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Clinicians Perspective: Impact of Biotechnology

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Welcome to the MOOC NPTEL course on bioengineering an interface with biology and medicine.In the last few lectures, we have discussed about gene cloning, polymerase chain reactionand various DNA tools.What is so interesting to learn that currently the revolutions in gene and genomic technologieshave started paying directly in the clinics, lot of patients are getting benefitted becauseof these genomic technologies.In order to illustrate some successful examples and to give you message that how biotechnologyengineering technologies have started making its impact in the clinics today we have inviteda distinguish guest Dr. Nikhil Phadke to interact with you.Dr. Nikhil Phadke obtained his PhD from the Molecular and Cellular Biology, Universityof Michigan, Ann Arbor in USA.He is currently the founder and head of a company GenePath DX in Pune.He and his team is involved in development of over 250 molecular assays in a state ofthe art lab in GenePath in Pune.He has years of experience in multidisciplinary collaborative research including clinicalmolecular genetic diagnostics, development of approved diagnostic tests and platforms.He has various collaborators and therefore his projects involved in different areas ofoncology and hemato-oncology as well as infectious diseases.I definitely felt very impressed with Dr. Nikhil Phadke’s work and realize that whateverthe latest advancement we see happening in US clinics where patients can directly seethe benefit of looking at the gene mutation, the sequencing analysis, those things Dr.Nikhil has brought very quickly back here and those have been started making revolutionin patient care.Today, it is great pleasure to have Dr. Nikhil Phadke with us who is going to talk aboutclinical and commercial applications of molecular genetic diagnostics, the HandyLab and GenePathDX story.Welcome Nikhil.Our story actually starts in a class very similar to this.Two of my colleagues who were the cofounders of the company one is from IIT Mumbai andone is from IIT Chennai which is it is really a personal story.I have been teaching classes in medical schools and international research and I am told thatthese are some of the brightest students in the country.So I am sure you are all going to be superbly successful at whatever you do.It would be nice if you could also enjoy yourself by being while being successful and make apositive impact on society while doing that, so if all these things happen then it is absolutelyfantastic.I will talk a little bit about myself.I trained at University of Michigan, Ann Arbor.I worked in the medical school there and in the science and engineering schools.And Ann Arbor always has those pretty pictures up there in the brochures but really mostof the time it is just full of snow.So we have two seasons, winter and construction anyway.So after working there for slaving day and night in the lab, I got together with a bunchof my friends, many of whom were from IIT and we set up a company known as HandyLaband we built a molecular diagnostic platform.Molecular diagnostic platform that integrated engineering principles, science principlesand medical principles all together.So really a melting of minds of different areas of specialization and after we weresuccessful with that I came back to India and started a company known as GenePath, whichis focused on doing pretty much the same thing that we did in the US but catering to theneeds of Indian patients and Indian doctors okay.I come from a medical background.There is 4 generations of doctors in my family on both sides and we have a hospital thatwas started in 1945 in Pune and my lab is actually based out of there.So there was a motivation to get into medicine but not actually do day-to-day medicine theway they had been doing it.I was also interested in engineering, so nothing better to combine all those areas into one.My doctoral research was at the University of Michigan and I worked on genome sequencingbefore the Human Genome Project was completed and if you look at the people on this, sothere is Craig Venter there, that is me there, this is one of the projects that we did beforethe Human Genome Project was completed and we took almost 2 years to finish the sequencingof this genome.It was a lot of fun but we were totally clueless at that point in time but the two years isan important part to remember.Anyway, throughout my PhD work, I did a lot of work on genomics, bioinformatics and proteomics.And the nice thing is that even after all these years many people are still quotingour research.It remains relevant even after all these years.But after I finished my PhD a question that I had to ask myself and a question that youguys will be asking yourself at some point.What is it that really drives you and what is it that makes you really happy becauseyou are going to spend more hours in a day doing that than not doing it.So find out what really drives you okay.If it is photography that is great, if it is making boatloads of money that is greatwhatever it is but find out what it is and then go after that and if you need to takesome time off to figure that out, do it.So mine were I was really interested in interdisciplinary state of the art work, so I did not want todo just medicine, just science, just engineering.I liked all that stuff coming together.I am a geek basically and I also wanted to be able to impact individual patients andthere is no joy like seeing a patient come up to you and say you know what sir I hadno idea what was wrong with my kid but you guys have figured it out.There is nothing like it, I think that is an amazing sort of motivation to keep yougoing through the day and the night.So these were my two motivations, they