're
studying and use that to determine whether you have a false negative or false positive. a lot of people in the field are starting to adapt to using this, particularly for facilities where they want to confirm a large number of hits from a
particular screen, so i put this for you to consider, if you're wanting to have additional controls for any additional experiments. so what i'm now going to do is turn to some of the cases where we've applied rnai in the lab, using some of the -- thinking
about some of the features that i've talked about, making sure we have really good assays in place, so we're using lots of rnais, and we're using the right models. what my lab has done over the last few years, summarized in this one slide, we've developed
s-rna in a way to screen these with an individual in each well, with all the appropriate controls and do lots of different assays that are similar to what you would do in the context of drug screening. what we've done is instead of now knocking -- using drug
inhibitors, we're now just knocking out every gene up to the genome, as i'll describe at the end. assays that we've run routinely at my lab, it's very -- the cancer community is keen on killing cells. that's a key parameter for them.
we've also done studies in apoptosis. then what we've done, if we've had an individual few genes of interest, we've gone on to do complex analysis of that particular hit. in some cases we've combined rnai screening or rnai
analysis approaches with this particular therapeutic intervention. we've been studying drugs and conducting drug analysis in combination with rnai. the first case is breast cancer, the aim was the identification of protein required for cancer
cell survival, our hypothesis being we might be able to find new strategies, a broad aim, for a number of labs, a lot of screens have been done in the rnai field. so just to make sure everyone know what is we're talking about, breast cancer we know
it's a heterogeneous definition for a group of cancers in effect because no we know the molecular basis for different breast cancers is hemming on the site of origin and genetic makeup can be differential differentiated. er and/or pr positive, amplified erbb 2 and triple negative,
these are important because this defines now how breast cancer is being treated, whether you're going to attack the dependence of these tumors on estrogen, whether you can use antierbbtreatments, and if you're triple negative surgery and chemotherapeutic.
this is one of the things we've been working on with a number of collaborators to attack this problem. critical need for targeted therapies in all of these. none of these are perfect. we want to know, but there was a particular emphasis to begin
with in triple negative breast cancer. this was a study started, a collaboration with the chief of the women's malignancy branch, started by an incredibly talented person, i'm sure done well, i've lost track, and a postdoc in stan's lab.
what we're seeing here is where we have silenced in triplicate with rna screens against the human tyrosine kinome, a major focus of stan's work. we focused on that. this is done in the triple negative breast cancer mb-231. we scored the relative viability
of this breast cancer cell line, versus a known control. and this was a gene, rrm 2, depend the on the ribonuclease. this was our maximum effect. if we could find any proteins that when we silenced them gave a better effect than rrm2 we knew we had a potential winner.
we've scored everything, when the genes were silenced we see an effect on growth. these cells are inducing -- are not as much as the positive control. the gene we following up at the bottom of this list, here we're looking at more details, three
breast cancer cell lines, a nontumor genic mammary epithelial cell line. this is very key to the success of this study. it's not always feasible in the context of cancer research as i'll mention, but it was very important in this study to
compare this to a line that grows in culture, transect, but nontumor genic, a p-16 mutation, it can grow but basically nontumor genic. if we find something that has an effect on these but not these we may have a differentialal effect, a selected effect.
i hope you can see here we have silencing. we see when we silence, this is just showing data for one sirna, we see the silencing of wee 1. we know we're silencing wee 1 but there's no effect on the mcf-10a.
what is wee 1? well, it's critical in regulation of the cell cycle. entry into mitosis is regulated by the cyclin-dependent kinase. if you review wee 1, remove wee 1, you're probably altering that.
entry into mitosis is dependent. it removes this phosphorylation from cdc 2, there's a relationship when the wee 1, cdc 2 puts it on. we can look at this, at this what was key was fortunately the inhibitor of wee 1 has also been generated.
we were able to with the drugs move to even more cell lines. we're able to show actually all of these breast cancer lines is representing er positive, herr 2 amplified, basal a or basal b, based on gene expression protein, sensitive to the inhibition of wee 1.
i'm not going to go through the drug's whole name. what we see is inhibiting phosphorylation of cdc-2, reducing the viability of the breast cancer cell line only. now we've been able to take an rnai experiment and confirm that pharmacologically.
