 Thanks very much. Thank you for the invitation to come here. I'm operating on about four hours sleep courtesy of American Airlines Okay, so I'm going to talk today based on practical experience as an NIH funded principal investigator Who has gone through this process? So first of all, what does the world look like from my standpoint we get happy news from NIH your grant has been funded We formulate a proposal for our IRB to give us authority to do human subjects research Because it involves return of results into a medical record We need to have a laboratory developed test that conforms to the clear standards In California, we have the joyous duplication of this effort whereby the state also regulates us independently and Authorizes our LDT results return and now most recently we have the FDA presubmission and IDE mechanism As you can see, that's a lot of stuff and if you want to get research done in a hurry. This is not the way to do it I'm not saying that there's a problem here. It's just that's a lot of stuff if we're going to make progress towards the realization of genomic medicine Before it goes direct to consumer, which is our great fear So our hypotheses that we were funded to explore were relatively simple They were that genome sequencing if performed rapidly in acutely ill infants in two locations Neonatal intensive care pediatric intensive care units who were thought likely to have a single gene disease Would increase the rate of diagnosis Decrease the time to diagnosis and improve the precision of acute medical care The backdrop is that there are literally thousands of these diseases And so a new strategy was needed that would be comprehensive as opposed to traditional testing Which as you can see below is sort of an on a case-by-case basis Decided in consultation with medical genetics and really not something that as as a nation as a health care industry People can practice in most health care settings So most babies do not receive this type of testing and likely will not get a diagnosis During their hospital stay and may not get a diagnosis before they die These diseases are the leading cause of death in the NICU in the PICU and in infants in general So September the fourth, I believe it was we Started our U-19 grant U-19s are different from other NIH grants in as much as they give a huge amount of oversight of the program by the program manager and by our peers, so there were four funded centers and we were on phone calls at least once every two weeks where we discuss Permanent aspects of our research. So this is a highly regulated mechanism for doing clinical research About a month after the grant started we had a call from the FDA to ask Could we enlighten them in terms of the work that we were proposing to do and Subsequently and our other three centers had similar phone calls and subsequently our program managers were notified That we would need to go through a pre submission inquiry and maybe an IDE none of us really knew what that meant and so we collectively put on our thinking hats we had numerous phone calls we shared materials and We submitted our Inquiry at month six So let's take a look at what that involved This is the process as David has shown you for us. That was a 13-page document with the following contents Specifically a device description Let's show you that This is our device We had many many many conversations about what the device meant because as academics We don't think device We think experimental design. We think technologies. We're familiar with the term laboratory develop test But device is not something that we build We typically have many components which we hook together Typically informatically to give an end result I'll walk you through this incredibly fast and so we have an enrollment process Followed and before that an ascertainment process Information is collected in a red cap database We collect a blood sample of the patients are enrolled Extract DNA. These are standard processes turned that into a sequencing library. This is PCR free And then sequenced them and in our case. This was on a high-seek 2,500 predominantly in rapid-run mode the resultant information then is fed into high-performance disk and Then into a compute cluster where it goes through these transitions in file formats Giving us a list of variants that may be causative of the infant's symptoms Those are annotated with a separate software tool in parallel with this process We enter the clinical features of the baby and correlate those with the 8,200 known genetic diseases to prioritize a differential diagnosis and these are then integrated using a software system that allows rapid variant interpretation The information similarly goes to a database a master database Ultimately as part of the mechanism of award We have a requirement to deposit this in public databases In terms of interpretation reporting by and large We perform a confirmatory Sanger test to confirm what we see based on our genome sequence but in exceptional circumstances We provide a verbal report to the ordering physician In advance of that confirmatory test and I'll explain a little bit more about you. So that's our take on the device The proposed or intended use This is a randomized controlled perspective study Inclusion and exclusion criteria are listed the group sizes Parents and clinicians were given pre-test questionnaires To understand the milieu if you will in terms of their hopes and fears regarding this novel type of testing and Then rapid genome sequencing was performed Return of diagnostic results was either verbal if the child was about to die And the result could potentially change the outcome But in the vast majority of cases weighted Sanger confirmation and with standard standard report placed in a chart some cases the results were not clear in terms of Whether this was a diagnosis or not In some cases we did not make a diagnosis and there was the opportunity for Physicians to cross a patient over into the WGS arm if they felt that the patient had to get that Which increasingly they did during the study and then after test results and at some point later There were additional follow-up questions to get information related to diagnostic and clinical utility and also again parents social legal ethical type information And another part of the device was to understand had it been validated and This was a device that was in Development the grant had three components and component one was to