Myriad Live - Let's Talk About Emerging Biomarkers for Li Fraumeni Syndrome Screening

Myriad  Live episodes are recordings of an open-forum webinar hosted by Dr. Thomas Slavin. The opinions and views expressed in this recording do not necessarily represent those of Myriad Genetics or its affiliates. To participate in a future recording, visit https://myriad.com/live/ for a list of dates, times, and subjects.

References for this episode:
Genome web article: https://www.genomeweb.com/sequencing/early-cancer-detection-li-fraumeni-syndrome-patients-enabled-liquid-biopsy-test

Abstract: https://acmg.planion.com/Web.User/AbstractDet?ACCOUNT=ACMG&CONF=AM22&ssoOverride=OFF&ABSID=12124

CHARM consortium https://charmconsortium.ca/

NCCN Guidelines: https://www.nccn.org/professionals/physician_gls/pdf/genetics_bop.pdf

Tumor-Based Genetic Testing and Familial Cancer Risk https://pubmed.ncbi.nlm.nih.gov/31570381

https://liftupstudy.org/

https://ascopubs.org/doi/abs/10.1200/JCO.2020.38.15_suppl.1514

Transcript

[music]

0:00:13.3 Dr. Thomas Slavin: Welcome this episode of Inside The Genome is a recent recording of Myriad Oncology Live a webinar hosted by me, Dr. Thomas Slavin, chief medical officer for myriad genetics. The opinions and views expressed in this recording do not necessarily represent those of myriad genetics or its affiliates to participate in a future recording, please visit myriad live for a list of dates, times and subjects. I look forward to exploring the world of genetics with you all.

0:00:40.3 DS: Hello, everyone. Welcome to Myriad Live. Thank you for spending your day with us. Sorry for the, I guess, two things, one the admission [chuckle] so that's a new thing I'm trying to figure out. We've been changing some licensing around, I believe in our organization and for whatever reason right now I'm having to admit people through Zoom. So thank you for being patient and it will probably throw me off as people come and go. So I pre apologize for that also the music. Yeah, that was a little melancholy. I thought at times I was gonna go into some Christmas in July, but there [laughter], but it's okay. I try to keep it usually more upbeat. So bear with me there, but it's a... We'll fix it for next time. Speaking of next time, I have here, I don't know if people can see my screen. Can people see my... All right. So...

0:01:40.6 Lauren: Yes.

0:01:41.4 DS: This is the, if you Google, Myriad Live, you'll get to this, but it's myriad.com/live. This shows the upcoming, sorry, I'm still having to admit people. [laughter] This shows all the upcoming...

0:01:53.8 Lauren: TJ, do you wanna make me a co-host and then I can just admit people while you're talking.

0:02:00.4 DS: Yeah, that'd be great. Let me see, make co-host. All right. Thanks, Lauren.

0:02:06.0 Lauren: Yep.

0:02:06.3 DS: All right. Hopefully yes. There's one last admission, at least on me. All right. Thank you. [chuckle] All right that was a question. I didn't know if other people can see who's coming in the waiting room. But yeah, thanks, Lauren. But you see here the upcoming events. So we have today we're gonna be talking about emerging biomarkers for leaf or mini syndrome. I'm really excited for this topic. So I have not met Derek Wong, but excited to talk to you. I see you're on, thank you so much for taking time outta your day to come educate all of us. And then we have June, we're gonna be talking about this will be post ASCO.

0:02:40.7 DS: We have an abstract at ASCO, those of you that are there, please stop by and I'm a coauthor on it, Alicia Hughes is first author and it's talking breast density as a risk factor for breast cancer. And we're gonna be bringing in polygenic risk scoring and Syracuse modeling in addition to breast density as a more comprehensive way to stratify individuals. So that's gonna be a poster and then we'll kind of debrief after ASCO on that poster and what's to come there. Alicia Hughes will be the special guest, and if anyone's met her, she is wildly smart. We cannot have a better person working on our polygenic risk scores. She has co-authored countless papers over the last three years, either first author or a co-author on polygenic risk score. I think she's becoming a very respected person in that space.

0:03:38.3 DS: So please come listen to her and I banter. And then in July, we have let's talk chemoprevention for breast cancer. We are gonna have, Holly Peterson is going to come on and she is a master of her craft when it comes to chemoprevention. So I wanted her to do a little debrief. It was suggested by Eddy Smith. Thank you, Eddy, if you're on, because she is just so knowledgeable in the space. So if you take care of patients with at high risk for breast cancer, wanna understand more about chemoprevention. That is your day, July 22nd. We're also filling in some other slots, probably one each in June and one in July just don't have them up yet. I'm trying to do this roughly every two weeks now as the current canons trying to bring in special guests, if this is your first time here you'll understand pretty quickly that this is a fairly relaxed format.

0:04:39.0 DS: We are here really for you all. I mean, the point of this is to have an open forum where people can pop on and ask any question they want. We do them theme based but we're here to educate, learn, and keep the branding generally to a minimum. We also have been putting all of these up on inside the genome, so they are recorded. So encourage you to still ask questions, but if you don't want to, because it's recorded, completely understand, and you can send those messages to Lauren, who is now the official co-host. So thank you. And we have been putting a lot of things up. There's tons of content up on inside the genome. You can get this anywhere, Apple, Spotify, whatever. And we've just... We just posted one that I did with Judy Garber going over Olympia, 'cause she was one of the Sentinel authors on that.

0:05:37.8 Lauren: I don't think you mentioned, but I think we have over 4000 downloads of that Myriad Live podcast, which is really exciting.