what lindsey and seretia proved is this is -- if you look at dna damage, wee 1 silence or inhibition induced dna damage only in the cancer cells. we have a basis for the mechanism of this which related to how wee 1 is required for the cell cycle.
actually another function, a similar effect in another triple cancer cell breast cancer line in the same year. we found we identified wee 1 function required for viability of multiple cell lines but only breast cancer cell lines, not nontumorigenic lines.
suggests breast cancer cell lines are sensitive to wee 1 loss of function. another observation a number of groups have made about wee 1, for this presentation what i wanted to get across to you is how we were able to take a sort of -- we focused on group of
important proteins, kinases, we're able to go down to one protein we think has a critical but selective effect but we had to take it from one cell line to a number of cell lines. and very importantly we're able to also use a drug to confirm those data.
we've used that consistently throughout, if there's a drug we can confirm we will. now this point, a different case study where we've done even more focused screen but have focused on the comprehensive micro analysis downstream of a limited number of genes to really
understand why a particular set of cells might be dependent on that protein for cancer cell survival and use that to understand potentially the normal and cancer-related functions of a protein. this has been done in the context of colorectal cancer.
this is still a major cancer in terms of morbidity and mortality so there are 150,000 colorectal cancers diagnosed in the u.s. annually, even with improved screening we need to have a better understanding of this disease to develop new therapeutic strategies.
a lot of molecular changes have been well characterized, particularly in the early stages when you're going from a pre--- from polyps to a full cancer, but in advanced cancers, colorectal cancers, there's a lot of room to understand the changes that are occurring at
this point. this is what my collaborator thomas reid, who is in this building over there, from the genetics branch, has been studying for many years. i've done a number of studies with him over the last few years.
this is from a paper published last year in cancer research led by a postdoc in thomas' lab and a fellow in my group, jason. so what they came to me, and we had to go through -- i put down the steps to get this paper to work. what they came to me with was
originally this concept that there were a lot of candidate genes in a particular region that showed copy number changes and changes in gene expression they wanted to study, and i will go into more detail in a moment. we needed to validate the model, screened these, did two screens,
then we did the molecular profiling and a hypothesis generation, a follow-up on one of these genes. so the concept they came to me with was that in advanced colorectal cancer, thomas was saying i'm not allowed to make the vague statement, you always
see amplification of chromosome 13. he thinks he's diagnosed it. but the question is we don't know why. what are the driver genes within this amplified region, other genes here that are required and i'll continue to drive the
development of colorectal they are integration focuses on 116 genes and what we needed to do, by this time very sure what we needed was a very good model to study this in. and so they had to do analysis on a very large number of colorectal lines to find the
best model, at least a significant portion of these genes showed the same type of changes at a genetic level, amplified and overexpressed in these models. importantly, we needed to see the manipulations in the cell lines, transect them and do the
sirna study. we got to two cell lines of interest that expressed high levels of 67 of these genes. we then managed to find sirnas that were effective to 44 of these, but we felt were robust, and then what we're seeing here is a primary screen
where we're looking at cell viability for one of the cell lines, and another one, dld-1. here is our positive control down here. if we put this sirna into cells this will kill everything. then we have others where we have one or two -- we did two
sirnas to start with, at least one of them induced a significant effect in one or both cell lines. from this set we got down to 17 genes, we went in with more sirna, and we got down to 8 genes of interest, which is shown here, which to be honest i
wasn't exactly wowed by in terms of using cell viability. so some clearly the cell lines required these genes for functioning, but some were very interested, we were only seeing very small changes. one of the important things at this point to bear in mind, we
were using a very broad phenotype, using cell viability, still not convinced this was the right assay and we should have used another assay but we didn't know what to use. that's the problem, intrinsic problem looking at these things. we also saw a lot of variation
between cell lines and i now realize two was not enough. we should have done, four, five, six cell lines. what was critical about this study in comparison with the study i described earlier, there was no control cell line. there was absolutely no
colorectal cell line that's not transformed so we hadn't got any way of seeing whether there was cell activity for these. and we were assuming single gene effect, i told you earlier this is a very large amplified region with lots of genes that change and we're looking at one.
