Let's talk about process a little bit.Dr Hanson and Dr Rush, you are doing simultaneous tumor-normal testing. How do you counsel patients about this? Traditionally, we'd go through this whole formal pretest counseling process for germline testing. How much of that do you incorporate?

For good or bad, what I tell my patients is at a fairly simplistic level. It actually starts with me; I don't usually refer them to the genetic counselor to have the discussion about whether they should test or not. It's usually happening with my newly diagnosed patientI mostly do prostate cancer. I do this for all the men whom I deem incurable, so basically all my patients with stage IV cancer at diagnosis. For all my patients with locally advanced disease, I encourage it as well, so it is discussed with some of the men diagnosed with higher stage III cancer. I tell them, “You have this cancer. It's definitely a lethal cancer. It is your major life problem. The goal is going to be containment. Cure is a hard word here. What we want to know is everything that led you to develop this cancer, and we want to know everything that drives this cancer. My feeling about the best way to do that is to understand your genetics.”

Prostate cancer is common among the population, but there is at least 12% germline variability.I would say to the patient, “We want to understand your genetics, not just for yourself and opportunities for yourself, but this can affect cancers of the breast, ovary, pancreas, and even male breast cancer. We also want to know everything we can about the tumor.We want to send off your tumor for comprehensive analysis to understand what's coming from your tumor, what's coming from you. They're going to do a hygiene panel of the tumor and of your germline. But we also want to drill down deep into those genes that we've known over time, about 50 genes that are related to multiple other malignancies, and screen you for them. If we identify something, we will send you to a genetic counselor to talk about what the implications are and find the malignancies in you and in your family.” That's the way I lay if out, for good or bad. I don't go too detailed into anything like, “Look, we're going to test for this large expansion of genes, and that means that we might find more things, not just incidental, but some things of questionable significance.” We don't really go deep into it.

Again, I don't know if that's the right approach or not, but, logistically and pragmatically, I find it to be a very good approach. They're usually very engaged at that time. There are some minor things with younger men that I talk about for the germline testing, because they'll sometimes ask me, so I bring up life insurance and things like that. But that's the way I approach it. It's not as ideal as sending them to someone like Dana, a genetic counselor. It's certainly not as ideal as having a dedicated tumor genetics program, where there's someone who's a tumor geneticist looking at that, as well. But it's the only way to make the workflow work for me. Then I see the genetic counselors afterward.

How about you, Dr Hanson? How do you deal with it?

Basically, our hereditary cancer clinic deals with all cancers.Unlike what Ashley's doing, we don't come from the standpoint, “I'm your oncologist for this, and I need to figure out what we're going to do here, particularly from a therapeutic angle.” We kind of start more from the germline angle. Just to back up a little bit, I find that patients are about equally invested in what this might mean to their family—that they have this cancer and what this means for their treatment. We talk about the germline, and then I tell them that adding the tumor has really important added value. It can help us figure out if we get a funny result on the germline testing. It can help us figure out whether that's important or not. Sometimes, I tell them the story about a recent patient that we had. We did testing on both the tumor and the germline, and, lo and behold, there was a 

 mutation in the tumor and not in the germline. We knew exactly what the spelling error was in the 

 mutation, and it was not in the germline. It was the best of both worlds. Now the patient had a treatment option that arguably there is a stronger argument for, and she didn't have to worry about the fact that this could affect her family. It's a really, really powerful example of how doing the paired testing helped this one particular patient so much.

We start from the germline, we talk about it, and then I add in the tumor conversation immediately thereafter. It's always very seamless. I do talk to patients about whether they want to do it or not, but I have yet to have a patient tell me, “I don't want to do this.” These people are invested in this issue. You can argue that I'm too direct about it, but I don't think so.

Well, you wouldn't. Dana, you're on the other side. You have this challenge, because you're coming from a conversation about a tumor only and then you're being asked to confirm. Do you find difficulties getting reimbursement for that if the people are not actually meeting criteria otherwise?

: We used to when we first started this program. It was, I want to say, 2016, and for noncanonical tumors, people were certainly paying out of pocket. I remember a woman had anaplastic thyroid cancer, and they found a 

 mutation in her thyroid cancer. She had no real significant family history that would meet any sort of criteria, she had Medicare, and she was considered unaffected. Her daughter actually paid for her to get germline testing, and it did, amazingly enough, confirm in the germline. So, yes, in the beginning we did. At least for the 

 that have been added to NCCN [National Comprehensive Cancer Network] guideline, if you identify a somatic 

, some of the insurance companies are covering it. Medicare is still a real sticky wicket. Luckily, the cost of genetic testing has decreased enough that people generally agree to pay at least no more than $250 out of pocket. But we still have to deal with insurance considerations.