do not have to be your motivation.I am just saying figure out what yours is whatever it is is fine but take the time tofind out.So the first thing we did was HandyLab.So HandyLab was developing a microfluidic lab-on a chip diagnostic platform.So lot of the stuff Sanjeeva has been teaching you about how you work with DNA, etc.These things are done in a lab and there are lots of people in the lab with lots of expensiveequipment.If you look at a lab from the top, it is basically unit operations of fluid moving from one placeto another.So you take some liquid a blood sample, it goes into the labs.Somebody might heat it up or cool it down or centrifuge it, stick it on the machineyou measure the pH or you measure the conductivity or you measure the optical properties youcombine it with something else.These are all unit operations.What we did was automate all those unit operations on a microfluidic cartridge that is a networkof tubes and pipes inside a thin little chip.Unfortunately, I did not bring it with me today and the idea was to have a diagnosisin a completely automated manner at micro scale in a very low time.So one of the first test that we developed in the US was a test for Group B Strep.Now Group B Strep is an infection that infects pregnant women around the time of deliveryand it can cause meningitis in the child and the child can die.The thing with that diseases that it can be treated with a simple single dose of penicillinup to four hours before the child is born but the older test took 72 hours to do bygrowing it on a culture microbiological culture.So you had to go in between 38 weeks, 37 to 38 weeks to get a swab, 72 hours later theywould tell you if it is not and then they would give you antibiotics.Now you can lose the infection in that amount of time or gain a fresh infection.So that is a little bit of a problem.So we develop this test that gives a diagnosis in 45 minutes.It takes the sample, extracts the DNA from it, mixes it with all the reagents which areinside that cartridge, optics everything, built-in and you get a result positive ornegative in 45 minutes okay.So that to actually pull off something like that we needed lots of diverse areas of expertiseto come together.So one is microfluidic cartridges, designing them and manufacturing them.They have to be something that can be built for individual patients not something thatonly works at 1 million dollar lab.Patients have to be able to get it.All the stuff that we moved on the chips was through thermo-pneumatic propulsion and control.So that means we would heat up areas of the chip, air pockets above that would expandand push liquid.By firing many of these different types of pumps on the chip, we could mix liquids together.We had gates and valves, so phase transition materials that are solid at one temperaturebut when you heat it they melt and then you push an air pocket through, so you can opena channel that was closed and the reverse when you have a closed channel or you canclose a channel by forcing in wax from the side.So wax that is solid at normal temperature but liquid when you heat it, so wax gates,valves, etc.So this is like I do not know if you can see it on this particular video here but yeahcan you see that that is the heating profile of the chip.So we are heating different areas of the chip, it is almost like one of those predator kindof movies but this is a thermal image of the chip and we are pumping different areas, mixingliquids together.This is thermal cycling for PCR.Here is some liquids mixing, right there you can see liquids mixing on the chip.So basically what is happening in a big lab is now happening in an automated manner outthere.As I said again I keep stressing interdisciplinary, so people from physics, people from electricalengineering, mechanical engineering, chemical engineering, molecular biology, business people,genetics all working very tightly together to build all this technology.There was a huge number of patents.I have just shown 4 out here.Large number of patents that were generated along the way.And we took nearly 10 years to build this, 9 years to build this.So hard work, there is no substitute for hard work but at the end of the day you can maketo be polite a shitload of money.So it is well worth doing and if you are interested in this kind of work, there is a huge rewardat the end of it.The technology that we created was globally valuable.So Qiagen which is one of the world's largest nucleic acid sample prep companies actuallylicensed our patents for their current generation of products.So the idea is that people who are sitting here are going to make great things in livesin their lives and you know these are examples of things that you can do to be world leadersnot just the best in India, you will be world leaders with the kind of work we do.So Becton Dickinson bought over our product after we took it for an FDA and then now marketrate globally under the BD MAX brand name.So having done that I wanted to continue doing that but to do something that was more relevantto our patients here in the context of Indian Healthcare and in India unlike in the UnitedStates patients pay out of their own pocket and so they cannot afford the same kind ofthings that they afford in US.So we wanted the same high quality, it had to be reliable but they needed to be relevantto the problems here and they had to be affordable and so we wanted to start by offering servicesand then go on to building devices and platforms.So go back to building what we did similar to the US but do it in the context of India.So our company in Pune is now in the service mode and we have just embarked into buildingdevices okay.So service mode is there, it is a big market.We do this, we have over 350 molecular diagnostic tests in our lab right now but we are alsostarting to build products.Keep