Hello, welcome to the Florida Society of Pathologists Precision Medicine Academy lecture series. Today the topic is hereditary genetic changes and somatic mutations in precision oncology. I am Dr. Marilyn Bui. I serve as the president of Florida Society of Pathologists. So this is a CME earning activity. Physicians can claim one AMA PRA Category 1 credit. This CME is provided by FSP in conjunction with American Academy of CME, Inc. So this program is supported by educational grants from AstraZeneca and Dianqi Senko. Next slide please. So today it's a very historical moment. This marks the very first time FSP is utilizing this brand new learning platform called FSP Learning Center. So this is a significant milestone in the history of FSP education. So everybody has registered online. If you're an FSP member, you use your login, and if you're non-FSP, you create your login. So today, this lecture is the number two of the lecture series. This academy is intended for educational enrichment. The curriculum is specifically designed for precision medicine, including topics of molecular diagnostics, genomic medicine, bioinformatics, digital pathology, AI, and personalized treatment strategy. And also, we will facilitate multidisciplinary communication and collaboration. So this academy not only shows the FSP leadership in education on the specific topics, it also provides professional development opportunities for our faculty and trainees. At the same time, create additional CMEs in enhancing the benefits for the members. Next slide please. So just one brief word of this Learning Center. So today, you're using this platform to attend this live lectures. And this lecture will be available a week later as a recorded lecture. If you're an FSP member, you can go back and access this for free. And you can see, this also hosts other educational activities, including our annual meetings. So without the further ado, let's move to the next slide, introducing our faculty. Today, we have Dr. Terry Boyle and Dr. Christine Wackel. Dr. Boyle is an associate member in the Pathology and Thoracic Oncology Departments of Moffitt Cancer Center. Her expertise is molecular genetics. And she has been really instrumental in developing Moffitt STAR, that's a Molecular Testing for Solid Tumor Program. And also she's instrumental in quality control of many of our products. She's the lead pathologist for several clinical trials and also a medical advisor to the Personalized Medicine Group, which interprets genetic results for direct care of patients. Her research is focusing on lung cancer. Dr. Christine Wackel is a senior member of the Pathology and the program leader for Precision Medicine. And also she is on the Precision Medicine Clinical Service. Her background is a PharmD. She has fellowship training in translational research in precision medicine. She's also a co-chair of the American Society of Clinical Oncology's targeted agent and profiling utilization register and the Molecular Tumor Board. And she's also the Southwest Oncology Group precision medicine trial experts. So with that introduction, you can see more detailed information on their Moffitt website. So next slide, please. So we're going to have some pre-lecture polling questions. Dr. Kim is going to run those questions for us. So the first one is what is the recommended action if a somatic test identify an incidental finding of a potential germline mutation? You choose single best choice. Second question, how can loss of heterozygosity scores inform clinical decisions in ovarian cancer? The third question, RNA-based NGS testing is more sensitive at determining what type of genomic alteration. All right, so please enter your vote and let's move on to the lecture. We'll give everyone about three more seconds to respond. All right, let's move on. Thank you, Dr. Bui for that introduction. I am really, really excited to be here with you and Dr. Boyle to begin our discussion today where we're gonna delve a little bit into germline variants, contrasting them with somatic variants, but hopefully give you a comfort level of what to do whenever we see these. We're also gonna talk through kind of how we approach it. And we really look forward to your questions as we go through all of this. And we will have a patient case to take us through. So just to set the stage, again, what we're going to be delving into are comparing germline versus somatic variants. We know that cancers have started out as healthy cells that acquire genetic variation over time. So they carry with them the germline variants. And we can often see these bleed into the results whenever we are looking for somatic variants. But just to remind all of you, our germline variants are inherited genetic variation. These are the ones that are present in every cell in our body, including the egg and the sperm cells. They can increase susceptibility to certain cancers. And that's one of the big reasons why we look for these alterations. Very common examples are BRCA1 and BRCA2 being associated with breast cancer, as well as pancreatic, prostate, and