we made a lot of assumption when we moved forward, but the feeling of the group was that that's what we wanted to do. so we took eight genes and decided to do expression profiling in one of the colorectal cancer cell lines to find out what was the functional
space, only one had previously been associated with colorectal cancer, in some cases there was minimal information as to the functional role of these genes. so this is what -- i'll blow one of these up, but one of the things my lab has done quite routinely and still continues to
do is when we silence an individual gene we then do gene-expression profiling to discover in an unbiased fashion what it is that protein is doing in those cells at a tran descriptional level, works well with genes as part of a transcriptional response.
we're seeing a correlation of the gene-expression profiling when we silenced a gene with one sirna, another, we see very good concordance, when we optimize, when we know which sirna we want to do, we get very good concordance between them.
what i'd like you to get from this slide is one thing, but sometimes in silencing this gene we see almost no changes significant. just a few. but this gene, silence this one, part of a proteosome we change half the genome as a consequence
of it. so this allows us to get a sense and build a hypothesis of what a protein is doing. discussion of one, lnx2, this is a blowup of a figure here where we're looking at the change for one sirna versus another. some genes don't change in the
same direction but we see critical correlation and can now use this to make a hypothesis about what this protein might be doing. so a little bit was known about lnx2's function in normal setting, it's known to be involved in the notch pathway,
and in fact if we look at our expression profiles we could see clear silencing. we saw clear downregulation of both notch and notch1 targets genes. so we could find that and confirm at a protein level. we were also -- the whole point
was discovery. we were interested to see if we could find a way to look at these profiles and make new discoveries of the function. lnx 2. what jason pitt did in my lab and i we went back to express profiles and curated known
targets of known transcription factors involved in colorectal cancer, for example n f kappab, beta-cattenin. there was huge enrichment of target genes in the signatures when we had silenced lnx2. we have a hypothesis that links it.
what is tc 7l 2? this is required for beta-catenin. if we silence lnx2, it induce as change in a phenotypic reporter assay of tcf7l2 activity. we can go back into publicly available data looking at the expression in colorectal cancer
and show the gene is regulating downstream of tcf7l 2, differential expression between tumor and mucosa, linking a protein that's amplified and overexpressed with two pathways we know with also in colorectal cancer. the last case study, how we
combine rnai with looking at a -- some other intervention. so over the years we've looked at chemotherapeutic agents, radiation, we've looked at other cancer agents that act through different mechanisms, and what i'm going to talk about today is the trail-induced apoptotic
pathway. the reason was a number of reasons, rna can give you a way of looking at synergistic targets, a huge amount of cost goes into looking at combination trials for drugs. particularly now, you usually have to use multiple drugs and
those are very expensive trials. if we can get some sense of a good combination this may accelerate that process. you can use lower concentrations if there's some sort of additive or synergistic effect. we've done a number of studies looking at these types of
studies overcoming drug resistance. also you have the potential of taking a drug that you know works in one context and expanding it into another cancer type. so again this is a collaboration with stan, a postdoc in his lab,
seretia, continuesed by a graduate student in stan's lab. tumor necrosis factor-relate the apoptosis inducing ligand, trail, has been known because of its ability to kill selectively cancer cells over normal cells. what we're seeing here is this is part of the apoptotic
extrinsic pathway. well, if trail will trigger this pathway through pro caspase 8 and caspase 3-7 and trigger many triple negative breast cancers are more sensitive to trail than other breast cancer cell lines. the determinants of this
relative sensitivity and resistance to trail are not understood. so there have been phase 1 studies that have demonstrated trail 1 is safe. but there's not been much clinical efficacy. so we need to identify
predictive biomarkers, identify which patients will be sensitive and which will be resistant to trail. and if possible, identify additional genes or pathways so you can target in combination with trail to impart efficacy. these were the goals of the
study. we did parallel rnai screens of the human kinome and three screens where we measured activated caspase 8, activated caspase 3-7. i'm coming back to controls. we use caspase 8 and flip. let's go back for a second.
what we can do is this, if we inhibit this activity, caspase -- trail caspase activation will go up. if we take out caspase 8, we block this pathway, and so it will go down. but that is shown here. perhaps best here
with this caspase 3-7 screen. when we add flip, if we inhibit flip, we can increase the activation, and this is showing our real positive control, that gives huge amounts of caspase activation. we see the inverse effect in
cell viability. these are rather complex slides and we're going to pick out one to walk you through, the paper was published earlier this year. what we were looking for here was to show that in the presence of trail, the first thing to take is that when we have no
trail we only see a few genes inducing apoptosis alone. so genes you anticipate will trigger apoptosis, when we add trail we see significant activation of caspase 3-7 or 8. there is a correlation across the different combinations of the different screens.
