Hello, everyone. Welcome from the University of Iowa. My name is Dr. John Burnett. I'm a clinical geneticist and clinical molecular geneticist, and I'm welcoming you to this month's ACMG Genomics Case Conference. Title of the conference today is, RNA Genetic Testing in Hereditary Cancer Improves Variant Classification in Patient Management. This is a disclaimer from the ACMG. It states that this activity is intended for educational purposes only. I'm fortunate today to be joined by four presenters that we'll be conferencing in. I have Dr. Huma Rana, who is a geneticist at Dana-Farber Cancer Institute, and then three genetic counselors, Sarah Neshed from Rutgers Cancer Institute, Deborah Collins from University of Kansas Cancer Center, and John Lee from Cedars-Sinai Medical Center. These are our disclosures. I receive research support from Edorsia, Pfizer, Protalyx, Sanofi Genzyme, and Takeda, and serve on advisory boards with Sanofi Genzyme and Takeda. None of these are relevant to the content of today's talk. Dr. Rana receives an honoraria from Ambre Genetics. Mr. Lee receives an honoraria from Envitae, and then Ms. Neshed and Ms. Collins have nothing to disclose. And then these are the disclosures of our planning committee. I'm also the vice chair of the ACMG Education Committee, and then this is our staff who have nothing to disclose for today's conference. Educational credits are available. When you sign up for the conference, this session is approved for one CME. For those who paid for a CME, you will receive an email with evaluation questions and a Certificate of Completion at the end of this session. These are the overall learning objectives for the Edgenomics Case Conference for the entire series, and then we also have some specific objectives for today's conference. We are looking to review the basics of RNA genetic testing and its role in variant detection and classification. We'll be discussing specific case examples of variant resolution with RNA genetic testing, and we'll identify examples of how RNA genetic testing can impact patient management. For those of you on the live conference, this is an interactive session. If you'd like to ask a question, you can type your question into the Q&A box at the top right corner of your screen, and we'll be answering all of the questions verbally at the end of the presentation. So, I'll be starting off with a brief introduction on RNA genetic testing, and I need to take a second to acknowledge the work of Dr. Rashid Karim. Dr. Karim is the Director of the Ambre Translational Genomics Lab, and we'll be – and he actually was – his lab was involved in testing all four of the patient cases that will be presented today. Dr. Karim wasn't able to present with us today, and so I'll be doing this introductory material and moderating today's session. So, this slide just represents the timeline of advances in genetic testing, in this case particularly for the hereditary cancer syndromes. Once a gene is identified, Sanger sequencing was available to identify pathogenic variants, and then with deletion-duplication analysis through various methods, we were able to identify additional pathogenic variants that weren't able to be elucidated by Sanger sequencing. With the advent of clinical next-generation sequencing and with additional genes identified for different syndromes, cancer panels could be built and allow for efficient testing of a number of genes. However, as you test more genes, more variants will be identified, and a number of variants of uncertain significance could be found, and RNA genetic testing is one way to help characterize some of these variants of uncertain significance. So, this complex slide is just to illustrate that RNA splicing is quite complex. You can see that there are a number of cis-acting and trans-acting elements that play a key role in splicing of the messenger RNA, and I won't go into any detail of the particular machinery, but I'll call your attention to the bottom of the slide that represents alternative splicing. And this can take place in a number of different forms. For example, exon skipping is characterized by an exon that can be included in some transcripts, but not others. The use of alternative splice sites, either the 5-prime or 3-prime ends of exons, allow for additional material to be included. Mutually exclusive exons allow for some exons to be included in some transcripts, but not others. And then, intron retention allows for a smaller intron to be included, provided that it remains in the same reading frame. Now, with all of these, the question is, what effect does it have on the actual gene product? So, in many cases with alternative splicing, a premature stop codon can be included that will give a premature end to that particular transcript, and then it goes to the nonsense-mediated decay pathway. Inclusion of additional material can also result in a new functional domain, so a new function for that particular transcript. So, this slide just characterizes a few different RNA sequencing strategies, and again, I won't go through this in great detail, but a few key points I'll point out here. If you do total RNA sequencing, so the equivalent of whole genome sequencing, you're sequencing all RNA transcripts in a particular tissue or sample. This will result in a lot of common RNAs being well overrepresented. So, for example, over 95 percent of transcripts in humans oftentimes represent ribosomal RNA, and so you'll sequence quite a bit of this. You can do steps to reduce the amount of ribosomal RNA that you sequence, but you still may not have your transcript of