0:05:43.7 DS: And cruising. So thanks. Yes, I will not go back and belabor, sorry I lost internet. But fortunately I have multiple ways to get internet [chuckle] so I'm on AT&T network at the moment, but thank you Derek for coming on. Let's just jump into it. So Derek, I stole your ACMG bio [laughter] from the meeting. So Derek Wong is a postdoc fellow at the...

0:06:14.5 DS: Princess Margaret Cancer Center in Toronto. And he has a large research interest in cell-free DNA and fragment-omics and proteomics. He completed his PhD with Steven Yip at BC Cancer Research Center in Vancouver, Canada. And now he is in Toronto and sir, we came across your article in ACMG... At the ACMG conference, made some a good splash and was on genome web, which is where... I forget if Shelly saw it first or I saw it first, but either way, one of us saw it [laughter] and we were like, "This looks really interesting." Would love to get you on. And I would... At the time I actually didn't look through the co-authors, but I do know a few people on here. David Malcolm actually was a mentor of mine for some time. So you're proof that he's a great person to work with. So tell him, hi, if you see him for me. And really impressive research and would love just... If you wanna give people a little bit more of your background, I probably did not do it justice.

0:07:24.7 DS: And tell people how you got interested in this research, and maybe if you could go through a little bit about the ACMG paper, and then we can take it from there. And I know... I think Shelly prepped you a little bit and we just take questions as they come. So we try to keep it pretty fun and informational. So for those of you in the audience, if you have questions, feel free to interrupt us. It always makes it more fun if we get some good crosstalk going. The last thing I will say is Li-Fraumeni Syndrome, I don't tend to get on a soapbox a lot, but Li-Fraumeni Syndrome is really near and dear to my heart.

0:08:03.6 DS: As a medical geneticist, Pediatrician, I think back in my life of seeing patients even though I'm not seeing patients currently, but some of the saddest outcomes I have seen in my clinical practice has been from Li-Fraumeni Syndrome. I've been involved in, unfortunately, multiple people that I've taken care of that have passed away with Li-Fraumeni Syndrome. Proportionally way more than any other hereditary cancer syndrome that I can... And this is from personal experience. I know many are horrible, like FAP, et cetera, but Li-Fraumeni Syndrome is a special kind of horrible sometimes for families and patients. So anything people can do to increase survival for these kids, their families, I'm all for it. So thank you for doing this impressive work and you're at the right-center to be doing. You're at the right-center to be doing it and working with the right people.

0:09:05.4 Dr. Derek Wong: Thanks. So just as TJ mentioned, I did my PhD at BC Cancer, the research center in BC, Vancouver, British Columbia. And there I was working on brain tumors, so more just Proteogenomics, so more Biochemistry. But because my mentor was Steven Yip, who is a Clinical Scientist, I got more exposure to clinical and translational research. And that sparked my interest in Clinical Genetics. So from a postdoc, I kind of switched gears and went straight into the genetics. And Steven and Trevor who's my current supervisor, are our friends, so they train together and they know each other. So I got in contact with Trevor and he told me about this charm project, which is a big Canada-wide study, looking at cell-free DNA testing in a whole bunch of hereditary cancer syndromes.

0:10:07.9 DW: And so that really aligned with my interest with clinical genetics and that's how I got started with my postdoc. I guess if people aren't super familiar with the Li-Fraumeni Syndrome, so these patients have germline mutations in P53, which make them super susceptible to cancers. And this is why it's so tragic. Some of these families, they have so many occurrences of cancer and nearly, I think the statistic now is nearly 90% of paid female Li-Fraumeni Syndrome patients develop a cancer by the age of 30, which is very, very significant. And so this just makes early detection so much more valuable in this patient population, compared to say the general population where cancers are diagnosed at a much later age, yeah.

0:11:03.4 DS: And what kind of... How do you get going down the... So you spoke a bit about the charm research or network, and is that how you... Were you already doing a little self free work at that point? 

0:11:16.1 DW: So no, most of my PhD was all expression-based analysis, but I got my first dip into software DNA when I started my postdoc. And I just jumped straight into the water, and really just immersed myself with CTG. And it's such a rapidly developing field with so many new technologies coming out in the past couple years, especially in Fragmentomics. And that's also why I'm interested in Fragmentomics because it is so rapidly developing and it is such a new concept and it seems very powerful, because of the multitude of technologies that people are looking at with Fragmentomics.

0:12:00.0 DS: Can you explain that a little bit more for our audience? Most people on here... We have a good mix of largely people taking care of clinically genomics geared providers, people taking care of patients with these syndromes. And then some people that are lab-based, but I don't know if everyone knows what the field of Fragmentomics really means. So maybe if you wanna dive into that a little bit more and explain, I think that'll help then put it into context all the research that you're doing.

0:12:31.6 DW: Yeah. So I guess with traditional kind of germline using lymph besides like Buffy coat and tumor sequencing, your DNA is in very, very long strands 'cause it's genomic. And then in the sequencing pipeline or the assay, you sheer the DNA into smaller fragments, which makes it easier to sequence on platforms like Illumina. But the difference between cell-free DNA is that it's already fragmented and that's because when cells die... So the normal turnover cells causes release of DNA fragments into the bloodstream. And these fragments are not genomic, not really genomic DNA. So they're not long fragments they're already fragmented. And the interesting thing about Fragmentomics is that these fragments are not released in a random array. So basically the fragments are kind of... The release of the fragment is dependent on the chromatin architecture of the cells. And so nuclear zones, because DNA is wrapped down in nuclear zones that kind of helps protect those regions of the DNA because they're not accessible for nucleuses to cut.