we were just confirming internally that on the whole, the screens were acting as predicted. we found much to our surprise. huge numbers of putative regulators of trail. we found 83 kinases, 4 phosphatases, and 63 others.
circles in black show no trail, red shows trail, and we're seeing here a known inhibitor of trail that when we inhibit bcl-xl we see activation of these are known inhibitors of trail, and again we see pretty robust activation when the genes are silenced.
this proved to be a very robust screen but we got way more hits than we anticipated. we worked through some of them for the publication published a few months ago. this is showing some of the more detailed data for bcl-xl, and we see these effects consistently
with multiple sirnas, we were able to do is put these within a pathway and now we've confirmed in multiple cell lines many of these genes as being significant, including cell lines resistant to trail. importantly, we could also copy this again with a drug, s so
where we used these and in all cases when we combine this with trail we were now able to sensitize multiple breast cancer cell lines including trail resistant cell lines to trail. this is now giving people a rational route to try to exploit trail mediated apoptosis.
but as i said earlier, we ended up with way too many hits, and we make prioritization difficult. the decision was made, to prioritize this we needed more data because this would hopefully give us a more essential of functional networks
involved in this. this is an expanded project, this is recently accepted into the pipeline at the trans-nih facility, allowing me to segue into very large scale screenings. i've thought about screens relatively small where we're
looking at maybe a thousand or so genes at a time. false screenings are genome-wide. this is ncat. you have to get into big robotics and get crazy about how you're going to do this and analyze this.
so a few years ago a core facility was said up by my former trainee, scott martin, and i've stolen slides from him. they are trying to make as much of this available, the information that comes from these screens, are all
collaborations with investigators within the intramural program but we're trying to move a lot of this information more broadly and methods involved more broadly. so you can do a range of assays there, these are just snap shots of the projects that are
currently being published, or currently ongoing in the facility and as you can see there's quite a number in the context of cancer. i'm deeply involved in ewing sarcoma, there are a number of others involved in - i'm told i have to point out the ebola
virus at the moment. these are snap shots from four studies published by the group. and here are included for you to look at more detail, i've talked about this one, the technology development, but then these are three recent papers from last year, large genome genes to look
at proteins involved in ganglion cell death, proteins required for morphogenesis and parken, involved in parkinson's disease. we've been pushing as the data is publishes in publicchem as well as the sequences, the data is useless without that
information. just in the last two minutes, i want to just give you some context. i talk about rnai what my lab does, but alternative ways of manipulating gene expression, to ask about functional -- to do functional genomic studies, gain
of function is an important tool in our arsenal as is other ways of inducing loss of function such as dominant negative. what i want to mention in the last couple slides is the new things coming into the forefront, gene editing tools, that i've listed here, zinc
finger nuke reaces, talens, and crispr and cas9 systems. so i've got a couple slides here that i stole from scientists in my lab, that stole them from somebody at the university of pennsylvania, which is just to try and highlight some of the key features of these tools that
are being developed. so what you're seeing here at the top one is a representation of how zinc fingers are supposed to work. these facilitate genome editing by creating a double-stranded break and then you can get homologous recombination or
nonhomologous to alter the dna sequence. each finger is a change of two-finger -- i've got to make sure -- the nuclease domain here is where your -- if you define this sequence you should be able to edit this gene specifically. talens, you have a different set
of domains used here based on the transcript activator sequences from a different host, i have it there because i can never say it properly. and then the new kid on the block that most of you have heard of is the crispr. crisprs are based, again, an
rna-guided editing platform that uses, again, a bacterially derived protein, cas9 shown here, and rna will define the sequence where this will cut. these are pro dominantly being used to do manipulations that you can't use rnais for, particularly for inducing single
nucleotide changes, if you want to mimic snps that you see in gwa studies, targeting long noncoding rnas which we cannot target by rnai or to complement rnai studies. crisprs, one is being published, but there are some issues with one of the issues with crisprs
is they may be subject to just as many if not more off-target effects as rnai. i've used this slide here to summarize some of the features, compare and contrast these, added rnai on this. the key thing about these, these are permanent gene editing
change, rnai is transient. there's a case of people moving over to some technologies to compare and contrast with the key one at the moment is still this off-target cutting, not well defined particularly for crisprs, so if you're thinking of doing this i would
urge you to look at different options, and you'll see in the last slide, yeah, i've included in today's presentation reviews to go back and look at these in more detail, some of the best that are out there at the moment, looking at the crispr/cas 9 system, and it
may be the talk will shift to other technologyings in the coming years but i didn't think i would make that swap today. so i've also highlighted a couple other studies, papers, all reviews, but if you're interested in the other features of how you can use rnai to
look at in particular this work on rnai therapeutics, which i have included in this talk before but it means they go too long so he cut it today. an enormous people have been involved. i want to highlight a lot of the work i described today involved
jason pitt in the collaboration on colorectal cancer with thomas reid, breast cancer all done with stan lipkowitz and people in his las, and my lab, probably other people, and scott martin who was in my lab now runs the facility at ncats. thank you.