interest in sufficient representation. So, there are other techniques, such as polyselection, which allows you to select just for mature RNAs, as well as cDNA capture, which then allows you to better select the transcript of interest. But then, of course, you will only find the transcript that you are selecting for in the capture method. So, all of those have advantages and disadvantages. Now, in terms of the utility of RNA sequencing in disease, there's a number of different roles that it can play, but I'm going to focus today on the last two, variant discovery and alternative splicing analysis. So, starting off with an example of variant discovery, to better characterize tandem duplications that are found. And this is a publication from Dr. Karam's lab that looked at pathogenic MSH2 tandem duplications in Lynch syndrome patients. So, most of the time, if a sample is sent for – if a genomic sample is sent for testing and an intergenic duplication is found, these will be characterized as variants of uncertain significance, because it's not known what effect that will have on the particular transcript. So, this study looked – did RNA sequencing to identify how the duplication was assembled and what effect it actually had on the particular transcript. And you can see on the left, there are five different cases that were examined here. The breakpoints were identified, and then they were able to actually identify what the gene product would be. In each of these five cases, this resulted in an out-of-frame change and, therefore, premature degradation of the product. And so, for all of these five cases, the VUS was able to be upgraded to likely pathogenic. I'm now looking at alternative splicing analysis to – and how it affects variant classification. So, I think most of you will be familiar with the five levels of variant classification, from a pathogenic mutation or pathogenic variant, all the way down to benign. Pathogenic meaning that there is enough evidence to show that – to substantiate that this particular variant can cause a disorder. Benign meaning that it's not going to cause a disorder. But then we also have some in-between classifications, likely pathogenic, likely benign, and a variant of unknown significance. And with the VUF, we don't have enough evidence to really call it either way, if it's going to be likely pathogenic or likely benign. This next slide shows some data that was provided by Ambre Genetics. In this case, looking at 307,000 patients that were tested for cancer predisposition syndrome. And of those patients, 51,828 were identified to have a variant of uncertain significance. Of the VUSs that were identified, 11% of them were thought to possibly affect the RNA sequence, so possibly affect splicing in some way. And of these particular RNA variants – or potentially – potential RNA variants, some of these were at the splice site, some of these affected the 5' UTR. Looking at the same data for variants – likely pathogenic variants, you can see that the majority of these, 55%, were thought – suspected to potentially affect RNA splicing. And the majority of these variants were at the splice site itself. So, to further characterize these variants, Dr. Karam's lab developed a technique called CloneSeq. And this solves the representation problem by doing a targeted RT-PCR for gene products of interest to better characterize the particular variant that they're wanting to look at further. In this case, RT-PCR for a variety of possible products is performed, and then these products are cloned into plasmids and libraries are created. From this step, then, massively parallel sequencing is performed, and then this goes through a bioinformatics pipeline to determine what the effect is on the gene product. Any particularly interesting variants can then be Sanger-confirmed as well. So, one example of this is a particular family with a BRCA1 variant. This is actually going to be one of the cases presented later. This is at the plus-5 position, so a neutronic variant, five nucleotides from the splice site. And what you see at the top right here is the results of just total RNA sequencing. And this is what's called a sashimi plot, so looking at the particular reads for each exon that is sequenced. And you can see that the numbers that are characterized here are quite low, so reads between 10 and 20 counts, so quite low and difficult to determine what exactly is going on. But by doing clone-seq, the number of sequence reads increases quite substantially, so now we have thousands of reads to tens of thousands of reads, and you can actually show that there is alternative splicing in patients containing this particular mutation, and you don't have the alternative splice product in patients without the variant. So, with this testing, Dr. Karam's lab looked specifically at three different cancer predisposition syndromes, Lynch syndrome, a hereditary diffuse gastric cancer, and then hereditary breast and ovarian cancer. And these pie graphs just show the different genes represented in the particular variants and cases that were examined here. The bar graph shows the outcome of RNA sequencing in 64 of these cases. The colors on the bars actually show the different classifications of variants and then how this changed before and after RNA sequencing. So for the 64 variants in total, the majority of these were variants of uncertain significance, and then a fraction of these, 12.5 percent, were likely pathogenic variants. After RNA sequencing, you can see a number of these were able to be upgraded in classification. So, 6 percent became pathogenic, 43 