0:13:48.7 DW: And so basically as the cell-free DNA is being released into the bloodstream is being cut up by nucleuses. And these nucleuses can only access sites in between nucleosomes. So you get a really unique pattern of release for these cell-free DNA fragments. And so Fragmentomics is kind of looking at the size distribution of these fragments, as well as where these fragments lie along the genome, because it's non-random, we can kind of reconstruct the chromatin architecture of the cells that are releasing these cell-free DNA fragments just by looking at where the coverages, where the cut sites are, and where the... Or the size of these DNA fragments. Yeah. So it's a really powerful new and emerging analysis or technology type. And I think there's a new algorithm being published every week now basically with Fragmentomics it's really exploded.

0:14:49.1 DS: Yeah. Yeah. And how did you use that here? 

0:14:53.9 DW: Yeah, so in our LFS, so in the charm study, we have LFS patients from mostly Toronto through SickKids and Princess Margaret hospitals. And we kind of, once we draw blood from them and extract the DNA from their plasma, we go through a unique three-pronged assay basically. So the DNA gets split off into targeted sequencing, shallow hold genome sequencing, and cell-free methylate DNA immunoprecipitation, which is also another cell-free or new and emerging cell-free DNA technology. So in terms of Fragmentomics globally, one of the interesting things that we found in our Li-Fraumeni patients that hasn't really been reported yet is that Li-Fraumeni patients seem to have a more fragmented cell-free DNA than normal non-carrier patients. And so this is kind of interesting and significant because in the normal populations in sporadic cancers patients with sporadic cancers seem to have more fragmented cell-free DNA as well.

0:16:06.3 DW: So it's kind of suggestive that maybe these shorter fragments in Li-Fraumeni syndrome suggests like it kind of supports that they are already more predisposed to developing cancer because they inherently have shorter DNA fragments. And that kind of suggests that they haven't altered chromatin accessibility landscape in their normal cells. So that was one of the findings from the abstract is that Li-Fraumeni or LFS patients have shorter DNA fragments. And I guess another interesting fact is that LFS patients with cancer have even shorter DNA fragments. So it's kind of like a conserved process. And then yeah, cancer patients have...

0:16:51.7 DS: That's interesting.

0:16:53.4 DW: Shorter DNA fragments.

0:16:55.4 DS: And you're finding this just for our audience here. I mean, this is through blood-based testing? 

0:17:02.3 DW: Yeah. It's wholly through blood-based testing. So we don't have... So these patients don't have, most of them don't have tumors. Some of them do have tumors. But yeah, we just draw blood from them and then extract the DNA from the plasma. Yeah. And I guess I...

0:17:18.7 DS: Are these patients being followed yearly right now in this program? Like once or every six month blood draws or something? 

0:17:25.1 DW: Yeah. So I can explain the CHARM program a little bit more, which will kind of answer that. So basically CHARM is a Canada-wide study where we recruit hereditary cancer patients. So currently we're following patients with hereditary breast and ovarian. So BRCA one and two, neurofibromatosis type one, Lynch syndrome and Li-Fraumeni. And we have started recruiting for other syndromes, such as like VHL, P10. But those syndromes, we don't have as large cohorts because they are rarer. But basically when these patients come in for their routine clinical screening, we just take an extra tube of blood from them and then we bank it. So the frequency is different depending on what type of syndrome, just 'cause certain syndromes don't require as intensive screening. For our LFS patients we do have blood draws as frequent as every three months. But for other syndromes, like BRCA one and two and Lynch syndrome, it can be around once a year, but yeah.

0:18:33.0 DW: Yeah. And that's just part of their regular imaging and biomarker analysis and everything that probably doing anyways is just part of their routine clinical care. And I know the network has been really.

0:18:49.4 DS: Yeah really been, you know, the leader to some extent, maybe not the charm network itself, but I think of like sick kids and, you know, Dr. Malkin and, you know, what has come out of that work over the years of like the original Lancet paper and things like that, you know, talking about screening for you know, Li-Fraumeni syndrome using really innovative techniques at a time, like whole body MRI. So, you know, you have all that clinical data and, you know, with that, there's a lot of you know, expense. There's a lot of you know, on patients, you know, particularly ones that might not be in research studies. You know, it takes a lot of time to do brain MRIs, whole body MRIs get all the blood draws. You know, you sometimes it's almost like exsanguinating someone with all the blood draws, especially for the children that are needed for the hormone biomarkers. So you know, definitely an area that's ripe for an improvement on biomarkers in general.

0:19:52.7 DW: Yeah. And so the goal of the CHARM study is basically to see if we can develop a blood-based test that can compliment imaging because sometimes imaging, it's hard to sometimes as things are fuzzy, you can't really make things out definitively. So if we do have a, if we find a signal in the blood, then we can kind of look back at the imaging and see if they correlate and kind of see how far back can we go, or how early can we detect these malignancies before they kind of truly manifest with clinical symptoms and stuff like that. Yeah. So that's kind of the whole goal of CHARM and it like CT DNA, isn't really there to replace any other screening. It's more just to compliment and to kind of give clinicians more robust predictions for these patients.

0:20:45.0 DS: Yeah. Yeah. That's a good point that it, yeah, we have a long way before it would maybe supplant some of the things that are out there now. We do have a question Karine, I saw your chat. You know, if you wanna unmute yourself and ask a question.