stressing multidisciplinary but here is our team core team.We have MD DM people, we have PhD’s.Karthik is from ITB and from Hopkins, I am from the University of Michigan, Meenal andKetki are MD path and clinical geneticist, Kavita is a mixture of scientists and a clinician,Peter is a bioinformatics person.So we all work very, very closely.We also have PhD students in our lab.So now to get to some examples and a little background, so Sanjeeva has been teachingyou about the basis of you know DNA and the basics of molecular biology.I will just go through it really quick.In human information systems, information usually flows in a direction, usually thereare some exceptions.Information goes from DNA to RNA to protein.So DNA is like a hard drive for your system like you buy a new computer, all the informationthat you need is on the hard drive.But when you want to use a program like I am using PowerPoint right now, the informationthat is required is taken up into the RAM.Similarly, in biological system the information that is required at a given point in timeis taken up into RNA and finally the effector molecules like the outputs in a computer systemlike you have a printer or a display, similarly the actual work is done by protein molecules.So information flows from DNA to RNA to protein.There are some exceptions; retro viruses like HIV change the game.They go from RNA back to DNA and mess up the system.We also have interaction with the environment through processes known as epigenetics andepigenetics actually reversibly writes through the DNA and modulates how the DNA is convertedinto RNA.So there is more and more understanding.This is a very exciting area of research right now, so just keep that in mind.So where is this DNA and what is this DNA basically?So it is like an encyclopedia for each one of us okay.It is like an encyclopedia with all the information you need in your life essentially how yourbody is going to react to its environment and this information is a very long message.It is 10 to the 6 base pairs or 10 to the 6 letter of an alphabet and the alphabet hasgot four types of letters A, T, G and C unlike English which has 26.Now this big long messages actually divided into 23 volumes from your father and 23 volumesfrom your mother.So think of it as an encyclopedia that is broken up into many volumes, 23 volumes fromyour father, 23 from your mother totally 46.Now each one of these volumes is a chromosome, so you heard of the word chromosome.The volumes are the encyclopedia of the chromosome.Within every chromosome or within every volume, there are paragraphs that make sense and therest of the paragraph that do not make sense.The parts of the paragraph that make sense are the genes.So we have about 25,000 to 45,000 genes in our system, so that many paragraphs in there.They are distributed amongst the 23 chromosomes and you have 2 copies of each of these chromosomes,one from your father, one from your mother except for the sex chromosomes.Males have one X and one Y, females have two X's.The interesting thing is that in this entire encyclopedia only about 2% of it has knownfunction today.This 2% is known as the exome and 85% of all mutations in our disease sort of the diseasesthat we know and understand are located in that 2%.So that 2% becomes very, very important to study.So every time a cell divides this entire encyclopedia gets replicated okay.Six billion bases are getting replicated every time you make a copy of a cell.Mistakes will happen, we have machines to correct those mistakes but sometimes thosemachines fail and you have the mistakes carried over.So what are the things that can go wrong?One is that so I have an English sentence, the dog bit the cat, the cat ate the rat.There is two copies of it, one from the father, one from the mother.This is how the sentence should normally look.You can one have an infection, you can have something coming from outside cow, pig, asswhatever that is not supposed to be in there, if it comes and messes up the meaning of thesentence.So that is foreign DNA or foreign nucleic acids coming in.You can have spelling mistakes which have single nucleotide polymorphisms SNPs.This is when a single alphabet or a single letter in the alphabet changes.So in the top case you can actually understand the meaning of the sentence, so it does notreally matter so much but in the lower one because the C has changed to a B, the entiremeaning of the sentence has changed.So this SNPs can be bad, they can be harmless depending on where they happen that is important.You can have insertions and deletions that is addition of a letter into the message,these are almost always bad.Conditions like Fragile X and Huntington's disease they are really nasty diseases, theyare often caused by insertions and deletions and this is because when you say added theC there, the complete meaning of the sentence changes.Similarly, when you remove something the meaning of the sentence changes, you cannot recognizeit anymore.You can have copy number variations where entire messages or parts of the message arereplicated.You can have inversions where the message is flipped over.You can have translocations which is like buying a cheap pirated book on the street,you are reading chapter 9.Suddenly it goes to chapter 22.The sentence in itself makes sense but overall the story is making no sense.So that sentence makes sense makes a protein that does not really work well, hell breaksloose okay.Loss of heterozygosity is very complicated, I will skip it further.So there is a lot of mistakes that can happen, you need to use the right tools to figureout what the mistake is.Somebody will want to use the most complicated tool available and charge a huge but thatdoes not make sense.So the right diagnostician has to understand the context of the disease, the molecularand the clinical