[applause] [low audio] i can try. rnai therapeutics at the moment, there's something for ebola in development. i think sirnas in the context of viral inhibiting viruses is coming, a potentially
dilateds approach. one study came out of cal tech but i have not seen a lot since. it did look promising, that's a struggle. i think where we're going to see the greatest effect moving forward, rnai looking at sirna and microrna in viral
to start with. we'll learn a lot from those and then we may go back to cancer and other fields. [off mic] it is, yeah, a paper in cancer discovery -- i can't remember the group that described their application of wee 1.
thank you. >> okay. so in traco some of you visited the tumor boards where you saw case reports, and today we're bringing the case reports to you. we have dr. olaku from the division of cancer treatment and
diagnosis, and he's going to be doing several case report studies. we go from the basic research back to the clinic. >> hello. we're going to talk about case reports, much easier than what you just had.
and what i hope to achieve at the end of this short talk is to describe the history of case reports to recognize potential rules of case reports, describe case reports, outline pertinent information for a good case report. this is one definition of case
case report is the formal summary of the unique patient and his or her illness, including the presenting signs and symptoms, diagnostic studies, treatment course and outcome. we're going to give you a bit of background, how case reports
started. probably one of the oldest examples of preserved medical literature contained in the text from ancient egyptian papyrus at 600 b.c., probably written from centuries before. among them were 48 cases discussing injuries or disorders
of the head and upper torso. there's a belief that some hippocratic physicians wrote case history that were retrospective accountings emphasizing action sit descriptions of only clinically relevant findings. one of the hallmarks are focus
on objective description of findings and observation of the course of the disease. they include the both mental and physical findings although they focused more on physical than mental. and the patient's version of his or her complaints was for the
most part absent. and then we come to the gallennic case reports. he placed himself in the context of the first person and was an active agent in the case description. he included working day, doubts, tentative diagnosis and
interactions with other physicians as the disease unfolds. we come to the middle ages. it seemed like at the time that medicine in the western part of the world remained dormant and there was a lot of literature from the islamic medicine.
those case reports were similar to hip to hippocratic and galenic. in the 17th centuries case records adheres to the conversational tone of the galenic case reports but put more emphasis on patient's subjective experiences.
at this time the authors employed dramatic devices to delay the moment of diagnosis or outcome of the story. what they wanted to achieve was heighten the tension and degrees of physician involvement with the suffering patients. this one says a girl 3 years old
remained a quarter of an hour under water without drowning, appeared in a case report in philosophical transactions in 1739. 19th cently, there was less emphasis on the patient accounts, the focus was on clinical findings, with
demographic details and outline of clinical course of events. and they introduced more medical terminology, it became more prominent. now, we look at how this was reflected in cancer case reports. milos jenicik, some were traced
back to the ancient egyptian records where the first cases of incurable tumors of the breast were described. what are the potential roles of case reports? one of them can be recognition and description of immune disease.
for example in 1999, in new york, you have the case of west nile encephalitis, and the case reports, the few cases they saw led to them being able to identify which groups of viruses were responsible for this disease and how the disease was transmitted.