0:21:02.8 Karine: Yes. Thank you for this talk. I have question about testing for germline TP53 mutation in patients with high burden of somatic TP53 mutations. So in our practice, we see a lot of patients with either unknown primary or patients with various malignancies when one would do next generation sequencing in order to find targetable somatic mutations. But then on several occasions, they came across patients who had very high LDL burden on somatic testing. They had 60 to 70% frequency a low frequency of TP53 mutation. And I was wondering if and with negative family history, otherwise to suggest, look for Li-Fraumeni syndrome or any other hereditary cancer. So with negative family history, but high somatic burden, burden of somatic mutation what would be your approach? How would you recommend to, would you recommend to proceed with germline testing for those patients or not? 

0:22:20.1 DW: Yeah, that's a great question. So, and I assume with that you have not done germline testing on.

0:22:27.6 Karine: I have attempted to do it on one patient, but she was denied because, like she didn't have any family history and she didn't have one of the cancers that she, hers was cholangiocarcinoma. It was not a pancreatic or any other cancer that would be supported by guidelines. I dunno if you are familiar with Dr. Antonarakis, he's a expert in prostate cancer and he is advocating for at least consideration of germline testing for patients with high burden on somatic testing, but it's not on any guidelines. So it's, you know, it's hard to convince someone that you.

0:23:11.7 DW: Yeah. I mean, what I will say, I can take this one cause this is a good question. Karine what kind of provider are you? 

0:23:18.9 Karine: I'm general medical oncologist.

0:23:21.1 DW: Oh, okay, great. Yeah. So, you know, I could point you to at least the NCCN hereditary breast and ovarian cancer, or now called the breast ovarian pancreatic cancer genetic assessment guidelines because on crit one now you know, there is a bullet that says, you know, testing is clinically indicated in the following scenarios. And the third bullet down on crit one is a mutation identified on tumor genomic testing. That is clinical implications of also identified in the germline. Now that is fairly vague, especially since, you know, we have a lot of overlap of our you know, tumor tests and clinic and germline genes. But you know, at least it's something to go off of. And yeah, I mean, you know, 60% is pretty high in the tumor. You know, especially in somebody that you know, it's interesting, you know, it's, I have many comments on this.

0:24:20.2 DW: I mean, you know, sometimes, you know, often I would say, you know, TP53 is an early event. And so sometimes you can see variant allele fractions in the tumor that approach, those kind of levels you know, and some patients have sneaky you know, family histories or might not have the most robust you know, family tree to identify if there's cancers in the family. So that's the potential, you can also have Denovo mutations in Li-Fraumeni syndrome, but it sounds like at least in the case of cholangiocarcinoma that's not a core cancer per se you know, I don't know how old the individual was, but yeah, I'd still think you're right to start thinking about germline testing in that scenario, because it could be incredibly meaningful if you find it in that individual for, you know, not only them, but you know, maybe, even if it was a Denovo mutation in their offspring.

0:25:19.1 Karine: Yes. I guess the barrier is is getting off coverage.

0:25:24.1 DW: Yeah. Yeah. So, I mean, I could... I would say that that's probably the best thing that I could point you to is that NCCN and CRI one page, at least if you're, trying to, convince a insurance provider [laughter]

0:25:38.4 DS: Okay. Thank you.

0:25:41.4 Karine: Yeah.

0:25:41.7 Lauren: TJ, can I...

0:25:42.3 Karine: And then since you mentioned, oh yeah, go ahead. Sorry.

0:25:45.6 Lauren: No, go ahead. I'll make my comment after.

0:25:47.9 Karine: Yeah. Since you mentioned the novel mutations, are you talking about like a patient who has no family history and he's the pro band? He's the one that...

0:25:55.4 DS: Has the first person to have a Tp-53 variant.

0:25:57.9 Karine: Okay. Is it a common...

0:26:00.2 DS: In the germline? 

0:26:00.3 Karine: Is it a common event? Like, do, do you encounter that frequently or.

0:26:04.8 DS: It's much more common in Li-Fraumeni syndrome than it is in a lot of the other hereditary syndromes that we tend to think about, like BRC one and two. You tend to almost never see that. But, Li-Fraumeni syndrome. Geez. I used to know this off the... I'm embarrassed that I don't know the statistic.

0:26:20.3 Lauren: I have it TJ from gene reviews. I was juggling it. Yeah. Seven to 20%. You got it. You still got it all.

0:26:28.2 Karine: Oh, wow. Okay.

0:26:28.7 DS: Yeah. So it's, it's it's enough. Yeah. I mean maybe one on 10, one in five patients.

0:26:33.6 DW: Oh. So even if with negative family history, if you have, young patient with sarcoma or several malignosis. Okay. Interesting.

0:26:43.4 DS: Yeah. It's, it's definitely something to think about, especially if people meet, Li-Fraumeni criteria. I, I will say too. I mean, I'm always skeptic when I, when it comes... Germline is... Or, or Denovo mutations, in Li-Fraumeni syndrome sometimes are confounded by, one other thing, which is, apparent clones, which I'm assuming at some point where we'd get to in this top... In this lecture anyways, but, these, like low level clones in people's blood, especially as they get older, it's just very frequent to see them in Tp-53. And, it, a lot of times has nothing to do with, having like a germ line, like mom and dad, genetic mutation in, in Li-Fraumeni syndrome. It's just kind of like almost like a pre-leukemia, type clone, so that people with those that tend to be a little bit higher risk for leukemia and, a lot of research going on, but potentially cardiovascular disease and things.