I do counsel people a little bit differently when we're dealing with someone with advanced metastatic disease versus someone who's having predisposition testing. There are certainly a lot of different conversations but we definitely have to have those conversations. The interesting thing—getting them on the back end, too—is that they knew something was going on with somatic testing. I don't think they clearly had a great understanding of what it was, sometimes. They're more concerned about their cancer. Then they say, “What does that mean for my cancer? Are you going to treat me with anything?” “Well,” I answer, “your oncologist has to talk to you about that, and then I'll talk to you about this.” There is a little disjointed conversation there. I, unlike Dr Henson, because maybe he's a better salesman than I am, have had people say, “No, I don't feel comfortable with this. I didn't know that that was being done, and now I'm not sure about it.” Eventually, the oncologist may say, “Can you just talk to his daughter? He's not budging.”We talk to the family members sometimes. But we've definitely been rejected. I think that's one of the pitfalls of tumor-only results—you're sitting on a 

 mutation, and the patient just will not, for whatever reason, come in, and it's tough.

 Anxiety provoking. We have 2 questions that I just want to ask. One is probably pretty quick. What was the name of the website that you used, Dr Henson, to look at the United States?

 VarSome. You don't have to sign up for it, but it's good to register. It's free. I find it to be quite powerful. It actually calculates the American College of Medical Genetics and Genomics risk or variant classification for you, based on the rules. It's quite helpful.

 Thank you. Then the other is sort of an EMR [electronic medical record] question. This person has Tempus [Tempus, Inc] fully integrated into Epic [Epic Systems Corporation], so everything's coming right in. Universal testing with Ambry [Ambry Genetics Corporation], and those come right in, and they have a genetic module where everything comes together. Can you share your experience in bringing discreet genomic variants from multiple sources labs in the EMR? I cannot.I don't know if any of you have experience with that? We don't have direct feeds at our place.

 Yes, Tempus does offer direct transmission into Epic. I don't know what other programs they offer. I'm pretty sure they do anyway.

Yes, we do. We have the same. We have Ambry coming right into the EMR. We're working with other labs, so some of our gynecologic oncologists are using Myriad [Myriad Genetics, Inc] for HRD [homologous recombination deficiency] testing, so we're working with them. I know we're working with GeneDx (GeneDx, Inc]. Invitae Corporation just launched from a germline standpoint. I'm not sure about Guardant [Guardant Health, Inc] or anything else. I don't think we've had any success there yet. But a lot of the germline labs are now wanting to feed anything to make it easier to make their company more attractive.We just launched Invitae, we have Ambry, and we're working with Myriad right now.

 Clearly, this idea of more transparent integration, ever-closer union, is part of what everybody's trying to do. It makes sense. We don't actually have a disclosure slide, so I don't know if any of the speakers would like to make any disclosures? I personally don't, but I wanted to see if anybody wanted to disclose a conflict.

 I speak for, have spoken for, and have worked with Tempus for many years, either collaborating with them or speaking for them in the last year or so.That's my only relevant disclosure. Then I partner with a lot of pharmaceutical companies that make targeted therapies that can be matched to patients through genetics. That would be my second disclosure.

 I certainly do that, too, for a number of pharma companies that use genetic information. I just feel like that's an important thing to get out there.

Mark, I have a research project with Tempus looking at HRD in nonsyndromic tumors. I haven't spoken for them.

 OK, very cool. Thank you. Dana, anything?

I'm on the speaker’s bureau for AstraZeneca [AstraZeneca Pharmaceuticals, LP]. That's about it.

 Cool. We are out of time. Thank you all for your participation. It was really energetic and enjoyable. Thank you for engaging.

Videos

Mark Robson, MD; John Henson, MD; Ashley Ross, MD, PhD; and Dana Farengo Clark, MS, LCGC, discuss operational processes and techniques for counseling patients about germline testing.

Operational Processes and Challenges

I want to touch a little bit on this new technology and get your vibe on how you are handling cell-free DNA (cfDNA) profiling. Of course, there are a number of different companies that are doing this now, so-called liquid biopsy or whatever you want to call it. And it's not infrequent that you find low–variant frequency alterations in cancer susceptibility genes. You get your cfDNA test, and there's a 0.2% 

 variant or something like that.How do you go about that? Dr Ross, let me pick on you first.

: Well, there are a couple of considerations. This comes up in prostate cancer, as there's sometimes bone-only progression. There is nothing easy to biopsy. And you're now trying to think whether you can assign this person a therapy. And with 

, as you mentioned, you think you can now qualify them for a PARP [poly adenosine diphosphate-ribose polymerase] inhibitor, but is that their next line or not? But could this be spurious? Could this be hematopoiesis of uncertain significance and just something that happened in a CHIP [clonal hematopoiesis of indeterminate potential] phenomenon? Could this be real? Should it guide treatment? When you don't have options, you use it as leverage to get the drug you want through clearance for insurance. When you have other options, you kind of think, “Well, I'm not really sure. I've had no guidance on how to interpret the allelic frequencies of cfDNA.” I would say I'm often bamboozled in using it the way I want to leverage the next line of therapy. So that would be my 2 cents. And now, to the more sophisticated answers from Dr Henson or Dr Clark.

: It is a complicated issue, because whether or not you're finding subclonal alterations, whether or not you're just finding CHIP … our assay happens to be also a plasma/buffy coat assay, so we subtract CHIP from ours. But that's not the case for most of the available assays, and it is a challenge. Dr Henson, we were talking about F1. What do you think about the issue of mosaicism that you're picking up on ctDNA [circulating tumor DNA] or cfDNA?