detection of drug side effects, drug retractions, case reports led to investigation. one of the success stories from the industry was the result of an unanticipated side effect of an antihypertensive drug they were developing. also for people who had nicotine
withdrawal symptoms, led to antidepressants being used to treat for nicotine withdrawal. now, also case studies can be used to study mechanisms of disease, for example in maternally inherited diabetes associated with deafness. this report could be useful in
medical school, they use that a lot. it can be used as the recognition of a real manifestation of a disease. recently a colleague of mine in minnesota published a case report of a lady that had recurrent acute pancreatitis,
and one of the things they found was it was due to increased level of deoxycolic acid. it can be important, exemplified by the recent ebola virus infection, we see how the cdc made changes to their policy as a result of seeing what happened in
cases that came to the u.s. case reports describe new disease or diagnosis. case reports of medical known a were described by hippocrates, and in 1832 what is now described as hodgkins disease. and a tumor in the jaw of the african child now known as
burkitt lymphoma, and in 1990 hepatosplenic t-cell lymphoma. good examples of case reports. this is a case of a 60-year-old man who did not smoke and was diagnosed with stage iv lung adenocarcinoma with unknown egfr status, treated with erlotinib after failing to respond to
first line chemotherapy. patient had partial remission in october, the disease progressed in 2010. however, his performance score remained at 1. sorafenib was added with aelotinib added as salvage therapy, patient developed
cough, dyspnea, fever and fatigue. a chest ct in march 2010 showed a mass in the lower lobe of the right lung with obstructive pneumonia, a diagnosis of interstitial lung diagnosis was made, combination treatment was
discontinued, patient passed away march 2010. now, this was the ct scan pre erlotanib. you can see when he had the recurrence, and this was following when they added the other. this is a 49-year-old lady with
stage iii nasro pharyngeal carcinoma, patient was treat the with radiation they were of cisplatin, followed by fleurocil. she subsequently developed low back pain and radiologic assessment identified diffuse blastic bone lesions and
metastatic disease. she had palliative radiation, extended systemic therapy, but after that she progressed again six months after. the second line of chemotherapy was added, gemcitabine, days 1, 8 and 15 for four yokes, weeks,disease maintained for eight months.
subsequently changed to denosumab. she developed axillary and iliac adenopathy. she was put on a trial, disease controlled for four months, again she developed a new palpable painless mast in the left breast, irregular mass at
10:00 o'clock position of the left breast, bi-ads category 5. r a d s c ategory 5. that's the lesion there.
the pet scan identified a new left breast mast, thought a primary breast cancer, they thought she had two types of biopsy revealed malignancy cells consistent with primary breast cancer, no amplyication of her2. they looked at all the biopsy
and found the pathways were similar. primary tumor were epstein-barr, changed to progressive mat static to the breast. at the time of the case report she was doing well and on a fifth line of chemotherapy. that's showing the similarity.
this is a 16-year-old lady with a three-month history of mass, acne, hirsutism, menstrual irregularities. diffuse tender and palpable mass. testosterone levels were normal, every other test was within normal limits.
ultrasound scan reveal the ascites with well vascularized mass with cystic and solid components. and a mass, multi-ocular mass, no significant lymphadenopathies. she had a laparotomy, the ovarian mass weighed 1945 grams
and measured 24 by 21 by 7 centimeters. cut surface showed multiple nodules. it was a spinel shaped sertoli and ill defined tubules. neoplastic cells exhibited nuclear atypia index. this is the specimen from
surgery. this is the histology showing the spindle cells. this one shows the mitotic figures. so the adjuvant chemotherapy regimen, with cisplatin, 3-month follow-up, no evidence of recurrence.
this 31-year-old lady had pain in the lower abdomen and back, ultrasound scan revealed cauliflower-like mass. ct scan revealed a mass with extensive calcification in the pelvic cavity. four years prior she was admitted
for fertility and amenorrhea. diagnostic laparoscopy revealed absent upper have a vigia, two irleg shapes near the pelvic ligaments. the left kidney was small, hypoplastic, scoliosis change in
the spine. at surgery they removed the pelvic mass, the left side of the pelvic cavity with multiple peritoneal seeding, omental invasion and rectal wall adhesions, ovaries slightly enlarged by normal. they removed everything, and did
some node sampling and performed peritoneal washing cytology, normal histologic features, numerous carcinoma cell implants on their surface. immunochemistry results revealed the serous tumor had developed in the third ovary. she received postoperative
chemotherapy, 16 months after surgery was stable with no evidence of recurrence. pathological findings again evidence of duplicate uterine bodies, the largest tumor was 1.3 centimeters in diameter. both uteri had separate uterine cavities and blind ends.