ovarian cancers. VHL can be associated in the germline setting with renal cell carcinoma. The other thing that we consider whenever we are looking for these cancer susceptibility or hereditary syndromes is where can we intervene? So not only is this information important for the patient who we are working with themselves in terms of considering if we need to do additional screening or perhaps preventative surgeries or other interventions, but this information is always really important for the family as well. And so those discussions are in the wheelhouse of our genetic counselors and our cancer geneticists who are very integral to our precision medicine team. The other thing that we do look for that germline variants can help us with is something that's a little bit near and dear to my heart as a PharmD, which is predicting drug exposure and toxicity. So the Clinical Pharmacogenomics Implementation Consortium Guidelines provide gene drug pair recommendations in terms of different dosing for drugs based on inherited differences in how the drug may be metabolized and or cleared. Very common examples of this are DPYD for our patients getting floral pyrimidines, UGT1A1 for our patients who are getting a renin-tican. And the way that we test these germline alterations in non-tumor specimens is blood, we can use buccal samples and nail clippings. So this is where we're really looking for those healthy cells and what are we seeing in that setting. To contrast this with our somatic variants, these are acquired over the person's lifetime. Initially in single cells, and then they can expand out and ultimately become the cancers that we are all here to fight and hopefully treat and prevent. Ultimately, these can provide information to guide treatment decisions. These include both prognostic and predictive biomarkers. So prognostic in terms of helping us to provide information about how the cancer may respond over time, independent of therapies, are these patients going to be higher risk, lower risk, have longer overall survival, shorter overall survival, things like that. And then obviously what we focus on a lot, which are the predictive biomarkers, which will help us predict responses to certain therapy. And this is where we test the tumor tissue and we can also utilize cell-free DNA, which is beyond the scope of discussion for this talk. Try to make sure that this advances. I think it may be stuck. I tried hitting it. Oh, there we go. Oh, sorry. Sorry, there's a little bit of a delay here. Trying to get it back to go and I don't want to hit it too far. Okay. It's on the next slide, so you may continue. Okay, thank you. So it's important to remember that whenever we are looking at somatic cancer testing assays, they are not optimized for detecting germline inherited alterations. I think that is one of the biggest takeaways from everything that we're going to discuss here. We're going to give you some examples of that, but a dedicated germline test is really required to verify the presence of a germline alteration if you are suspicious of that. That includes even if you've had a matched tumor normal or a cell-free DNA. There are some assays that do analyze matched tumor normal. So this allows us to subtract out the alterations that are being seen in the germline that are likely not tumor drivers and that we are likely not going to be targeting. We can, as we look at the results of these assays, allele frequencies that are near 100% or 50% or commonly may indicate a higher chance of an alteration being germline in origin. However, we really have to put that into concept with the tumor purity that we see in the specimen. So just because we see an alteration that's at 50%, we need to look at the other alterations that we see and also consider the tumor purity. We can look up the pathogenicity of alterations. There are different databases that help us do that. The one that I go to a lot is ClinVar. So whenever we do genetic profiling on the tumor tissue, we see a gene alteration that is suspicious for being germline and we're wondering if it is pathogenic. I go into ClinVar and you can put that information in ClinVar to see if this has been reported in the germline before and has it been classified as pathogenic, likely pathogenic, benign, likely benign, and then you can also see the different groups or laboratories that have reported it. And many of them will also give kind of a little rationale of why it is classified in the way that it is. So this can be a very, very important resource in terms of kind of teasing out what may be germline. But again, the big take-home is if you see a alteration that you suspect may be germline, we always wanna verify that in a dedicated germline test. And with a dedicated germline test, you also get the counseling that comes along with it. So patients are able to make an educated decision about whether or not they want to do germline testing, what genes they want to know more information about, or perhaps do not want to know information about. And then when those results come back to put it