 In most hereditary cancer situations, mosaicism is not as big a phenomenon. What's even worse than an F1 [first filial generation] is an F2 [second filial generation], where mosaicism is a huge issue. When you see a variant allele fraction in plasma that is on the order of a few percent to 15%, that's very common with what you see with ctDNA. CHIP is supposed to be less than 20% or 30%; mosaicism is under 30%. Mosaicism in most solid-tumor situations is actually rare, and I would encourage people not to spend too much time worrying about mosaicism unless there's some reason. With CHIP, on the other hand, the genes involved are fairly well known. If you have a tumor that has an 

variant at 30%, that's not a common CHIP gene, so I'm not thinking CHIP in that situation. And then you really have to think about is whether this is something that's being shed out of the tumor. If the level gets up to 50%, obviously, it's different. But there again, and I think Dana mentioned this, the variant allele fraction (VAF) is very tricky, because it could be that you've lost the other allele, and now you're only amplifying the 1 allele that's mutant. But if it does go up to the 50% range, then you need to think about the possibility that the patient has a germline change. Obviously, the canonical percentage would be 50%, because 1 allele of all the lymphocytes contributing to the circulating DNA is going to be abnormal. So low frequency, it's probably going to be a tumor. There are all kinds of problems with these assays, like low metastatic burden. Certain tumors don't really shed that much tumor DNA.It's helpful in really advanced cancers. And in the early stages, I think we have more work to do. Once we get up to 1000x coverage, we may get a lot more information. But anyway, it's a complex subject.

: And just so it's clear, and not to poo-poo cfDNA, I found it very useful in prostate cancer. We're often targeting the antigen receptor (AR), and we will sometimes see cf DNA emergence, resistance, or alterations in AR that are actionable, so that's very helpful. I've also been very impressed with how much they've been able to push the envelope on cfDNA, such that they can even start to report things like if they're not MSI-high [microsatellite instability-high], you won't really know. Sometimes they're doing a 100-gene panel, and they can start to look at some of the MSI or even at beta testing or tumor mutational burden. There are certain things to learn about resistant clones that might be out there and about some of this immunogenicity. And that really speaks toward the idea of doing serial testing throughout the patient's treatment lines, so I think there is a role. So just for the audience, there are caveats, but I'm glad that it exists, and it has a role in patient care.

: Dana, have you seen cases referred to you on ctDNA?

 Yes, we sure have.Penn Medicine uses Guardant [Guardant Health, Inc] a lot, and so what we've done is partner with the Guardant people in our region, because the ability to get any germline information from a commercial liquid biopsy lab is pretty difficult. We've asked them if they could provide us with any additional information. They will send ones that they think they can't put on a report and send us a little heads-up email saying, “We think you might want to bring this person back in.” We're trying to backdoor it a little bit. But I do think the VAF is tough to interpret, especially with different levels of tumor and how much they're shedding.They've told us not to rely on that in the least bit. We've asked them, “Could you give us a clear list of genes? And could you make these reports a little more obvious for the average person?” We are continuing to work with them to see if they can give us any information. And they do have some pretty sophisticated algorithms to try to tease out what is in the germline, but again, it's not going on the report, so it's of limited value unless you dig a little deeper.Yes, we've certainly seen them coming, and these are mostly a lot of lungs and noncanonical tumors. When they see a 

, people just don't know what to do, so it winds up in genetics. That's OK.

: Everything does. I think one of the important take-home points for the audience is that these ctDNA assays are not germline assays. I think we all have been taking that as a given, but I think we should underline that, just like not finding a 

 mutation on tumor testing doesn't necessarily mean there's not one present. It's even less of an exclusion if you're just looking at a ctDNA assay. You can't assume that it gives you the same kind of information. I will also kind of tease you. We've gone through actually a substantial proportion of our impact database. Among 20,000 or 25,000, we did find a very small number of mosaics. 

, perhaps not surprisingly, is the most common. In situations where you really would not expect it, you see “de novo” 

 mutations all the time. But how many of the “de novo” 

s are actually from parents who were mosaic and then transmitted it?We submitted that for publication somewhere. I don't remember.

 We're looking into those as well, all of our 

mosaics. That's become challenging. The germline labs are not doing a great job, because, as Dr Henson said, anything with a low frequency (under 30%) they call “mosaic.” I think it's between 10% to 30% that they'll call “likely mosaic.” Some 

get special interpretations, so when someone who's even in genetics gets one of these reports, I think it's very confusing. I think the world of mosaicism is becoming interesting.Just trying to figure out whether people have Li-Fraumeni or not is becoming more challenging.

 This is a little bit off topic, but just pragmatically, you find these alterations that somebody says, “Maybe this is a mosaic.” How do you deal with that? Because we can just give them biopsies or whatever, other tissue sampling to try to confirm it, but what do you think the most practical way of dealing with that is with regard to the family? You have an idea? How do you deal with that? You can't resolve it, necessarily.