percent likely pathogenic, 14 percent remained variants of uncertain significance, and then 36 percent were able to be downgraded to likely benign variants. Looking a little bit more closely at just the likely pathogenic variants, the next two bars show that of the eight likely pathogenic variants, four of these were upgraded to pathogenic, two remained likely pathogenic, and two were downgraded to variants of uncertain significance. Of the VUSs, of which there were 56, about half of them were able to be upgraded to likely pathogenic, and then 41 of them were able to be downgraded to likely benign, while a small number remained unresolved. And then the further bars show how these break out for the different disease types. Now, what impact did this change in classification have in patient management? So, looking at the first bar on the left here, this shows a variant or patient cases where the variant was upgraded from a variant of uncertain significance to likely pathogenic or pathogenic. In this case, eight out of the 10 cases resulted in an increase in patient management based on this upgrade, whereas in two cases, there was no change to patient management. For downgrades from a variant of uncertain significance to likely benign, this only decreased the management in one case out of eight. And this, you might surmise, is because the patients themselves have a cancer, and they're still going to be managed based on recommendations for that particular cancer and not for this variant that we now think is not probably causative of their cancer. For other family members, though, who are not symptomatic, for an upgrade from a variant of uncertain significance to likely pathogenic, this resulted, again, in an increase in management for most of these patients, whereas a downgrade from a VUF to likely benign resulted in a decrease in management for more of these patients, for more of these family members who were asymptomatic. So, with that, we're going to focus more closely on four cases of where RNA genetic testing was performed in cancer predisposition syndromes, and we'll see how this changed the classification of these particular variants and effects it had on the particular patients and their families. And with this, I'll turn this over to Dr. Rana for case number one, a case regarding Lynch syndrome. Thank you. Thanks for that overview. So, I'll tell you about a patient who I saw in the clinic where the technology was incredibly helpful in informing care for her. So, the consultant was a 54-year-old woman at the time that I saw her. She had a history of basal cell carcinoma, which was diagnosed at age 47, and she had a history of a colorectal adenoma that was found on her first screening colonoscopy at age 51. Other than that, she had no personal history of cancer. She did have a sister with endometrial cancer diagnosed at age 54. The endometrial cancer had identified loss of MSH6 on immunohistochemistry, and the sister had a variant of uncertain significance or unknown significance in MSH6. As you can see, this was a deletion of four intronic nucleotides. So, the consultant came to see myself and my genetic counselor, Sam Culver, and her unaffected 55-year-old sister also came to see us later as well. She underwent genetic testing, and the consultant, we'll call her S, S was found to carry the same variant of unknown significance. If we look at the family history, we can see that there are some Lynch syndrome-associated malignancies in the family, but this certainly is not a classic Lynch syndrome family. Her father had died of lung cancer at age 70. There was a paternal uncle who had thyroid cancer at 40, non-Hodgkin's lymphoma, colorectal cancer, and a kidney cancer. The colorectal cancer and kidney cancer were synchronous at age 75, so relatively late age of onset for classic Lynch syndrome. There was an aunt with an osteosarcoma, another aunt with a sex cord stromal tumor, and then a paternal aunt with an endometrial carcino-sarcoma, also at a later age than is typical of classic Lynch syndrome, so at age 60. Furthermore, the histology of this type of endometrial cancer is not what's typically associated with Lynch syndrome. As we know, it's endometrioid-type endometrial cancers that are associated with Lynch syndrome. The consultant, S, wanted to know what to do with all of this information, and so we had to come up with a plan for her. The first step for me is always whether or not this family meets clinical criteria for Lynch syndrome. We looked at Amsterdam 2 criteria, and there are three or more Lynch syndrome or Lynch-associated cancers in the family, two of whom are in first-degree relatives. That's in the sib ship in her father's family. There was a paternal grandfather with stomach cancer, so the colon, the RCC, and the stomach get you there. If you don't want to count this odd histology endometrial cancer, it was two or more generations, but there was not a person affected by age 50. For those of you who see a lot of Lynch syndrome, I think you know that with MSH6 and PMS2, though, that these criteria are not always met, and, in fact, they're as often met as they're not met in families with attenuated Lynch syndrome. Our Lynch syndrome assessment was really that there's not a clinical diagnosis, but that's not completely unusual in MSH6. What gave us some pause was that there was this immunohistochemistry in the sibling of the consultant, and immunohistochemistry showed loss of MSH6. That could be reflective of a defect in translation suggesting aberrant protein function, or it could be double somatic hits, which we now know are not