0:27:36.0 DS: And, to this whole effect. I mean, if there's, there there's a lot of research studies, I mean, we're obviously talking about, CHARM and, and Dr. Wong's research, a project, that, I was involved when with we, secured an R one on at, city of Hope, with Dr. Garber and, Dr. Wessel at the time. And, Chris Amos is, the life consortium, the Li-Fraumeni exploration, consortium grant. So, they're, they're doing a lot of, work to unravel. What, what variants mean in TP53 across tumor, across colonial metabolic, problems across, germline features of, Li-Fraumeni syndrome. So that is a, NIH funded grant now. And, we could probably put something in the, chat just to point people if they have patients that, might be interested in getting involved with that study.

0:28:35.4 Lauren: Yeah, I can, I can definitely put it in the chat. What I was gonna comment is as, as myriad has been starting to do our own, molecular profiling, I've been noticing a lot of TP53 mutations, in, in patients with various cancer types. So I think it's important and there's another resource that I can share here. A paper that was actually published by two genetic counselors about tumor based genetic testing, and, and germline assessment based on molecular tumor results. Because I think that we, we find TP53 in a, in a lot of tumors, but it doesn't necessarily warrant all the time. Or most times I would say a germline follow up who I think obviously family history is a piece of it, personal history, age of diagnosis. And then there's some other factors like TJ mentioned chip other things that make it complicated.

0:29:27.6 Lauren: And then, on, on top of it, I was gonna actually ask TJ your thoughts on just the complexity when it comes to variance interpretation, when it comes to TP 53 sequencing and how we do it at myriad. But I don't know if that was part of, what Dr. Wong, the, the research that was done, like the germline testing for these patients. I think sometimes we have special interpretations because if their VAF is between like 10 and 30, we kind of call it out as not straight out, Li-Fraumeni syndrome because it could be a chip situation or something else. Some sort of like, TJ said like a leukemia precursor. So I didn't know if TJ or Dr. Wong had any further comments on that.

0:30:12.0 DS: Yeah. And I'll just clarify, yeah. Chip being clinical [0:30:23.8] ____ potential. So that's kind of that whole, like, what is this, it's a clone flowing around in somebody's blood it's, don't know exactly where it comes from. And usually at leads like a workup that sometimes includes, even doing skin biopsies to make sure it's not in that person's, germline. So, but yeah, good question. I mean, I, I don't know Derek, if you wanna comment on, on, yeah. Like, the germline testing and how the individuals, at least you're looking at, they have, Li-Fraumeni syndrome. We do, at least in my, clinical work, I, I was in the habit of, verifying with skin biopsy. There are some, various publications suggesting that that is a good approach to, I, trying to make sure it is really, truly in a someone's germline before you really, mom and dad genetics before you really commit to, lifelong screening for that individual, because it is these, these clones sometimes when they're floating around in someone's blood, especially like, Lauren, as you brought up, if they're like not where you'd really think they're not kind of at that, 40 to 60%, level that we tend to think of as, coming from, either mom or dad or being like on one allele around 50% of the time, they can be really tricky. So Derek, I don't feel free, I don't know if you have any other commentary on this.

0:31:40.9 DW: Yeah. So basically all the patients in our charm study have been confirmed Li-Fraumeni syndrome. And yeah, we usually use like a cutoff around 40%, VAF just to make sure that it is, and then there are.

0:31:53.6 DS: And do you confirm them through usually alternative sample, like skin biopsy or, yeah, we were, we were even playing around with eyebrow follicles. I knew some people use nail clippings. I mean, there's a lot, lot out there. [laughter], some's more in the research real.

0:32:08.2 DW: But there are like... I think one of the interesting... Some of the interesting cases are there's like the rare occurrences of chimeric, patients who do have a TP53 mutation in, I guess, part of their germline because they are chimeric. So I think we have one...

0:32:24.0 DS: Yeah, Mosaic. Yeah.

0:32:25.1 DW: Oh, yeah, mosaic. Like one of our patients then charms that he's mosaic. And yeah, that definitely complicates analysis sometimes.

0:32:34.7 DS: Yeah, yeah. Very much, yeah. Yeah. So Derek, I mean, if you wanna go through, what you find you did say... You did... How many patients did you look at and what were the results of your analysis? 

0:32:48.8 DW: Yeah, so we have about a hundred-ish patients in our cohort now. And some of them, we do have longitudinal samples, so I'll just share like an interesting one case here. So you can see this, right? 

0:33:08.8 DS: Yes.

0:33:09.1 DW: Yeah. So for some patients that we do have longitudinal samples, so this is looking at their targeted sequencing. So we do have a CHARM specific targeted panel where we look at hereditary cancer gene. So TP53, BRCA1 and 2. And it's basically just there to see if we can detect second hits or somatic mutations in their blood. So we have a algorithm basically corrects for sequencing error, and we can cut down variant allele fractions down to about 1% in the blood. So this patient, patient 22, they had three time points, three bloods, one in May, one in January, one in September. And in the May time point, we actually detected a somatic TP53 mutation in their blood. And then in November 20...

0:34:05.6 DS: And this is someone with a germline. Yeah, I see. You have their germline. So they have one germline variant, and then you found something else.