 The best way to get started with it is to do triad testing. If you test mom and dad and you can't find it, although there are certainly other explanations for why you can't find it, it is very likely that the individual has a de novo mutation. Of course, the only place that you see mosaicism is in de novo, because mosaicism is a postzygotic phenomenon, maybe at the 8-cell stage or the 100-and-whatever cell stage. But if you want to make the mosaic argument, you should not be able to find that mutation in the parents. That’s the first step. If you find it there, then that would be difficult. You wouldn't find it. That would break some fairly significant rules. I think the idea of triad testing is where you go first if you're trying to make an argument about mosaicism. Then you can look at white blood cells and try to figure it out. RNA can be helpful to see what kind of transcripts are coming off the genes and whatnot, but it's complicated.

 We've taken a very pragmatic approach. If we can't exclude mosaicism, we test the kids.

: If you can't exclude it, test the kids.

: This is pragmatically easy to do, and that way, if the kids are negative, it doesn't matter whether they're mosaic or not.

 We have a very low uptake on skin punches.

: Yes, we don't do too bad. I just wanted to show a couple more. This was the other abstract that was given to us to look at, which is this idea of using ctDNA to guide germline testing. This was interesting. A number of samples were analyzed by Guardant. Those that had either a high allele frequency or those with 

 were then referred out for testing. There were a number of patients who actually did have germline mutations with a high MAF [mass air flow], and they turned out to be pathogenic. Some of them were actually of unknown significance. So there also are a number of other 

s at low frequency. Not all of these actually turned out to be germline. You can see CHIP in just about everything, actually, and you can certainly see somatic alterations in just about everything. But I agree with what you said, Dr Hanson. If you have a high allele frequency or if it's in a gene that's particularly meaningful to you, then that's certainly an area to chase dedicated germline testing.

Mark Robson, MD; John Henson, MD; Ashley Ross, MD, PhD; and Dana Farengo Clark, MS, LCGC, discuss the evolving role of circulating tumor DNA (ctDNA) in germline testing.

Role of ctDNA in Germline Testing

But can we talk a little bit more about this? How do you decide who to send for germline testing? Dr Ross and Dr Henson sort of fixed the problem because they were doing simultaneous tumor normal. But if you do tumor-only, and you find a variation, how do you decide who to send for confirmation? How is that?

 It's been my work for 4 years, so I will say the process continues to be refined. When we started, there wasn't much out there on any sort of algorithm We don't love to use allele frequency because obviously, that has its own problems. You can get ones that are very low that confirm. You can get ones that look around 50% that don't.We kind of threw that out the window. We looked at TCGA [The Cancer Genome Atlas] data of the genes that seem to have a high germline to somatic ratio, so we didn't want things like p53 that you find in every tumor. We also used a lot of the current guidelines put out by ACMG [American College of Medical Genetics and Genomics]. There was a paper by ESMO [European Society for Medical Oncology] last year.We do try to look at what everybody else is doing in terms of what the standards are. We kind of basically decided that the [Penn Center for Personalized Diagnostics] CPD would send a notification email to me and a notification email to the ordering provider that something that is potentially germline may have been found. It's clunky, but that's what we've been doing. And so, the obvious ones; anything 

 was an automatic referral. Any Lynch was an automatic referral. And then we really tried to use some of the, as kind of a playing ground, some of the ones that have NCCN [National Comprehensive Cancer Network] guidelines that we thought, "Someone with a RAD51 CD might have their ovaries out," so something that was clinically actionable in our minds. And of course, that's coming from a clinical genetic standpoint as actionability. So that's kind of how we started. We just reconvened, and now we're adding things that, phenotypically, you would expect to see in the germline, things like 

. And we really just are using our clinical judgment a little bit, and that's kind of how we've bumbled through it a little bit. But we keep adding genes as things evolve in terms of literature. And again, the high-risk genes, such as Lynch syndrome and 

, those seem to confirm much more often than not the majority of the time, so we're continuing to look at those. Some of them come up so infrequently, but things like p53 now, per ESMO, we're only doing people with a diagnosis under 30 years of age. So, there's a lot more caveats now, I would say it's not easy and doing what you guys are doing seems a little bit less messy. But we're trying to follow published guidelines and then kind of go with our clinical gut.

 It's a little less messy in some ways, but we'll talk about process and flow later on. It introduces complexities there as well.I'm curious, either Dr Ross or Dr Henson, if you do tumor-only sequencing, and you don't find a pathogenic variant, let's just say in 

, do you feel like that's good enough that they're off the table?

 That is a difficult question. For me, I would say that practically, my heart would say no, and I'm mostly doing prostate cancer where germline testing for the high-risk in metastatic disease is endorsed and often covered. When I see no pathogenic variant, I still sometimes push for the actual dedicated germline sequencing. I think that the NGS [next-generation sequencing] reads a 600-plus gene panel. I sometimes don't trust that they're actually identifying all the variants are there. And then I can't remember exact specifics, but I think there's been a couple times where there has been something in the germline that has been missed. There are tricky genes, 

, some other ones that are tricky to identify by standardized NGS, and so I guess it's not enough for me. If there's a strong indication for germline, I'm going to a dedicated one. As Dr Henson said, X-Gene and GeneDX makes that kind of easy, but again, I'm the least genetic savvy on this call, so I'll see what Dr Henson says. I don't know if I'm doing it the right way or not, but I think I have to be belt and suspenders when it's indicated.