uncommon when you have mismatch repair or loss of expression. Overall, this clinical picture gave us some pause. In terms of the molecular results, this is a variant of unknown significance at the time, and it wasn't terribly helpful to have this result. We were unclear on what this meant and whether we would have any real true negatives if we tested for the variant, although we ended up doing panel tests on the consultant and her sister because of the striking family history. Prior to the RNA study, the management questions, which were many, are delineated as follows. If this is Lynch syndrome, we would recommend colonoscopies every one to two years. If this is not Lynch syndrome, then this would be pretty invasive. While one could argue that based on her own history of a colorectal adenoma, she should be screened more frequently, I don't know that any of us would necessarily be comfortable with every one to two years, as is the NCCN recommendation for Lynch syndrome. Upper endoscopy, screened for gastric cancers, there's not really great evidence supporting the efficacy in Lynch syndrome. In this case, yes, no, maybe you could go either way on this. There was the paternal grandfather with a stomach cancer, but I think that without a Lynch syndrome diagnosis, this is sort of, again, an invasive procedure. Transvaginal ultrasounds and endometrial biopsies can be done, but we know that their sensitivity is not perfect, and for individuals who have increased risk for female genital cancers and endometrial cancers related to a genetic predisposition like Lynch syndrome, this leaves much to be desired in a preventive hysterectomy and salpingo-oophorectomy, and really the standard of care, which was discussed with the patient. However, she was not interested in undergoing a surgery of this nature without a definitive molecular diagnosis, and I think that's fair, although the counterargument could be that she had an aunt with an endometrial carcinosarcoma and a sister with an endometrioid-type uterine or endometrial cancer. She was already having skin exams every year due to her history of basal cell carcinoma. Urine cytology, we sometimes do in Lynch syndrome when there's a family history of urothelial cancers, and that paternal uncle did have a RCC, but there's potential harm, and that's in everyone because we know that individuals who have this type of screening have more cystoscopies. There were no small bowel cancers, so we wouldn't have recommended capsulendoscopy or MRCP anyway in aspirin as part of the standard of care for Lynch syndrome, but as you can see, there was a lot of, at least on my part when I was making the medical management recommendations in this case, there was really a lot of hemming and hawing, and we could do this or we could do that, but I wasn't really sure what the right thing to do was. So we ended up consenting. The consultant asked, as well as her unaffected sister, A, who had both seen me in clinic, for RNA genetic testing, or RGT, to clarify the familial MSH6 variant. And it was sort of shocking in the sense that RGT data led to the rapid reclassification of this variant as a variant likely pathogenic. And then based on this new classification, the diagnosis of Lynch syndrome was made. And we then recommended that we adhere to NCCN guidelines for screening and risk reduction. I must say that the consultant and her sister were both actually very happy with the turn. I mean, it was a shocking turnaround time. I mean, you can imagine if someone asked you to give blood for a research study, you don't necessarily expect that there would be results in a change or shift in a recommendation so quickly. So this was the consultant, and so she was positive. Her sister was also positive. And the affected sister with endometrial cancer obviously had this VUS, which was now variant likely pathogenic, as did this uncle and this aunt, who were able to be tested. And so this is now a family with Lynch syndrome. And post-RNA management recommendations were far more definitive on my part, or at least as definitive as one can be about MSH6. So colonoscopies every one to two years, that was unequivocal. Upper endoscopy, yes, we should do it every two to three years. Transvaginal ultrasounds and endometrial biopsies, yes. But we really should move to a risk-reducing THBSO as quickly as possible. And then we added the urine cytology based on the family history. And so in summary, we were able to make more clear evidence-based management decisions. We were able to definitively recommend surgical risk reduction and inform the frequency of colonoscopies, as well as when to initiate surveillance in the unaffected family members. And I think that's incredibly important because this, I'll just go back, what this meant was that all of these people could now have predictive genetic testing, so the nephew, her two daughters, to inform their care. And then one could also identify true negatives, which I think is so powerful in the past. When we've had loss of immunohistochemistry or been concerned about Lynch syndrome, we would call it Lynch syndrome NOS and follow the whole family in perpetuity, not knowing who actually was at risk and who wasn't. So this allowed us to differentiate those people. And then a definitive diagnosis could be provided for the extended relatives, so those who have active malignancies. And as we know, there's potential therapeutic impact for those individuals, and they may be candidates for immunotherapy. And so this was a case that I think really highlighted the power of RGT, and it was incredibly rewarding for all of us