0:34:13.9 DW: Exactly. Yeah. So they had the germline variant, which we found at roughly 50%, which is expected. And we also just use that to see if there's a sample swab. So just like a validation to make sure we're looking at the right patient right there. And then we found like a second somatic TP53 mutation that was at around 1% variant allele fraction. And we just checked it then in May of 2018. And then in November of 2018, the patient actually got diagnosed with a lung cancer. So it kind of shows the power of target sequencing in this deep targeted sequencing in this cohort. So we sequenced down to around 20,000X, so it is quite deep, and we use the error suppression algorithms to make sure that we're capturing true mutations instead of just random sequencing errors, 'cause when you're calling down to 1%, that's kind of in the area that sequencing errors do pop up. And so you really have to be confident that these aren't just random sequencing errors that you're capturing and it is an actual mutations found in the blood. And then, so this patient after the lung cancer was treated with surgery and chemo and then we took a time point at in January of 2019 and in September of 2019. And you can see that that somatic TP53 mutation is no longer there.

0:35:36.9 DS: Yeah, that's interesting.

0:35:38.6 DW: Yeah. So this is one of our interesting patients and to kind of like hit on the topic of chip...

0:35:45.6 DS: Yeah, that's what I was gonna say 'cause another thing that's probably gonna confound this over time is... Yeah. And I know this is even what the research we were doing at City of hope was trying to answer is just how common is chip... Like are people with Li-Fraumeni syndrome from their mom and dad genetic germline, the actual syndrome, are they more predisposed to making clonal hematopoiesis, this is... Can you have a germline susceptibility to it? It's been well described in RUNX1. I was doing some research with the people at City of hope and Jeff White and so on... ETM in particular, and then we were kind of getting into whether that is also potentially the case for Li-Fraumeni syndrome.

0:36:30.5 DW: Yeah. So I'm not sure if I know anyone really knows the true answer to that question yet.

0:36:35.3 DS: Yeah.

0:36:36.4 DW: But it is... Like chip is always something that you really have to look for, especially when you're looking at mutations in cell-free DNA. So one thing that you can do is fragment chimeric analysis. So looking at the fragment length of these mutant molecules, so because chip happens in your normal hematopoietic cells, when they turn over and release their DNA fragments into the plasma, it kind of conforms to the normal, healthy fragment release that your normal non chip cells release. So... And this is kind of... I'm using Li-Fraumeni syndrome patients as an example, so if you look at their germline mutation, so this patient, patient 009, they have an R 181 H mutation in their germline and the fragment... So this is a fragment size distribution and it's just the length of the fragment. And how many of these fragments are that length? But basically the germline wild type and the germline mutant molecules they're around the same frequency distribution suggests that germline fragments are kind of the same length as your normal wild type fragments just floating around. And it's only really when we have somatic mutations. So fragments of somatic mutations are actually shorter. As you can see in this one patient, we detected an R 273 C somatic mutation and these fragment... All these Mutant molecules are actually shorter compared to their wild type and others.

0:38:13.3 DS: Yeah, it's interesting.

0:38:14.2 DW: Yeah. And so I don't have an example of chip here, but we do have some patients that do have chip and the chip molecules. So the mutant molecules that are chip are actually around the same frequency distribution as their germline...

0:38:30.4 DS: It should be longer. Yeah.

0:38:32.1 DW: Yeah.

0:38:32.6 DS: Just because it's also coming from like more Buffy coat white blood cells, a room for idea.

0:38:41.3 DW: Yeah. So this is kind of like one of our QC metrics that we use for variant calling in our LFS patients is to make sure that these somatic mutations are shorter fragments. And it just kind of gives you more confidence that you are detecting a somatic mutation rather than a chip or a low frequency chip, or just random.

0:39:02.4 DS: Yeah, that's really interesting.

0:39:03.5 DW: Yeah, sequencing error and stuff like that yeah...

0:39:06.0 DS: And Karine on that last slide brought up a good question, which is, did you then... Did you find that other, that second hit in the lung tissue in that patient? Just to lock it all down... Tied up on a bow.

0:39:20.9 DW: Yeah. Unfortunately, we can't get tumor sample for every patient. It is a bit of a challenge getting tumor samples sometimes. So we don't have the lung sample from that patient for that time point. Unfortunately, it would be really good to just kind of like hammer it down. But yeah, like that patient we did it... We do have some other patients that we have tumors for. But we haven't sequenced it yet just due to funding. We're focusing mostly on the plasma sequencing. Yeah.

0:39:56.5 DS: So what were some of the things I mean, you say you found... Did you find anyone with cancer that was detected during the time of the study? Or they were under evaluation that... Clearly, you just showed that lung cancer case, but I mean, other examples where you were able to find something? I should rephrase this. So like I guess I'm trying to get at were you able to find any cases of cancer before imaging clinical symptoms, or other biomarkers would have identified using the circulating tumor DNA? So like, for that lung cancer instance that you just showed, did you start looking for why that... Did you start following... Did anyone start following that patient a little closer, because they saw that second TB 53 variant floating around? 

0:40:49.2 DW: Yeah, so right now, CHARM is more of a retroactive study. And we have applied for funding to do a prospective study of CHARM, where we do report the findings from our analysis back to the clinician and then from there, they can make a decision whether or not to follow the patient more closely. So yeah, currently, none of our results unfortunately, are affecting clinical management of these patients. It's more just a proof of principle to see if we can deter... Detect these cancers earlier than standard clinical workup right now. Yeah. But I think that is...

0:41:28.4 DS: It looks like you had a few examples on that PowerPoint of... Were they similar type examples that you identified someone with a second hit or some other mutation profile prior to them getting diagnosed with a cancer? 