in context with their own disease and then what else they may need to do for prevention and how they can communicate with their family members as well. One time and hopefully it'll work. So let's meet our patient who will take us through some of the discussion here. We have a 66-year-old female with stage 3C high-grade serous ovarian carcinoma. At diagnosis, she is found on an MRI to have a large cystic mass in the pelvis and her CA125 was elevated at 236 with a normal range being less than 35. She underwent optimal debulking surgery which confirmed involvement of both of her ovaries and pathology diagnosed a stage 3C high-grade serous ovarian carcinoma. She then started on adjuvant therapy with standard carboplatin-paclitaxel and bevacizumab. Her tissue debulking or tissue from the debulking was then sent for genetic analysis to determine whether or not she should receive a PARP inhibitor. So a polyribose polymerase inhibitor for maintenance therapy. This is one very classic scenario. And one, again, the question that we are asking is should the patient get a PARP inhibitor alone or with bevacizumab after she finishes on her adjuvant therapy? So just a reminder what PARP inhibitors are. So when we have DNA damage occur, specifically single-strand DNA damage, PARP1 is activated to bind to that DNA strand. This catalyzes addition of ADP ribose, so adenosine diphosphate ribose, to the DNA, the histones, and itself. And it does this through the reduction of nicotinamide adenine dinucleotide to the ADP and the ADP ribose. This enables, ultimately, the unwinding of the DNA helix for repair. It recruits base excision repair components to actively repair DNA. So this is what the DNA is going to try to do as it goes through its repair mechanism. So if we have a patient who has an alteration in a homologous recombination deficiency gene, especially BRCA1 and BRCA2, we can inhibit this process and also take advantage of the fact that they already have a repair process that is dysfunctional to prevent the DNA from repairing itself and ultimately cause death to these cancer cells. So PARP inhibitors were designed to resemble the nicotinamide adenine dinucleotide, or NAD, and they competitively inhibit the catalytic domain of PARP to prevent this DNA repair from happening. So again, I think it's a little counterintuitive of what we usually want to do as repair cells, but since we're talking about cancer cells, we want to work with the body's own biology in that it already has a repair mechanism disrupted and further disrupt that so that these cancer cells die. So we do send this tissue from the debulking for next-generation sequencing, and you see the results listed here. We have a BRCA2 truncation with a mutation allele frequency of 48%, and we have some other alterations. There is a TP53 alteration that we see very commonly in high-grade serous ovarian cancer so that matches what we would expect from the histology. You'll notice the mutation allele frequency is at 89%, and obviously we're talking about this in context of is it germline, is it somatic? We know that TP53 mutations are commonly seen in these high-grade serous cancers. Obviously, we would want to consider could it be germline, but the other thing that may be happening is that the wild-type TP53 may be lost, and what we're seeing is predominantly that mutant TP53. The microsatellite status is stable. The tumor mutational burden is low. There is a loss of heterozygosity score that is high at 22.4%. The threshold is typically 16%, and we have four variants of uncertain significance. So what we want to consider as we look at this case is is the BRCA2 a germline mutation? Is the TP53 a germline mutation? How should this LOH score be interpreted? And is additional next-generation sequencing testing needed? So one thing I also obviously, or that one thing I always like to discuss too is to remind you that just because we see a gene that you may recognize as being important in terms of prognostic or predictive importance, we do need to consider the specific mutation. And so this allows me to highlight a little bit of what ClinVar can help us do. So this is a different case. This is a patient who happens to have castrate-resistant prostate cancer. Tumor genetic testing was done, and it shows a BRCA2 truncation as you see here, and this looks suspicious for being potentially a germline mutation, but it's in a gene that could predict benefit from a PARP inhibitor. And so in cases like this, we do go in and look at ClinVar. This is a screenshot from if you enter this gene in ClinVar. So the K is the single letter abbreviation for lysine. This truncation is occurring at the 3326 amino acid location, and it is a truncating alteration. And so in ClinVar, we put in TBR for termination. And we see that this is a benign alteration, which may be a little surprising because it is a truncating mutation. Most of the time, we always think of truncating mutations as inactivating. However, if we actually look at the BRCA2 gene, this location is at the very, very end after the common most