involved. Thank you. And I'm going to pass the baton to Sarah Nishad from Rutgers. Thank you. So I'm going to go over case number two. The patient is a 44-year-old female diagnosed with a papillary serous carcinoma of the right fallopian tube in both ovaries. She had already had a hysterectomy and was undergoing chemotherapy when she was referred to genetic counseling. And when I had met with her, other cancer screenings she was doing was annual mammograms and a clinical breast exam, nothing else. This is her family history. So on the mother's side, it's really unremarkable. Mom was 67, and the only thing on that side was an uncle with esophageal cancer at 70. But on the paternal side, dad had a squamous cell at 72, but looking further, his mom had an early onset breast cancer at 33, and two of her sisters also had breast cancer. So there is some distant history. So after her consultation, the patient elected to undergo a 24-gene hereditary ovarian cancer panel, and she was found to have a BRCA1 VUS as well as an ATM VUS. So with the VUS, there was nothing different we could tell the patient to do just to continue with her planned care, and we didn't have any further recommendations. Her unaffected 38-year-old sister was with her during the initial appointment, and she was quite concerned about her own risks and whether she needed to have her ovaries removed prophylactically. With these results, we weren't able to help her any further. We discussed just getting screened according to personal risk factors and family history of cancer based on what her physicians were recommending. We didn't recommend genetic testing for the VUS, but because they were so interested in figuring this out, their family was submitted for family studies at the lab, and they were accepted to do family studies as well as RNA splicing analysis. So the parents as well as the sister did consent to have their blood tested, and through the RNA analysis, it revealed an abnormal transcript that led to the deletion of the first BRCT functional domain in BRCA1, and this led to the reclassification of the VUS to likely pathogenic. The father was identified to be the pathogenic BRCA1 carrier, and mom and the sister were true negatives. So how did their medical management change? The patient opted for high-risk breast screening in addition to her current treatment, and we discussed that later on she can consider pancreatic cancer surveillance studies and full-body skin exams, but that was really put on the back burner. And for the father, we had discussed considering prostate cancer screening and also recommending monthly self-breast exam as well as an annual breast exam by a physician, and we also talked to him about pancreatic surveillance studies as well as a full-body skin exam. And now the mom and the sister could now follow general population screening guidelines, and the anxiety of the sister, you know, it dramatically decreased for obvious reasons. And it was very – these studies were very helpful in his family, and now extended family members can also be tested. So now I'll give it over to our next presenter, who's Deborah Collins. I'm a genetic counselor at KU Medical Center, and I'm presenting the case that we have followed for the last seven years. The patient initially came to us as a consult when she was 21 years old. Actually, she was in the hospital. She had come in three months – to the hospital about three months earlier. She had weight loss, fatigue, some GERD, abdominal pain, and she was diagnosed with diffuse gastric cancer. On pathology, it had signet ring features, which is a feature of diffuse gastric – or – she also had metastases, you know, peritoneum, omentum – IHC had been done, and there was no evidence of Lynch. And at that time – this was in 2012 – we arranged to have the CDH1 testing done. She died a few months later. The family history involved three siblings that had a lot of gastric issues, as well as her mother. And her mother was estranged from her father, so we didn't have any family history on that side of the family. Diffuse gastric cancer was an issue. We were trying to figure out if she had the gene, because the VUS – I'm sorry, let me go back at the top – she did have the VUS in the CDH1 gene that was found. And we were trying to decide what to do with the rest of the family, because of the higher risk for diffuse gastric cancer. Her mother was 41 years old. She had these three siblings that had gastric issues. There's also the risk for lobular breast cancer in her mother. She was 41 years old, so she was kind of in that range. There was a lot of evaluations that you might want to do on this family, but we had a VUS. Mother then came in and said that with her stomach issues, we went ahead and did an endoscopy and colonoscopy on her when she was 41. The concerns was how far to go with this family with EGD or the H. pylori infection to see if she had that, because that can cause hypermethylation of the CDH1 gene. So that was another concern, and how many people we needed to do this kind of evaluations on, as well as the lobular breast cancer and the recommendations for that. She had a lot of gastric polyps, a variety of types. And because of that, we then decided to go ahead and do VUS testing, which we often don't do on families. But we did it on her to try to figure out if it was coming from her side of the family, because of all these clinical findings in the family. And she did have the VUS that her daughter had. So at that point, she was followed for another three or four years every few months with EGD and colonoscopy. And at age 44, so