0:41:42.2 DW: Yeah, so I'll show you another interesting patient. So this patient is one, like they had a very extensive history of cancer. So they had bilateral breast cancer, astrocytoma thyroid carcinoma, lung carcinoma. And then we got three time points, one time point, July 2018, one in February 2019 and one in January of 2020. Concern jet... So during that time point, they actually weren't diagnosed with any cancers. But we were able to detect somatic B 53 mutations in all three time points. So we actually... The interesting thing about this patient is that we actually detected three different somatic mutations. And then in April of 2020, the patient was diagnosed with a schwannoma.

0:42:36.9 DW: And then in August of 2021, they're diagnosed with bilateral renal AML. So it kind of suggests that maybe we can detect mutations up to a year... More than a year before these cancers show up clinically, because we just talked to three different somatic mutations in three different time points and they had two additional cancers as well as a previous cancer prior, like, we don't know exactly where these mutations are coming from in the body, but we do know they're floating around and we are detecting them prior to their cancer diagnosis. So yeah and that's kind of why we chose to do a three-pronged approach. So targeted sequencing is just one aspect. The other very powerful aspect is cell free methylated DNA immuno precipitation, so methylation profiling, of plasma and so...

0:43:38.5 DS: Yeah, can you explain a little bit what that is and how it differs from... Because that's really pretty cutting edge and not used really clinically right now. So...

0:43:47.8 DW: No it's not...

0:43:48.9 DS: Probably... Refresher. Yeah.

0:43:50.9 DW: It is very experimental, but basically... So we call it CF... And what you do is... Or so, the identity of your cells are dictated by methylation patterns. So, methylation results in the, I guess differentiation paths to different cells. So, each different cell in your body type will have a unique methylation signature. And when these DNA fragments are released into the blood, these methylation marks are preserved on the molecule. And so, I guess theoretically, you could then kind of determine what kind of cell these DNA molecules came from by looking at whether or not there's a methylation mark at specific positions on that DNA fragment. And so basically CF... What we do is we take an antibody and we pull down all of the methylated DNA in the plasma and then from there, we can build cancer specific or tissue specific methylation classifiers using methylation data generated from repositories such as TCGA or like...

0:45:07.3 DW: The brain classifier, the brain methylation classifier from DTFZ. And then from there we can profile the methylation marks in the plasma to see if we can detect certain specific cancer types or tissue types in the blood that we wouldn't normally see. So it's quite powerful in that you're enriching for a specific signal, in the plasma. And so, we do have one case where one of our LFS patients was presenting clinically with abnormal lactation, and imaging didn't really find anything and they didn't, there was basically no confirmation of a breast tumor, but when we did the CF media analysis on this patient, they actually scored very highly on the breast cancer signal. And then eight months afterwards. So eight months after that time point, they then presented clinically with a breast cancer. So CF meetup is powerful in that you can pinpoint where these mutant molecules or where these molecules are coming from in the blood. Yeah, so.

0:46:19.4 DS: Yeah, that's really interesting.

0:46:21.6 DW: Yeah. So we kind of use targeted panel, to detect somatic mutations and then CF meet up to see if we can pinpoint where these molecules are coming from in the body. Yeah.

0:46:33.1 DS: Yeah. And it would be great to have the tie in, as Karine brought up, I mean, really on both of these, as, cancers identified, to look and see if that same signal, whatever it is still there just to really solidify that in the blood. So you got next grant right there.

[laughter]

0:46:51.2 DW: Yeah. There's like so many ideas for CHARM and it is like, there's so much work to do on this project. It is like crazy. But it's very fulfilling and yeah, it's very interesting.

0:47:04.3 DS: Oh yeah. And, for those of you out there that have worked with Li-Fraumeni syndrome families, I mean, many are just the most appreciative families, period, because they've just seen cancer, just ravage their families and, are just so worried about their own risk and their children's risk and their nuclear family's risk or sisters, brothers. So, very motivated people, a lot of, good organizations, the LFSA out there has been, great working with this community over the years. So, yeah, but, it's a great group of people to work with. Let's pause there and see if there's any questions really quick. Any questions for Dr. Wong? 

0:47:46.1 Lauren: I didn't see anything else come through the chat, but people can feel free to unmute if they want to.

0:47:52.2 DS: Well it will make it awkward.

0:47:58.2 Karine: Yeah. Derek, am I, correct to assume that the CHARM study is only for Canadian individuals? 

0:48:06.9 DW: Yeah. So right now CHARM is just Canada wide. So we have, different sites across Canada that we recruit. But we are open to collaborating with other institutions and other countries. So we do have, collaborations in England with Manchester, and I think we're developing some other international collaborations right now. So yeah, like if you have, a cohort or, like a program at your institution and you wanna collaborate, feel free to message me or anyone else that's part of the CHARM consortium, like the organizers, like Trevor Pew or Raymond Kim. Yeah. And then we can potentially set up something.

0:48:45.8 Karine: Thank you.

0:48:47.8 DS: Yeah. Very helpful. So where's, where's all this going in your mind.

0:48:52.0 DW: Like the future of CTD.

0:48:54.4 DS: Yeah. In Li-Fraumeni syndrome, I guess we can keep it [laughter] focused.

0:49:01.5 DW: Yeah, like in LFS, as I said, before I do see it kind of, complimenting imaging and other kinds of blood tests, blood based assays. And I think, I wouldn't really say we're replacing imaging, but because imaging does take so long and is very resource intensive. I think one of the visions of CHARM is to basically have more frequent blood tests for these patients. And then if we do detect something abnormal in their blood, then kind of triage the, imaging for these patients. So certain patients will have more frequent imaging, whereas other ones will have less frequent imaging. And that would just kind of alleviate some of the, resources needed for imaging and time, because it can be quite daunting for these patients. And it's a lot of mental toll and strain to be sitting like in an imager for hours, can cry or something.