important functional domains. And so it has been demonstrated in clinical studies to be benign. So this is why we always do look this up. And again, just because you see a gene that you recognize to make sure that the alteration is as important as the gene whenever you're making your interpretations. So one, again, one of the big themes that we wanna talk about, and I'm certainly gonna let Dr. Boyle talk about this in more detail. But when we are reporting the somatic genetic test results of these tumor specimens, it can be sometimes hard to tell, again, is something germline and, or is it somatic? And the other question is, if we see these alterations, what do we do about them? And again, this is where incorporating with your genetic counselors, your medical geneticists in that discussion. But one of the really helpful references is that the American College of Medical Genetics has published recommendations for reporting secondary findings and guidelines for referral for when germline testing should be done. So if you are developing an assay and it are wanting to make sure that you're following these guidelines, referring to the ACMG published recommendations is really helpful. We do provide the website there. The list of genes is a little bit more than what we wanted to go into because it is encompassing and it also does include consideration of specific alterations as well as the age and some other clinical characteristics of the patient. But as you can see at the bottom, this is a sample of an NGS secondary finding statement that we utilize on our reports to first of all frame the results in the lens that we are looking for somatic alterations and the assay is not definitively able to distinguish between germline and somatic alterations. And then we reference the list and include the genes that are on the assay that if there are pathogenic or likely pathogenic alterations in any of these genes that consulting with a genetic counselor and considering whether or not the patient should be referred for germline testing should occur. And I think with that, I will turn it over to Dr. Boyle and I'm not sure if there was anything you wanted to comment further on that statement as well. The secondary findings. Well, I did wanna comment. This is an area where with Chris Walco's Precision Medicine Group and with us as molecular pathologists signing out these reports where it's been very helpful for us to discuss what should we put on the report when we have these germline or incidental findings. And it's really been helpful to have a Precision Medicine Group to help translate and communicate about these findings with the medical care teams. But I'm going to move on and talk about how are these mutations detected. We've talked a little bit about this patient case with ovarian cancer, having a series of mutations and how are we detecting BRCA and TP53 and all these other mutations and profiles. And it's really with comprehensive genomic profiling with targeted next generation sequencing that we most standardly detect mutations in cancer samples. NGS is also used for the germline alteration detection and the panel design though is different when you're planning to detect mutations in cancer samples versus from the germline. Olexome sequencing has also entered clinical sequencing but most places are still using the next generation sequencing. At times, single gene assays are still useful when there's a need for a faster turnaround time for lung cancer. We have an EGFR single gene test that can provide a result within hours. But usually you want to preserve the tissue for this next generation sequencing because it provides information on so many different genes that are clinically relevant for so many different advanced cancers. Even in the earlier stage lung cancers, the genetic profiling is becoming more important as they're finding that targeted therapy can be helpful after surgery. There are some genes that are important not just for one cancer type but multiple cancer types like BRAF. It started in melanoma but it's also important in other cancer types. Some of the hematological malignancies and lung cancer and colon cancer and the list grows. Ntrek has inhibitors FDA approved in all solid tumor types when an Ntrek fusion is identified and also RET, same thing when a RET fusion is identified there can be FDA approved therapy available in different solid tumor types. So I think I've emphasized that the importance of preserving the sample for the comprehensive genomic profiling and not using it all up for the single gene testing. There are times when the sample is too small for the comprehensive profiling or when there's a faster turnaround time needed. But in general, we follow the NCCN guidelines, the National Comprehensive Cancer Network guidelines which recommends broad genetic profiling for advanced cancers. And they recommend this because it really does result in better outcome overall for patient care. And this picture is just illustrating how you can take the DNA and fragment it and attach it to a flow cell for sequencing. And