background and pick the right test that is going to be relevant to the patientin a clinical relevant time.So the most all of our technology is based on two very major things.One is PCR, the polymerase chain reaction.Do you all know PCR?Have you all done it in this class?Briefly, so the polymerase chain reaction was invented by a total slacker known as KaryMullis who got the Nobel Prize and everybody is still scratching their heads about whyhe got the Nobel and how he managed to pull off something so brilliant.But it is basically a system for photocopying DNA, it is like a xerox machine, you takeone molecule of DNA it becomes 2, it becomes 4, it becomes 8, 16 and in a very short amountof time half an hour 45 minutes you can make a billion copies of DNA from a single copyin a test tube okay and that is really the basis of almost everything that we do in thelab.The Watson and base Crick pairing that he talked about where A pairs with T, G pairswith C is exploited by the PCR process.Again this class is too short a time to explain and introduce PCR but it is the cornerstoneof everything we do.You can couple PCR with a fluorescence event and monitor it in real-time.So when there is more DNA you get this S-shaped curve.The more to the left this is the more starting material you had, so you can use that to detectwhether infections were present, how much of it was present.If you had a mutation in a gene, what ratio of mutant to non-mutant, so this is probablyone of the most important techniques that we use in the lab day-to-day.The other one is capillary sequencing.In capillary sequencing again was a Nobel Prize winning effort for Maxam and Gilbertas well as for Sanger and it is a way to read DNA base by base one by one.Now Sanger sequencing has been the cornerstone of our sequencing abilities for a long, longtime and the Human Genome Project which everybody must have heard of hopefully was completedin 2002, it took 3,000 scientists, 13 years and a cost of 3 billion dollars to sequencethe first human genome.Many countries across the world they were all working day and night these machines runningby Sanger sequencing.Today, it is possible to sequence a single genome in under 3 days for 5,000 dollars.This has put Moore's law completely to shape and this is because of a series of technologiesknown as Next Generation Sequencing.Again explaining Next Gen Sequencing is beyond the context of this class but it is a parallelprocess.So basically what you do is you take all your DNA, you fragment it into small pieces, eachsmall piece you clonally amplify and you sequence it individually.So our sequencers in the lab today can do 25 million sequencing reactions in parallelon a chip of this size.And the newer models that are coming can be 10 or 100 times more powerful than that.So you break your DNA, you add adapter molecules to barcode them, you clonally amplify them,you sequence them in real-time, capture the data and then you put it back all togetherlike a giant jigsaw puzzle.So because of the increase in computational power this has been possible but just thinkabout it, 17 years ago what took 13 years to do, today can be done in under 3 days okay.This is just mind blowing as it just boggles the mind and even that is only the start.Now we have this single molecule sequences and we have some of these in our lab whichcan take a single molecule of DNA, thread it through a nanopore and read out the sequencein matter of seconds.So they are going to further change this.So we use all of this, now this is over 350 tests that we have but we use this for many,many different areas to actually manage patients and I am going to give you very few examplesof where this technology is used.So one very common use is in hereditary breast and ovarian cancer okay.There are I told you there are about 25,000 to 40,000 genes and every time the DNA getsa cell divides the entire DNA gets copied over.Now there are some machines that prevent errors and BRCA1 and BRCA2 are two examples of thosemachines that prevent errors.So when there are errors, they fix it.Unfortunately, sometimes these error correction mechanisms also fail and so if you have amutation or an error in one of these genes you have as high as an 85% chance of gettingcancer okay.It is a very, very high chance and nearly 20 to 30% of all breast cancers and ovariancancer seem to follow that kind of pattern.So what are the advantages of genetically testing for this?One is that if you know that you have this mutation, you can modify your surveillanceoptions.So you can go in for improve surveillance and this is not only affecting women, mencan also get prostate cancer, they can get breast cancer themselves which is far, farmore dangerous in men than in women.So we can have specific risk reduction measures.So we can have salpingo-oophorectomies, we can have bilateral mastectomies and advancedsurveillance.Depending on the mutation they have to decide whether they need to get radiation or notget radiation.Once they have the cancer, we can change the therapy that they use, so if you have a BRCAmutation, we can use platinum based salts, we can use PARP-inhibitors which are verynew exciting drugs.Some of our patients who have been using them were in sort of almost terminal stages andnow they have got a fresh lease of life and we can figure the risk to other family membersalso.I would not get into details of individual patients because I think we are running outon time.But just the summary is basically of all the patients we have tested nearly 40% of themhad a change in clinical management after they did genetic testing.So you do the testing and what you would have done by the old method of medicine is no longerdone, they would have some new treatment options.Another very nice example, use of genetic testing is in CML which is chronic myeloidleukemia.Chronic myeloid