 For me, I think if I get a 500-, 600-gene panel back on NGS from the tumor, and there's actually nothing there that fits the patient's expected profile of potential alterations, I do relax a little bit. There are weird things that can happen; for example, in a treated tumor, there's a small percentage of cases which will have revertant mutations, which is a really fascinating phenomenon where the initial driver event goes away because you're successfully treating those cells.There are things that can happen, but I still like the follow-up of the germline. It just makes me feel more comfortable, particularly if it's a tumor where I'm really anxious about the presence of a germline change. We had a case today in clinic where there was a large tumor panel that was completely negative, and we looked at the family history. It was very quiet. Big family. Not much to actually look at. And we actually accepted that as evidence that we weren't going to do germline. But most of the time, we do it.

It always strikes me when I get these reports. There's a lot of data that I'm either not looking for or is maybe not being brought up to the forefront as much. For this NGS on its own, for example, sometimes, they'll report out the low-coverage amplicons from that particular sequencing run. And then I never, as a clinician, take the time to look at, "Well, is one of my 46 germline genes there? And maybe I had lower coverage than I wanted, and that's why." And so, the solution to some of these problems is actually doing the dedicated panel so you don't have to do that. But we were talking a little bit before about nonsense-mediated [mRNA] decay and these VUS [variants of uncertain significance], and are they important or not? There's a lot of computational stuff that's also missing that could be added on top, even for categorizing the VUS, not just from genetic data banks, but also just from biology. I'd love to know if they're doing an RNA sequence at the same time, if my mutation was expressed or if it was a nonsense? I'd love to know, where is this mutation? If it's the amino acid level, where is it? Is it in the active site of the enzyme? Does it affect tertiary, quaternary structure? Or is it just nothing? I think there's a lot of rich data. I know it's off topic, but just grabbing the soapbox, I would say the solution to some of these problems is adding in the more testing. But I think that there's so much rich data that is not at the forefront of someone who is not a geneticist like you guys who's just trying to go through, with the patient, the report. And it's a blind spot for us sometimes.

 The RNA is very powerful. I think if you get normal RNA on the tumor, both in terms of expression levels and sequence, you're safer in that case, unless there's something really weird going on in the tumor. But germline testing is still, I think, very, very valuable.

We have a fairly wide experience with our simultaneous tumor-normal platform, and I don't think we've published this yet. Dana and I were talking about this yesterday. But we took a look at our patients who had germline alterations and tried to figure out how many of them were identifiable in the tumor sequencing. And it's about a 10% miss rate, and most of it is due to usually homozygous deletion.You have LOH [loss of heterozygosity], and then you lose the mutant allele, or you delete out the variant, so particularly, when you're finding hom dels [Homozygous deletion]. And 

 actually is a gene that has a reasonable number of hom dels. I think the take-home message for those of you who are listening is that tumor profiling alone does not exclude germline variation. If you have a normal tumor profile, or if you don't see a 

 mutation or some other mutation on the tumor, it doesn't necessarily mean it's not there. And I do think it's worthwhile considering dedicated germline testing, either as part of a package or as a separate standalone test.

Sorry, Mark. The last point in our bullet that we send to everybody in terms of tumor-only sequencing is, "If it smells like a mutation, send them anyway."I think sometimes, we have to go back to basics. And if it's a canonical tumor that looks suspicious, there's maybe family history at all, we always tell people, just use a little judgment as well. And I think that sometimes goes out the window. But basics do work.

 The 30-year-old triple-negative patients with a family history of breast history, and they don't have a 

 mutation on their tumor, you might want to send them anyway. It's just common sense.

The panel of experts provide insight on how they identify patients for germline testing.

Identification of Patients for Germline Testing

I think a lot of people have been doing, at least up to this point, doing tumor only sequencing. I just wanted to show what we talked a little bit about this before the program started. These are data actually looking at the identification of germline variance or potential germline variance using a computational algorithm on tumor sequencing. And this is a group of about 850 patients who had, CGP probably stands for comprehensive genetic profiling or something, by a commercial enterprise. And then they do have this fairly complicated algorithm that they use to try to figure out whether or not something might be germline. These were the results of this. It's interesting that they found nearly, slightly over a third of the patients had a potential abnormality. And then they took these to frank germline confirmation with a separate sample, presumably. Ninety-six percent of the variations that they identified were indeed confirmed to be germline, not unconfirmed to be germline. This particular algorithm supposedly works particularly well without a separate tube but it's interesting. And they found a lot of these germline variance in tumor types, again, without testing recommendations or without universal recommendations. Essentially using the tumor as a population screening technique. And perhaps not surprisingly, a fair number of the 

 tumor types. So, this is the idea of using the tumor, essentially, you're doing opportunistic population screening in cancer patients in an attempt, as Dr Ross said, to kick it out. Dana, what do you think, I have a couple of issues, or a couple of questions. What's been your experience because somatic reports frequently don't do the same kind of variant curation that we do on germline reports, particularly tumor only? What's been your experience with confusion around that issue?