it's a parallel sequencing that's why it's as fast as it is. And there's a fluorescent reaction with the sequencing that allows the detection of these mutations in multiple genes. Let's see now I'm trying to forward, so here we go. So I wanna show an example of how this broad profiling really does help with patient care and the survival curve on the black line you have the lung cancer patients who had a driver identified such as an EGFR mutation and receive targeted therapy such as an EGFR inhibitor. And you can see that on the black line the patients have a better survival than patients on the blue line where no driver was identified and much better survival than patients on the green line where a driver was identified and no targeted therapy was given. So this is more rationale or justification from the Lung Cancer Mutation Consortium for this broad NGS profiling to identify changes in multiple genes. So how is it performed? Really it's a team effort and oftentimes the patient comes in to see the oncologist and then a biopsy is taken or maybe a surgery is performed and the sample sent to the pathology lab and the samples cut and processed onto slides and stained and the pathologist looks under the microscope to make a diagnosis and they may determine that molecular testing will help with either diagnosis or therapy or trial enrollment. So they'll send unstained slides of the tissue with an H&E recut and that's reviewed by the molecular pathologist to annotate the tumor, estimate the tumor purity and maybe circle the region that has more tumor to enrich for the tumor cells, and then the DNA, RNA is extracted out of the tissue and sequenced, and there's pretty big machines these days that can do the sequencing in just a day, and then the sequence files go through the NGS bioinformatics pipeline and are processed and filtered into a report that gets reviewed by the molecular pathologist, and interpretations are added to any findings, and when the molecular pathologist signs out the report, the report goes into the medical record, and there's also a message going to the precision medicine group, and they review the report to consider how do these findings on the report help this specific patient? Are there any targeted therapies? Are there any trials? Do we need to get a genetic counselor involved if there might be a germline or a secondary incidental finding? And so there's communication going throughout the team, and sometimes the oncologist will communicate back to the precision medicine group that they need help to get insurance approval for off-label therapy, but that's another way that the precision medicine group helps the team, and they involve the PharmDs and the genetic counselors leading the group. So, let's see, next slide. There we go. So, when we're designing these next generation sequencing panels, one consideration is whether it should be a DNA based panel, an RNA based panel, or both, and traditionally, DNA was really the basis for next-generation sequencing because it's more stable in the formalin-fixed paraffin-embedded tissue, but, and it is still ideal for small variant detection, copy number detection. You can also detect tumor mutation burden, micro satellite instability, and homologous recombination deficiency with DNA based testing. But, more recently, technologies have improved and RNA extraction has become more robust, and RNA really is simpler for fusion and splice variant detection because the RNA is composed of the exons, and so the introns are spliced out, and so the changes like splice variants are simpler to detect at the RNA level. At the DNA level, there can be deletions of different sizes that cause a splice variant, whereas in the RNA, the change might be consistently exon 13 sequence going into exon 15 with abnormal splicing, splicing out the exon 14, and so it's easier to detect that change at the RNA level, and likewise with fusions, you can see the breakpoint for the fusions better at the RNA level, and really it is the DNA to the RNA which translates into the protein, so you're a little bit closer to the protein translation when you're looking at changes at the RNA level. So, our panel here at Moffitt integrates both DNA and RNA based NGS to have the ideal detection of all types of alterations, and it is helpful having overlap of the DNA and RNA sequence for some of the variants if the tumor cellular is low and BRAF has a low allele frequency in the DNA, it's nice to look in the RNA as a separate test and see that BRAF change in the RNA also. So, another consideration with NGS panels is the gene coverage, and I think Dr. Walco described the importance of whether an NGS assay is designed for germline inherited mutation detection or somatic mutation detection, and that there can be overlap in the genes, but it may be that the germline panel has better coverage of intronic gene regions, and as an example in BRCA, it's important to detect intronic changes associated with exon 15 deletion. And that might not be covered in the somatic mutation panel, and so these details that can be very