the summer before last, she had these other symptoms with her appetite and pain and things like that. And she was diagnosed with gastric cancer at age 44. At the same time, the siblings were being followed with various kinds of evaluations for the gastric problems. And we also then did her father, the grandfather, who had the variant as well, trying to find out which side of the family it was coming on. During that time frame, she got in touch with him and found out that her grandmother had ovarian cancer. So as you probably know, back in that time, it was a little easier to get the genetic testing done based on the ovarian cancer, but also the polyps that she had. The mother, that's my 46-year-old woman on there in the middle, she had a 29-gene panel and it found the variant. Then she had a total gastrectomy at that time. And the pathology on it showed the diffuse gastric cancer, signet ring carcinoma. At that time, all we had was VUS on all of these individuals. We updated the family history and we sent the pathology reports to Ambry, who then, a year later, we got the reclassification that it was the CDH1 gene. It was likely pathogenic. I didn't realize that the RNA studies were part of what was done at that time, but that really helped us with the family then because now that we had the pathogenic gene, we could test her sister. It took a few months to get all that done with insurance and so on, but the sister was 26 years old at that time. Her sister had died at 21 and she was pregnant at that time, so that complicated things a little bit. We did get the upper endoscopy done during the pregnancy, which was okay, and they did biopsies and she was evaluated for H. pylori as well. Her brother was 19 and was tested. The sister has actually been tested at 17 and she's negative, but then more family history was found. The paternal grandfather had nine siblings and we found that four of them had stomach cancer and three had breast cancer. Generally, for the management guidelines, the prophylactic gastrectomy is what's recommended for people and we were obviously with a VUS hesitant to recommend that on individuals early on several years ago, but now we can follow the guidelines to start doing upper endoscopies on the family, colonoscopies occasionally, and for the females, do breast exams starting at a younger age with 3D mammograms, possibly the MRIs, and hopefully pick up any kind of cancer at a very early age. The family is a little messier than it looked from those first few slides. There's a lot of liaisons and half-siblings and alternative people that we have trouble reaching. There might be a half-brother here and there, so we're still now pursuing other people in the family that might benefit from the testing. What's changed for us for management is the testing of all the siblings is possible. We have now three children that are under age 10 that when they get a little older, we'll be testing them. Generally, we test children 10 years younger than the youngest affected person, but given that this young woman was diagnosed at 21, we'll probably be testing them around 16 to see if they have the gene and to start any kind of screening on them. It's been a long saga with this family, but by having that gene reclassified that seemed to fit the clinical findings, it's been very valuable to us. I'll turn it over to John Lee. Hi, everyone. I'm John Lee. I'm a genetic counselor at Cedars-Sinai. I wanted to present a case about the other possibility with the U.S. reclassifications, basically a reclassification from a variant of uncertain significance to a variant likely benign. This is certainly very important, similar to the upgrades. Even though I think most of us understand that most variants of uncertain significance will be downgraded to likely benign, they still certainly present a challenging clinical situation. While they're still classified as variants of uncertain significance, as the other presenters have alluded to, it can make it difficult in terms of knowing exactly what to recommend for families. The case I have in question, a 51-year-old unaffected woman of Ashkenazi Jewish ancestry presented to the clinic. She presented due to family history, primarily of a paternal grandmother who had early onset breast cancer, diagnosed at 45. It was noted that her father also had colon cancer at 61, and on her mother's side of the family, a maternal uncle with a head and neck cancer at 68, and a maternal grandfather with prostate cancer at 84. Her parents, at that point in time, were not willing to do genetic testing. We did agree to test an unaffected patient. It certainly presents challenges of its own in terms of interpretation of results. She had opted to undergo CancerNext, which is a pan-cancer panel at that point, a 34-gene panel. She was not found to have any pathogenic mutations. She was found to have a variant of uncertain significance in MSH6. Similar to the case that Dr. Rana had presented earlier, the family history was not particularly striking for Lynch syndrome. Again, the caveat is being MSH6, a little bit less penetrant than MLH1 and MSH2. At that point, I made recommendations based on her family history. Essentially, she had a first-degree relative with colon cancer. Per NCCN, we had recommended colonoscopies every five years. Being 51 and with her family history, she was a very motivated patient. She had already had a colonoscopy through an outside gastroenterologist and reported that she had a history of no polyps. Certainly, again, this family history did not meet Amsterdam criteria or anything else suspicious, so didn't