 First of all, just confusion in the EMR [electronic medical record]. Things being listed as the patient is a 

, so there's a host of those sorts of issues. Just incorrect documentation of the actual etiology of the mutation. The other thing is just HDVS [Human Genome Variation Society], the standard nomenclature when you get a somatic report. FoundationOne has a lot of these issues of they're giving you not standard nomenclature. So, every time I get a report from our somatic testing lab, I vet it. I will look it up in the germline literature because obviously, the way people vet things in somatic testing reports is very different than how we look at them in germline. I'm kind of the genetic counselor who does that sort of work to see, A, has it ever been seen in the germline, and B, what does it consider? Because they will be switching over, but historically, I think somatic labs didn't ever call things likely pathogenic, so they kind of bumped it up or bumped it down based on their experience level. And we don't confirm VUS, or variants of uncertain significance, so those would never even reach me.it could've been something that was likely pathogenic that they downgraded to a VUS.I always make sure, if things look suspicious, to sort of re-read the report. Sometimes, for people that are out there that are getting reports that they don't understand, there's always someone at the lab that you can call to ask them for the correct nomenclature because some of these, if you go look them up in ClinVar to determine their germline significance, they're impossible to find because the nomenclature is just completely different. I would encourage anyone out there to lean on the labs and ask them. And I know our group at Penn Medicine has tried, "Please use HGVS nomenclature like everybody else does," so our somatic reports are a little bit better in that they report in HGVS. But again, just the classifications can be very, very different. And to speak to your other point about that abstract, when we looked, we sort of did an algorithm with looking at high-risk genes, so BRCA, Lynch, and we found that about 75% of those that actually wound up with a mutation in what we've called a high-risk gene in the tumor, that 75% of them did confirm. We sort of thought, "Our sniffers are OK." Without knowing anything else other than that this is a high-risk gene and high germline to somatic ratio, we did confirm a lot of those. But the somatic reports can be tricky. I always tell a new genetic counselor, "Just know what you're looking at. Know what genes were tested. Know the pluses and minus, del/dups, all that sort of thing. Make sure you really look at the report or have someone walk you through it."

 And on that point, you know what I like to do, is I use VarSome, the website, and I'm sure you folks use this as well. But it will help to clarify those issues. In fact, it'll even give you synonyms. And I'll tell you, any VUS that seems like it might be relevant, I run it through that, and it's remarkable how many times you come away with that sense that you better think really hard about ignoring that VUS. And VarSome, I personally find that to be a very, very valuable site.

I always have a very difficult time with VUS because in the surveillance setting, it's pretty straightforward if you're telling people to do an MRI or not. But here, if you're talking to family members about having body parts lopped off, it gets a little anxiety-provoking, especially if you've identified it through basically population screening, unanticipated finding in the setting of tumor profiling. It's awkward.

A panel of experts share their experiences with interpreting information on somatic reports and provide recommendations for verifying results.

Experience with Interpreting Somatic Reports

 I think you're exactly right about this. And this next slide, which comes from one of the abstracts that was suggested as part of the conversation this evening speaks to just how prevalent these changes are. This was one of the studies that was presented at ASCO [American Society of Clinical Oncology], this is from the MD Anderson [The University of Texas MD Anderson Cancer Center] group looking at this issue if you did both tumor sequencing and normal sequencing. How many times would you find changes in the inherited DNA and the germline that were potentially relevant? And were those findings just confined to cancer types where you already had an indication for doing germline sequencing? And as you can see, while there certainly was a greater prevalence of alterations in the cancer types that had already existing germline testing guidelines, about 11%. There is still a meaningful prevalence of genetic alterations in the cancer types that didn't have otherwise germline sequencing indications. In other words, you're finding things essentially through population screening. It's a complicated heat map type of slide looking at the different cancer types in the rows and then the intensity of the coloring shows you just how prevalent alterations are in the genes which are listed in the columns. This is YH which is a very common alteration that's seen particularly in European populations and in my view, at least as a single alteration, probably meaningless. It causes a recessive syndrome of polyposis. But the interesting thing, as you can see, that although the 

 mutations were more common in the cancer types that you usually think of as being associated, there are fewer such alterations but still a meaningful number of such alterations in other diseases. And then the light blue background just indicates how scattered, what a long tail there is of findings in other genes. Dr Ross, with that as background, I'd like to ask you, what platform, and I'm going to ask all three of you actually, what platform do you use for tumor profiling at your institution?