this shows the duplicate uterus, this is one uterus, this is another one. this is the ovary over here and this is part of a tumor, some of the things they removed. this is the normal ovary, and all this is deposit of tumor on the normal ovary.
two normal ovaries, tumor developed in the third ovary she had. it was a serous papillary carcinoma with multiple deficits of serous papillary carcinoma. now we go to rare cases. in rare cases, it's difficult to a study.
we don't have too many. and case reports provide the only means for the physician to come in contact with the patient. all you can go by is what somebody else has reported. this is the case of a 41-year-old man who presented at
the department with a history of two-month neck pain, healthy, unremarkable medical history apart from smoking 20 pack per year smoking history. ct scan showed osteolytic changes with bony fragmentations, transpercent processes and vertebral bodies
suggestive of pathological fractures by tumors. a scan confirmed osteolytic bone lesions and fractures. electrophoresis was negative. glandular pattern of neoplastic cells, almost completely replacing hematopoietic cells in the bone marrow.
cells are positive for aeis and ae3. pet/ct showed disseminated hyper metabolic lesion in the axial skeleton and lesions in the left axillary lymph nodes. he had ultrasound guided biopsy, adenoid cystic carcinoma. head and neck screening did not
find any area to look at the primary tumor. specifically these type arise from the salivary gland. physical examination revealed a small pallable and mobile nodular lesion on the left breast initially missed by the pet/ct scan.
it measured 1.7 centimeters, adenoid cystic carcinoma, tumor was p53 positive. that's c3, 5 and 6, osteolytic lesions, chipping of a bone there. from the scan there was evidence of fracture. some of the lymph nodes there,
that's the breast mass there. this is the histology. this is from biopsy. sorry, c5 is the bone marrow biopsy, evidence of tumor inthe lymph node, the breast lesion. this is a 23-year-old male, osteosarcoma of the heart. x-rays revealed a level pleural
effusion. cytology from the fluid did not real evidence of malignant cells. however, he had an echo cardio graphy that revealed a left atrial mass, measuring 3.8 by 3.8 centimeters in diameter filling the left atrium and
extending into the mitral valve. that's the mass in the left atriole. the presumptive diagnosis was cardio myxoma. the tumor was excised. pathologic examination revealed a high grade conventional osteosarcoma, mostly
osteoblastic in nature. bone scan and ct scan were normal. he had adjuvant chemotherapy with cisplatin and ifosamide. after the chemo, ct and echo were normal. nine months after the last cycle he presented with left
hemiparesis and abnormal speech, a central pontine mass 3.3 by 3 by 2.5. that's the pontine mass, with calcification there. a metastatic lesion diagnosed by radiologist. it was close to vital nervous structures, surgery was not
performed. he was given radiotherapy and treated with radiotherapy, and during the course of his hospitalization he lost consciousness and died because the tumor had compressed vital structures on the brain stem. and that is what was removed
from the left atril, irregular mass with discrete margins. this is from the osteoblast. this is 64-year-old male patient admitted with a history of progressive dyspnea and cough. undergone a right loboctomy in 2006. 14 months after, ct scan
revealed multiple metastasis and left adrenal gland metastasis. he was given palliative chemotherapy with carboplatin, progressed following two cycles of chemotherapy, received more, two more with your 5-fu, etoposide and carboplatin, and three cycle also of third line
chemotherapy with oral capecitabine. there was no objective response. chemotherapy was discontinued. patient had past history of hepatitis b in the past. miliar nodules in the lungs.
ct scan of february 2009 indicated miliary metastases. you can see the ct scan. at this facility he had follow-up every three months. he continued to have cough and dypsnea, no changes on the chest radiograph. however in september of 2009
general condition improved, cough and dyspnea stopped, all metastatic nodules disappeared. this was in september. the serum afp decreased. the patient took herbal medicine for approximately one week, recommended by his family. and follow-up did not reveal
recurrent or metastatic lesions. patient was alive and well without tumor. chest x-ray, june, the nodules. september disappeared. all cleared. spontaneous regression, it's difficult to say. this is a lady admitted to outer
east coast with six months history of left plantar heel pain. she had been diagnosed with chronic plantar heel pain six months earlier, in the local hospital. this pain was not relieved by antiinflammatory or analgesic
treatment. she didn't have any fever, no radiating pain to the lower leg, no night pain or numbless to the local skin. examination revealed alteration of skin color and soft tissue swelling around the left
calcaneus. no past history of smoking. radiographs showed purse in the left heel, ct scan revealed a space occupying lesion of calcaneus with bone destruction, chest ct revealed lesion in the posterior segment of the right upper lobe.