feel the need to recommend anything further at that point. Ambre genetics then offered to do family studies for the family at first, so we tested both parents. It turns out that the father did not carry the MSH6 VUS, while the mother did carry the MSH6 VUS. Therefore, it was concluded that it did not segregate with colon cancer in this particular family. Then, at a later point in time, Ambre genetics offered RNA genetic testing for the family, and we sent in samples. They had determined that RNA splicing analysis had found that no abnormal transcripts were identified, and this ultimately led them to downgrade the variant of uncertain significance in MSH6 to a variant likely benign. The impact on the clinical care here, while certainly not as necessarily drastic as some of the upgrades that you saw from the previous cases, I would argue is still very important. As Dr. Rana had mentioned, again, for Lynch syndrome carriers, females, we typically recommend prophylactic hysterectomy, total hysterectomy, and BSO. In this particular case, we could reassure her that that was not necessary. Again, the MSH6 finding was essentially negative at that point, and there was nothing else within the family history that was suggestive of her needing to have surgical intervention. More frequent colonoscopies are not indicated, so we had recommended that she stick with the five-year intervals or is based upon polyp burden, as it was similar with when it was still the uncertain results. And then she also has three children, and this was good news for them in that they did not need to do genetic testing. Certainly, of course, have to take paternal history for them, but there was nothing concerning on that side, so we could rule out the possibility of Lynch syndrome coming from this patient's side of the family. And I'll pass it back to Dr. Burnett for concluding remarks. All right, so thanks, everyone, for your input on these four very interesting cases. I hope that all of the webinar attendees were able to see different ways in which RNA genetic testing was able to better characterize these variants and provide more information and management recommendations for the affected individuals as well as their family members. Again, I just want to recognize Dr. Karam from Embry Translational Genomics Lab. It was his laboratory in which this testing was performed. And I think at this point... Let's see. My slides are going the other way. Here we go. We're at the Q&A session, so we'd like to ask...if you'd like to ask a question of any of the panelists, just please type your question into the Q&A box in the top right corner of your screen, and we will answer those questions as they come in. We have one question already that asks, which labs offer clinical RNA genetic testing? Of course, we're not trying to promote any lab over any other here. All of these tests were performed with their clinical testing with Dr. Karam's lab. Certainly, if I tried to make a list, I would leave some labs out, and so I don't want to do that at the moment. But I will say there's certainly a growing number, and I'd encourage anyone who's sending genetic testing, whether it's for a cancer predisposition syndrome or any other condition, to talk with the lab what options are available should they get a VUF. The lab may have research that they are doing on certain VUFs or offer additional clinical testing for that. So, certainly something to look into. I have another question that says, what if the gene of interest is not expressed in the blood? Does RNA genetic testing have to be performed in another tissue? So, yes, that is a very good point to make with RNA testing that, of course, you need RNA that's being expressed in a particular tissue that you are going to be testing. I think with next-generation sequencing being very quite sensitive to even low-level transcripts, if you're enriching for a particular variant, you may be able to pick up some transcripts from that. Of course, if it's an affected individual who's had a resection of a cancer, for example, there may be a tumor tissue that's available. Even though for all of these cancer predisposition syndromes, the other thing to keep in mind is that we are talking about genomic variants and not necessarily somatic changes. So, you may still be able to pick up those particular changes in whatever tissue you're looking at. Let's see. Other questions. How do you choose a patient with a VUS for further investigation by RNA sequencing? So, I think this is a matter of talking to the particular testing laboratory to see if they have research studies or additional clinical studies that are available to further characterize those particular mutations. It may also be, depending on your disease of interest, that there may be a different research group outside of the testing laboratory that is doing further research in variants of that particular gene of interest. Let's see. A question about, can you clarify what the RNA transcripts were for the abnormal MSH6 and CDH1 cases? So, that actually goes back to our different presenters. Why don't we start with Dr. Rana. I don't know if you can give us any more information about the MSH6 case there. I actually don't have that on me. I'm sorry. Okay. How about Deborah with the CDH1 case? Any more information? Deborah, are you still on the line here? Yes. I don't have the information on the transcript. It's probably possibly in the test report. I was going to say, though, about the previous question. For me, if the clinical findings really fit the VUS, that's when I pursue it in greater depth, particularly if there's