0:50:04.3 DS: Yeah. And, I think there's been some, at least I've been part of some debate whether, we need to be doing annual imaging or yeah. I mean, because if something's not found on that first year imaging, yeah. Do you need to do that 12 months later? If everything was caught, and scanned really, appropriately or could be weighed two years. Yeah. I think these are the, the types of new technologies that, hopefully can come into the mix and, and just help overall for the care and you know, exactly what you're getting at. Reduce the burden on, patients to whatever extent that we can, because, it's, bad enough obviously to have Li-Fraumeni syndrome much of the time based on the high cancer risk, but then you, add all this, screening, that patients really do need to keep up, with every three to six month, depending on where they're at in the centers. And, it is a lot, and it becomes really to some extent, a large chunk of their life, especially if they have multiple family members and, they're keeping track. And, so, and so goes on, these months of the year, and this person goes on this month, it's like, everybody's always kind of getting [laughter] screening sometimes in these families.

0:51:16.0 DW: Yeah, exactly. And so, like one thing that we have done with CHARM, because there are Structures that preserve CT or CF DNA quite well we've partnered with our national network of blood draws or phlebotomists. So they don't even really have to come into the hospital to get their blood work done. They could just go to any one of the phlebotomy sites across the country and then just get it done there. And then it'll be sent to our institution for extraction and everything.

0:51:47.0 DS: That's great.

0:51:48.0 DW: It's very powerful also just for more rural families, so then they don't have to come in every three months or something. They could just go to their local phlebotomy unit and then just get a blood draw. And then, yeah. So it really reduces the barriers for a lot of families and patients as well. Yeah. And then with the cost of sequencing going down, like I only see these kind of assays becoming cheaper and cheaper to run. It really is more the bioinformatic expertise that's now required to develop. And so, like developing these algorithms to make sure that we're catching the right things in these tests and assays. Yeah.

0:52:30.9 DS: Yeah. And you brought up too, how looking at multi omic strategies and things, you know, there's clearly a lot to do there. You brought up the whole field of fragments that's, you know, exploding right now and, and methylation and everything else. So yeah, we certainly have a ways to go to, but at the same time, I mean, I don't think the first versions that are being used commercially over time are gonna be obviously what we end on. It's just like everything else, it has to evolve and we're gonna learn more as we do more. So I see... Yeah. Karine, I see your, it's more of a comment just that you'd like to do more frequent blood screening you know, feel free to talk about the barriers that you're facing.

0:53:18.9 Karine: Oh, no, no particular. I mean, it's just would be and not specifically for many patients, but whenever it's something done off guidelines, we face this with insurance that they would not cover it, but I think I, when it comes to my particular patient, I will, can I contact you? 

[chuckle]

0:53:44.1 DS: Yeah. I'm happy to give advice and help where possible. We have a pretty good regional medical specialist team, and Lauren is part of the medical science liaisons too. So you have more than enough.

0:53:57.7 Karine: Okay.

0:53:57.7 DS: Help out there, but yeah, anytime, if you wanna bounce something off me, let me know and happy to, happy to help.

0:54:04.8 Karine: Okay, I will...

0:54:05.7 DS: We'll try to point you in the right direction.

0:54:06.6 Karine: Keep that in mind. Thank you.

0:54:09.5 DS: Yeah, because there's...

0:54:10.9 DW: Yeah, Karine...

0:54:11.3 DS: There's a lot of good resources. Oh, go ahead, Lauren.

0:54:11.5 Lauren: Yeah, I was gonna say, I'll get your email from the attendance and we will make sure to connect you with either myself or your regional medical specialist, that's your clinical support, and they can definitely help with some of your questions. Because I think it's a very valid question and in a lot of patient types trying to, to get testing that you feel is appropriate based on your clinical gut and your experience, but insurance maybe is not as willing to pay for it. So it's something that many of our previous clinicians that work here dealt with and are very familiar with kind of that conversation.

0:54:50.9 Karine: Yes, we have a regional specialist. I guess my question is in general, if I decide and if my patient agrees to have periodic somatic, I mean, periodic CT DNA for, to supplement her annual screening, I will get in touch with my regional specialist to see how it can be done.

0:55:13.2 DW: Yeah. And also with how rapidly the field is developing. I think anything that's being used now will probably be obsolete in like a couple years just 'cause the algorithms and the analysis techniques are developing so rapidly. Yeah.

0:55:29.2 DS: A good question. Yeah. Yeah. I mean that, there is a high likelihood the field's going towards [chuckle], you know, some sort of early detection biomarkers over time just because it, as you know, Derek's research is pointing out, it does look like it's useful. And I just lost track of time. So we are over. Sorry. [chuckle] but just wanna say thank you so much, Derek for coming on. This was fantastic, great food for thought. Really exciting research, tell Dr. Malcolm, I say hello, if you see him. And thank you Lauren for running the chat and hey, thanks everyone for coming on and taking some time to learn about advancements in Li-Fraumeni syndrome and some of the early detection technologies that are coming out in plasma and liquid. So appreciate.

0:56:16.6 DW: Thank you.

0:56:18.3 Lauren: And have, have a great day. Yeah. Thanks for the great questions, Karine.