 Thank you and thank you again for having me. At Northwestern, there's no mandate for the platform to use. Obviously, there's multiple next generation sequencing companies out there. But we tend to use or prefer partnership with Tempus. One of the reasons that we decided to partner with them is that, they do germline and somatic sequencing together and we found that to be a benefit. You touched on some of these concepts, also they offer RNA sequencing to help pick up fusions. And they try to do some data analytics as well that I think for some larger institutions can make a preferred arrangement also. To your point, I think that it's an important one and also to Dr Henson's point that finding an incidental germline mutation in a tumor that is not thought of as a typical hereditary cancer. It's crucial for cascade testing and I'm just a urologist, but I strongly believe that the way that we're going to cover the nation and figure out who's really at risk for, not just development of cancer, but development of lethal cancers is, that's going to be the way to do it. Sort of like, identify these index cases and then screen their family member. And so, I think the other minor point, and I'll give it away to the rest of the group is, some of my patients that have prostate cancer even when I talk about germline testing, the guy with metastatic disease, they're not super sold on doing germline testing. They do understand there's somatic testing. We understand that PARP inhibitors are approved, and they want to get into their somatic testing. They understand that doing germline testing is a way to make those somatic calls. And then when that gets flushed out and we tell them, you might have a germline mutation in the gene, we would like to confirm it with a very validated germline genetic test, then they're all for it. But it's been an interesting phenomenon I've seen in my practice. I saw that Dana was nodding so I'll let here speak to it as well.

 Dana, or Ms Clark, what's the situation at Penn? How do you do tumor profiling?

 Ours is a little more, I'll just say challenging. So, each group, as you said Dr Ross, there's no mandate on what we have to do. So, the gynecologic oncology group generally does a Caris type test where they're getting HRD [homologous recombination deficiency]. For the most part, we do have an internal center for personalized diagnostics. So, a lot of Penn Medicine providers will use that. The only problem or challenge is that it's tumor only. So, anything identified on that, the patient hasn't been consented, so there's a whole host of different things when you're dealing with tumor alone versus tumor normal. And so, every patient that we find anything, and we can talk later maybe about how we identify who is the targets of offering germline testing. But once, as Dr Ross, I totally agree with your point that it's a tougher sell. The somatic piece, I don't even know that patients actually get the option. They're told, we're going to do this test. I don't think they really understand it necessarily, but they know it's going towards their treatment so they're OK with that. My job is to get the downstream patients. When you're doing tumor only, obviously you have to confirm anything that you find. We have no inkling whether it's germline or tumor only. It is sometimes a hard sell and we looked at our group and even of patients that their oncologist talked to them about coming in and why we were calling, still a large number of them never showed up. They didn't want to be tested. They just declined germline testing for one reason or the other. So, tumor only definitely has its own challenges. A lot of providers are going with the paired. And I think the other thing that's starting to really take off is the garden testing. That's been another interesting arena in terms of germline findings. We're just starting to deal with that now.

 Certainly, I want to circle back to the circulating free DNA later on. Dr Henson, what are you guys doing?

 We, I would say, at the moment we're probably split evenly between Tempus and Caris Life Sciences. The advantage of both of those companies, obviously, that they do offer paired testing whereas there are certain circumstances come in without a history of cancer. They're worried about it because of family history. That's a different scenario from what we're talking about tonight. But probably in a large genetic center like ours, half of the cases are more- We've actually convinced most of our clinicians now to let us manage all of the genetic testing. And so, we go to either Tempus, which has sort of been our major partner or to Caris if the clinician prefers that. And we like the ability for those two companies to get the paired germline. Those are new developments, particularly for Tempus. I think a lot of the audience will recognize that Tempus now interacts with GeneDX as their germline partner. Caris interacts now with Ambry. And historically, we would have these tumor panels that would be a germline filtering program which I used in a completely different way. Not the filter but to identify germline. And I wasn't perhaps- That's not- That wasn't the intended use for that, but it was very powerful. Now that both of those groups are partnering with bona fide germline testing companies, this is just terrific. And so, we can send one requisition to either of those companies and get tumor DNA, tumor RNA, very, very powerful germline. And so, we use both of them. Tempus is the partner that I use the most.

 It's interesting, I don't use either of those for obvious reasons, because we have our own. Do you send a separate normal DNA? They don't take it off the tumor, right? You just send a separate one.

: Yeah. In fact, Tempus, for example, they get the tumor from the hospital. You send in a paired germline test which is their standard that they've been doing for a long time. And now there's an additional tube that goes in a separate box to GeneDX. So, it's an extra tube for the patient, but, it's really not that much more for them.

 The nice thing too is they'll do the paired normal, but then also if you don't get enough specimen out of your FFPE [formalin-fixed paraffin-embedded] block, you can have them reflex the circulating free DNA, for Tempus at least, off that same tube. Just like Dr Henson, when I'm seeing the patient, I'm doing exactly what he's doing. Sending a tube for the XG, which is our GeneDX. Sending one for their tumor match normal XT. And then I'm saying, if you can't get enough specimen off of that paraffin, please reflex the XF, which is their circulating free tumor DNA test. It's a nice one point of contact. I think it's made things very easy. If only it would communicate with our EMR [electronic medical record] freely, but otherwise, it's made things very easy.

 It's an interesting challenge, because the question obviously becomes, what's the gene set on the normal DNA, right?

Mark Robson, MD, reviews data from a recent abstract on incidental germline findings detected by tumor-normal matched sequencing and the panelists share which platforms they utilize for tumor profiling.