biopsy showed adenocarcinoma with all those things positive. left calcaneus, replacement with bone cement. she received palliative care but succumbed to nonsmall cell lung cancer six months after diagnosis. you can see osteolytic bone, the
lesion, the ct scan, that is the lesion, this is an mri showing the lesion there. and this is the ct scan showing the chest -- the lung cancer. she presented with heel pain. and from the investigation, the biopsy from the lesion of the foot that led them to find she
had lung cancer even though she didn't smoke and succumbed six months after that. this is the histology, tumor cells, nucleus, all this fibrous strands. this is tumor in the blood vessel that shows metastasis, tumor markers, staining.
so the case reports important in the era of evidence based medicine in some people feel they are reported in journals because it doesn't contribute so much into evidence-based medicine. and from a single case report you can't determine safety and
efficacy of a part line of intervention. albrecht and colleagues published 100 case reports in the archives of dermatology and they had concerns. one was publication bias in support of positive results. exaggerated claims of efficacy,
inadequate informed consent reporting, underreporting of patient centered outcomes. case reports are rarely cited creating a dilemma for journaled course, because they can negativively affect the journals. when people are planning to
publish they will want to publish in a journal with a high impact factor which in turns could influence where companies determine which journal they want to advertise their drugs. evidence based medicine tries to find the best clinical evidence, whether you apply a particular
therapy or therapy to the case reports have their place. it's also important in the progress of medical science and education. nayfield and gorin looked at 21 cases of tamoxifen-related ocular toxicity in breast cancer
patients. it highlighted the difficulty trying to attribute ocular toxicity to tamoxifen or other things that can cause ocular problems like retinal, macular and corneal abnormalities. they looked at primary carcinoma of the fallopian tube and
characterized, have a more appropriate patient characteristics and also be able to hypothesize on the underdiagnoses of cases, what are the causes of treatment failure, and the absence at the time of lack of controlled trials, usefulness of the second
look laparotomy mormon we looked at toxic effects. there were clinical trials of some of these, green tea, mistletoe, and others. what we found, many of the herbs with promising case report findings have not been explored or maybe they are not written or
reported in journals that are written in english. so we know from this review that what we can get is the characteristics and case outcomes. however, in situations where it's impossible to conduct clinical trials for whatever
reason, either because the numbers are small or for ethical reasons, the best of the first line of evidence should be taken into account. what impact have case reports had a medical research? we look at this, case reports followed by published trials.
only 17%, looking at 64 case out of that when look at outcome, 5% was failure. you look at the case, reports are followed by published rounds. when you look at the reported improvement or cure, 39%, treatment failure is 10%.
so if case reports are going to be useful in clinical research how do we write a good case report? if we can write a good case report, you may provide a template to conduct clinical last year the care guidelines were published.
a group of people were assigned to find a way to bring case reports, these are some of the things we suggest in writing a case report, it should have a title, key words, abstract, introduction, patient information, clinical findings, have a time line, either have a
figure or chart that outlines what happens when the patient was seen. for example, the year or the month, so at a snapshot one can have an idea. how the diagnostic assessment, therapeutic intervention, follow-up on outcome, have a
discussion. include the patient perspective. there's some controversy about that. and informed consent. so if journals will begin to require this list of things from the publishers of case reports in the future, time will tell.
case reports should be written in an organized and structured manner, still relevant in today's medicine. in fact, over the years there's been a lot of -- there's been some journaled specific for case you have case reports in medicine, oncology, surgical
case reports, and just in the last few weeks journal of family practice decided to publish in case reports again. it's a path to recognition of case reports. but there are instances clinical trials are not possible because numbers are not there or for
ethical patients, case reports would be needed. any questions? yes, ma'am? [off mic] currently it would be the clinician. we're hoping that case reports would become a template where it
can generate hypothesis to now conduct like studies, especially in this day and age where they are more personalized and targeted treatments. that's why having a standard would help, like consults, the other areas, so you can look at 200 cases and get good
information out rather than what we have now, write the way you feel. any other questions? thank you very much.
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