more than one person in the family that seems to fit clinically the VUS. All right. Thank you. And then how about John Lee? Any more information about your MSH? Unfortunately, so much of the other two presenters, I don't have more information about the transcripts, but I would echo what Deborah said in terms of, again, pursuing further, certainly, I think, asking the laboratory. When we have higher clinical suspicion based on the phenotype in the family, that's typically what we would do to try to exhaust all options that are available, RNA or otherwise. Okay. And another question is, is the result of RNA analysis always obtainable or interpretable? So, obtainable, I think, again, goes to finding the testing laboratory that's able to do that analysis. And then interpretable, of course, in genetics, nothing is always an absolute, and so there may certainly be cases that are difficult to interpret even with the additional information. I think one of the introductory slides I had showed that of VUSs that were tested with RNA genetic testing, there were still about 10% or so that remained a VUS even after the additional analysis there. Okay. We'll just wait a couple more minutes for any further questions here. and show them the VUS and ask them how they would interpret it. And they've been pretty helpful in that respect. Our challenge is when there's discordant between the labs, of course. But I've had one situation similarly with actually a CDH1 where it was a likely pathogenic, and I was a little concerned about insurance and doing surgery on someone that was a likely pathogenic. And then other labs had classified it as pathogenic. It wasn't with RNA, but that's another challenge for us sometimes where sometimes we send samples or family members have sent samples to different labs and they've gotten different results. All right. I think there's a question here that would best go to Sarah Nishad. The question is asking about research protocols for pancreatic cancer screening. And I think this is probably with the HVOC guidelines. Sarah, could you address that at all? Yeah. So there's a few universities around us who are conducting studies on what's the best way to screen for pancreatic cancer. So I typically just give information on those studies to the patient. I don't have like specific inclusion or exclusion criteria, but they oftentimes take BRCA carriers or AM carriers. So there's different criteria for each of those studies. But I would, if that's of interest, I would see what's located near your location at different universities. I can add to that. This is Huma Rana. There's the CAPS5 study is one of the large pancreatic cancer screening studies that's ongoing. And it's, there are sites sort of across the country and it's information about it. And the eligibility is on clinicaltrials.gov. And it's CAPS5. And here really it's anyone with any mutation in any gene that's been associated with pancreatic cancer. If they have a mutation plus a family history of pancreatic cancer in a first or second degree relative, they're likely eligible. And we would begin screening at 50 or 10 years before the first pancreatic cancer diagnosis in the family, whichever is first. And so I think that, as Sarah said, there are many, many groups studying this. CAPS5 is just one of the studies that I'm aware of. And it is a multi-site trial and it's led by investigators at Hopkins. Great. Thank you. So a question here, is there any guideline to determine which level was affected? RNA, protein, or cellular level? Does damaged RNA always lead to a deleterious phenotype? So I think this question is asking about functional effects of a particular variant that's detected. And, of course, there are certainly bioinformatics tools that can allow you to try to predict what an effect might be. But then I think at the end of the day you're looking at some sort of a functional study. And this particular testing, the RNA testing, allows you to at least see if that particular variant on the DNA level could have an effect on the transcript level. Further studies, depending on the particular gene of interest or disease type, could help to further characterize the gene product, if there's going to be an abnormal gene product made, and so on. So at the end of the day, functional evidence is going to be our strongest evidence here. Okay. Any final questions? All right. Then I think with that we will close. Just have a few closing slides here. So a reminder that the ACMG also has a few other educational events going on this summer. The 2019 Genetics and Genomics Review course will take place June 27-30. And then there's also the Alternative Pathway in Laboratory Genetics and Genomics for people eligible for that pathway. That course will take place immediately after the Genetics and Genomics Review course. Our Genomics Case Conferences will actually break for the summer. We resume in August. But if you're still looking for some educational events, the Undiagnosed Diseases Network hosts a Grand Rounds. And anyone is welcome to join for this. The next Case Conference will take place June 13 at 1 o'clock Eastern Time, and there is a link here that you can sign up for that. And that is it. A recording of the seminar will be available at the ACMG Genetics Academy, so you can look online for that. I'd like to thank all of my co-presenters for their assistance with today's seminar, and thank you for joining us.

RNA Genetic Testing

Hereditary Cancer

Variant Classification

Patient Management

University of Iowa

Dr. John Burnett

Dr. Huma Rana

Genetic Counselors

RNA Genetic Testing Impact