Tumor-Normal Matched Sequencing and Platforms Utilized for Tumor Profiling

: Good evening everybody. Welcome to this OncLive production, “The Evolving Role of Next Generation Sequencing and Germline Sequencing in Patients with Advanced and Metastatic Cancer.” I'm joined tonight by an excellent group of individuals. Professor John Henson, who is the director of the Hereditary Cancer Clinic at the Georgia Cancer Center in Augusta University. Dr Ashley Ross, who is associate professor of urology at Northwestern School of Medicine, the Feinberg School of Medicine in Chicago. And Dana Farengo Clark, who is a senior genetic counselor at the Abramson Cancer Center, University of Pennsylvania in Philadelphia. We're going to have an interesting conversation today about a relatively complicated area reviewing some information about germline testing and so called, incidental germline findings. And we'll come back to that in a second. We will provide you with some recent data on germline variation that's detected in the course of tumor profiling. And then wrap up with some pragmatic conversations about operational challenges and approaches to this difficulty. But what I want to start by doing is getting us on the same sheet of music when it comes to terminology because this is a complex area. We're working with DNA but we're working with DNA from 2 different sources. A lot of what we're going to focus on today is tumor sequencing, which is also known as somatic sequencing, which is the analysis of DNA from a biopsy of the malignant tissue. And of course, most of the DNA sequence that's in a tumor will be the same as the DNA sequence that the individual was born with. So called, constitutional DNA or germline DNA. The changes that occur as the tumor evolves are the kinds of alterations that we're looking for to identify therapeutic targets in particular. Also, sometimes assistance with diagnoses and the like. But most of the tumor DNA is the same as the inherited DNA. And the way you actually look directly at that inherited DNA, also called normal DNA or constitutional DNA is through germline sequencing. And this, of course, is the analysis of DNA from the normal tissue, which is usually white blood cells but increasingly now, we're doing testing on cells in saliva, which are also frequently white blood cells or epithelial cells. And sometimes you actually do direct testing of normal DNA that's dissected out from the tumor in a biopsy specimen. But most of this is either blood or saliva. A new and evolving area is sequencing of circulating free DNA. In other words, DNA that is actually in the plasma that shed from a couple of different sources. One is it's actually shed from dying tumor cells themselves into the circulation. And the other is that some proportion of circulating free DNA is normal. It's either derived from lysed white cells that shed their DNA into plasma or it occurs from normal tissue as you're doing the processing. So, ctDNA sequencing is an attempt to circulating tumor DNA but what you're actually doing is sequencing circulating free DNA, most of which is going to be tumor in a cancer setting. Other things that we're perhaps going to touch on but not in the same detail is RNA sequencing, which is the analysis of the sequence, of course, from the RNA that is transcribed from the DNA template. This is important because sometimes by doing RNA sequencing you can identify or characterize genetic alterations that have led to a particular form of damaged called nonsense-mediated decay which just means that the RNA is not working or not being transcribed which means the change that was in the DNA is meaningful. So, some laboratories are using RNA sequencing to help cure a, if you will, DNA variation as to its significance. And lastly, an important area in somatic sequencing which is not so important from the standpoint of what we're going to talk about tonight is fusion testing, where you're analyzing either DNA or RNA for particular genomic rearrangements that have created fusions between 2 genes that are driving the cancer. And this is particular issue in lung cancer, or a particular observation in lung cancer. But that's really not going to be much of what we're going to talk about. The other thing I want to touch on is 2 terms which get very, very confused. And we're going to launch into a little bit of a conversation about this because we talk about incidental findings and secondary findings. And incidental findings just technically are things that you find that are unexpected or unanticipated. And this term was first came into consciousness in radiology. You're doing a CT scan for, I don't know, lung discomfort and you find an abdominal mass or something like that. But it applies in genetics as well. When we're doing sequencing for one purpose and sometimes, we find things for another. Secondary findings are a different concept which is, when you are deliberately looking for abnormalities that were not part of the original reason for the test. And in genetics this really came up in the context of whole exome sequencing where people were doing exome sequencing for rare disease diagnosis or the like. And then certain professional societies felt that it was incumbent upon the laboratory to explicitly look for changes in particular genes that could be clinically meaningful. Secondary findings really aren’t important in the tumor space because we don't do a whole lot of exome sequencing right now. But incidental findings is a term that comes up all the time. And Dr Henson, I just would like to start the conversation because when you and I met a day or two ago, you had particular views about this idea and this term of incidental findings. And I was wondering if you could explain where you were coming from on that.

 Thanks Mark. So, the thing that worries about, or bothers me about the term incidental is it often means present but not a major part of. That's the thing that doesn't quite match up to my understanding or interest, particularly in germline findings. I think if by incidental we mean that it was unexpected, that's one thing. But often times incidental also means that it's a minor issue. And I think by the time we finish this evening, hopefully the participants will have, the audience will have a better understanding of just how significant germline actually is.

 Mark Robson, MD, and a panel of experts discuss key terminology and definitions related to tumor and germline sequencing.

Dana Farengo Clark, MS, LCGC, Penn Medicine

Articles

Operational Processes and Challenges

Role of ctDNA in Germline Testing

Identification of Patients for Germline Testing

Experience with Interpreting Somatic Reports

Tumor-Normal Matched Sequencing and Platforms Utilized for Tumor Profiling

Defining Germline Sequencing and Incidental Germline Findings