continue to develop the device during the five years of award as I say we didn't refer to it as a device but We started to And so with time analytic performance improved this is about its current analytic sensitivity and specificity and as you can see from the area under the rock curve Next-gen sequencing is maybe the most sensitive and specific Test ever to be developed in terms of its analytic performance looking at nucleotide variation that's both single nucleotide Substitutions and small insertion and deletion events we also had information on the diagnostic performance and its comparator to all gold standard methods combined and so we had reason to believe that the diagnostic performance Albeit, this is a retrospective study was superior and Then lastly did it make a difference to have that information? Yes, it did we had one life saved We had four cases in which there was a medication change sort of the definition of Precision medicine Three cases in which major morbidity was avoided This was maybe the most important slide Which was that there was not much room for time delays In a NICU or PICU environment that by 100 days of life over 50% of babies with a genetic diagnosis Would have died and therefore delaying return of results Was not in the best interests of the child And spoke to the relative risk and harms of returning an unconfirmed result albeit one with quite superb analytic specs Versus Sanger confirmation Taking about a week to perform. So after less than two months We had an initial FDA response the FDA responded to us with a series of questions And we had a teleconference shortly thereafter I'll just go through this very quickly. I don't want to take too much time But this shows you the actual questions and our responses and how we formatted this And so largely these were clarifications of specific items in our 13-page document that required additional explanation And for many of these we had to actually go back. This was a learning curve There was a different language being spoken and we needed to sort of get our heads around The FDA thinks about research, which is fairly different from say a clear cap lab directors thought or an IRB members thought So there were questions about When and where we would do confirmatory testing This was back in 2014 remember when that was normative in all cases Fda sought clarification about what was a high likelihood disease causing variant That we were able to show Relatively simply based on published a CMG criteria Process for determining whether a verbal disclosure Would occur and who did it and to whom and we walked through that And you know, this was a rare event. I think we had two of these events in a three-year period Bit more information about this a little bit more information about the actual information we were placing in the medical record Additional clarification about V us Whether there was any other mechanism to return an unconfirmed result This was something else Lastly there was questions about blood draws and whether these would be higher than what our institution allowed in Infants and some of the infants we were enrolling with premature Were there other any other? samples being retained And then lastly our question for the FDA was Do we require to proceed to the Investigational device exemption proposal We were notified in writing a couple of days later that no that was not required that this was a not significant risk Proposal our understanding was that there was risk. This was a Experimental device However in this somewhat unique setting of high acuity illness in infants with high morbidity and mortality That the benefits exceeded the harms And so we were then able to start enrollment I'm just going to finish up because that's not the end of the story. This is a five-year Project. I have now moved to a new institution. The technologies are now Rather different than they were back in 2014 For example, we can now decode a genome and return a result in 26 hours Previously the speed limit was 50 hours This is our device these days As you can see the level of complexity Has gone up at least an order of magnitude that these days we use genome or exome sequencing Interchangibly we have a variety of sequencing instruments and furthermore as is normative now We often outsource this to clear and cap approved External laboratories who will undertake these processes for us and we rely on their clear cap accreditation and Qc controls we do the analysis in-house Some of the components are similar, but as you can see there are a lot more of them now And as you can see they now involve both cloud-based solutions as well as local solutions and by and large There is a lot of duplication and redundancy that we are rarely using a single tool to make a diagnosis We still have the bottom line here that confirmatory assays are normative But increasingly we are giving our lab director Oversight of that as is generally the case around the country for diagnostic results return In part this is based on the improved analytic performance Which as I showed you has significantly improved as we understand the components better and partly by this fairly seminal paper where Leslie Beesiker and Colleagues showed that Sanger sequencing actually was not a good test to validate Next-gen sequencing results in as much as it was more likely to incorrectly refute a true positive Than to correctly identify a false positive And so information such as this is Making us Take this on a case-by-case basis in individual laboratories and relying heavily on our own experience I would say that we need to remember that this manuscript spoke only to Substitution events where we know that next-generation sequencing really does have 99.999 something sensitivity and specificity for indel events Paper did not speak to that and I would argue that it's still premature to To not confirm those by Sanger Confirmation, but I think this is an area where different labs will have different approaches So thank you very much. I hope I didn't run over my time Thank You Steven, and I think I really liked your analysis that That everyone is speaking a different language And I hope today's forum can act as sort of a translator and help to develop a dictionary for The different dialogues between research and and regulatory science Next we have Dr. Paula Cappasino who is a scientific reviewer in the Division of Chemistry and Toxicology Devices in the office of in vitro diagnostics at FDA You