leukemia up to 15-20 years ago used to kill over 90% of the people whogot it.Today, thanks to a drug known as imatinib, 98% of people have a normal healthy life.It is absolutely like magic but to prescribe imatinib, you have to show that your chromosome9 and 22 have fused and we have a genetic test to do that.We test the patients; find out if they have that fusion, if they have that fusion theyget imatinib, every 3 months we monitor them to make sure they are responding correctlyto that drug.As I said 98% of patients do very well but there are 2% of patients who do not do well.In that case, we sequence that translocated gene, we find different mutations in themand based on those mutations we switch them to other drugs.So some patients are switched to imatinib, some patients switched to dasatinib, somepatients have multiple mutations and for those you need a hematopoietic stem cell transplantbut this genetic information is actually being used for personalized medicine on a day-to-daybasis and that is pretty, pretty remarkable.So I give you an example from solid tumors and example from leukemia blood cancer.This is also used in endocrine system, so here is an example of a 2-month old baby,very high sugar at one month, initially being managed with two insulin shots a day whichis very, very painful for a you know family of a small child.We went ahead and sequenced and found that there were two missense mutations in thisABCC8 gene.We knew how this gene behaved, so the child could be switched to an oral drug sulfonylurea.The child stopped the insulin dosage and now is being orally treated, doing absolutelyfine.So this is an example of this in action.Another example in endocrine, two obese children profoundly obese 28.7 kilos at two-and-a-halfyears, another one 18 kilos at 1.8 years went in and sequenced the MC4R and leptin genes.One patient has an MC4R mutation.The other one had a leptin mutation.Recombinant leptin is now available, so the child who had leptin mutation we could actuallystart that child on recombinant leptin and that child is doing fine.So there is an example of that.Completely switching tracks, we also do a lot of testing for infectious diseases usingthe same kind of DNA techniques.Here is an example of a patient who had an eye infection okay.They were worried that this person would go blind, so they started the patient on antibacterials,antifungals and antivirus.This is like throwing the kitchen sink at the patient.In about 12 hours we showed that it was not a fungus, it was not a virus but it was abacterium and within 48 hours we showed it was Stenotrophomonas maltophilia.This means that the drugs that it has a different drug reaction profile, so they had to switchthe drug, patient got fine but they would have missed that because they only pick thatup after 5 days by conventional cultural methods.So these genetic tools are really, really important.Recently, there has been a spate of Chikungunya and Dengue in the state.In Pune, it is an insane epidemic and again conventional testing was missing in huge amount,using DNA and RNA based testing we were able to show almost 70 almost 80% of our patientswere positive and this can be diagnosed instantly like within a day after you get the infection.One of our patients was even Chikungunya and Dengue positive, so that is great.And then at the start I told you that when I started my career, we sequenced the genomeof Caulobacter crescentus as part of my PhD thesis and that took us nearly two years todo.Recently, we had a patient from Hinduja Hospital in Bombay with extended drug extensively drug-resistanttuberculosis and they said can you do anything for us and said sure we can try and sequencethe genome.So for about 12,000 rupees we sequence the entire genome of that patient, we found 9mutations that are corresponding to the XDR phenotype and we did this in under two daysokay.So what took millions of dollars when I was a student today can be done in days or like12,000 rupees is like 200 dollars.So it is just incredible.So I just snapshots of examples I can go on for hours but I did not want to waste yourtime.I have some take-home messages for you guys before I go away.Take your time to find out what you are truly passionate about and what really motivatesyou because you are going to be spending a lot of time doing it and cliched as thoughit might sound.There is absolutely no substitute for hard work and do not pay attention to people aroundyou who say you cannot do something you can almost always do something.When we started building that chip, everyone said sample prep is not possible, we succeededin doing it.Follow your heart and your instincts and do not follow the herd you know blaze your ownpaths.Multidisciplinary teamwork is the only way to go forward.Get do not stay side out in your small areas, get together with smart like-minded peoplefrom other areas no matter what they are.Modern healthcare is really a team sport and doctors do not know this.They think they are all superstars and they want to score the goal themselves but youneed a strong team, you have to all work together and Jugaad is not a long-term solution.Everybody in India loves to do Jugaad but it just takes us back.You have to innovate and do real hard work.Jugaad might take you 3 places ahead in the queue but the entire line gets pulled backwardsso that does not help and really figure out how you can positively impact society aroundyou rather than trying to reach out how you can reach the top of the pile because in along-run it is much more gratifying and evolving.And there can be win-win solutions in my experience you know people always think that you haveto screw somebody over to come up on top, you do not, everybody can win and be reallysuccessful, so I have learned that in my journey.So that is it really.Thank you for your time.Thanks.