important for patient care because you might actually miss a germline change if you test with a somatic NGS panel and not the germline panel. Another question, how well are specific genes associated with FDA-approved therapies covered? Panels tend to do pretty well with this. Does the pipeline report tumor mutation burden and or microsatellite instability? For tumor mutation burden, it does require a larger coverage of the genome to accurately report T and B. Are genes associated with homologous recombination repair covered? I'll show the list on the next page. BRCA is one of them, but there's many more. And is homologous recombination deficiency, which is caused by mutations and changes in homologous recombination repair genes, is that reported as a separate element? This is basically the effect of mutations in the HRR genes. Or is loss of heterozygosity reported, such as we saw in this patient case? So what is homologous recombination repair? What are these genes? These are genes that encode proteins that repair double-stranded breaks. And so these four genes are examples of genes that encode proteins that are involved in a complex that repairs double-stranded DNA breaks. And when you have a dysfunction or mutation of within these genes, that can lead to homologous recombination deficiency. And so that the repair does not occur and you can end up with quite a lot of mutations. And so how do we detect this homologous recombination deficiency? There's different ways. You can detect pathogenic changes in the HRR genes, the homologous recombination repair genes, such as BRCA2, BRCA1. Or you can look at the effect of these genes and look for loss of heterozygosity, allelic imbalance, and large state transitions. And so, or you can test for both. So this table shows some of the genes that are involved in homologous recombination repair. Some have clinical trial data to show that when a mutation is identified in these genes that patients respond to PARP inhibitors. And and for ATMNCHEK2, there's actually some evidence to suggest that there is not PARP inhibitor benefit. So the HRR genes are not all alike. I think there's a lot of confusion about these because there's so many and there's different lists and the clinical trials have used them differently. Even in the NCCN guidelines, this subset in bold is mentioned, but they, in the text, they say including but not limited to. So there's still work to be done to really define which of these genes are most important for targeted therapy. And it's really not enough to just detect the mutations in the homologous recombination repair genes because there actually are other changes like epigenetic changes and unknown causes of changes that can lead to this homologous recombination deficiency. So ideally you're also detecting the effect with the HRD score, the homologous recombination deficiency score, to really see the scar from the deficiency in the homologous recombination repair. And this scar causes these teloleric allelic imbalances, these large state transitions, and the loss of heterozygosity. And it's really not enough to just measure loss of heterozygosity or to just detect mutations in HRR genes. Ideally, you're also reporting the HRD score. And with that, I'm going to turn it back over to Chris. Thank you so much, Dr. Boyle. I think that gives a lot of the context as we kind of put all these pieces together and you think about a specific example of why detecting germline alterations, why we care about them, and then moving into an area that I just think is so exciting of the genes are important, but looking towards these genetic signatures and looking towards the actual phenotype of the cancer so that we can understand that underlying biology that's driving it and then match that to drugs like the PARP inhibitors that may take advantage of the cell's inability to fix its DNA and exacerbate that. So as we talk about these homologous recombination deficiency or specifically loss of heterozygosity first, whenever you are thinking about validating this as part of your assay, perhaps in-house, or just how you interpret these, it's important to realize that the loss of heterozygosity score, the homologous recombination deficiency scores that have been used in clinical investigation are continuous variables. And so it's not just, is it there, is it not? It is a continuous variable, and with that comes an interpretation of that number. And so I did want to provide two of the references that helped us establish the cutoff that we utilize for the loss of heterozygosity score. So the ARIEL-2 trial assessed the PARP inhibitor Rucaparib in patients with recurrent platinum-sensitive high-grade ovarian cancer, and they looked at patients who had BRCA mutations and who we know the PARP inhibitors benefit. And if you look at the Kaplan-Meier graph on the right here, you can see that those in blue are the ones with the BRCA mutation and do have the highest benefit from the PARP inhibitors. But we can also see that those who did not have an apathogenic or likely pathogenic alteration in BRCA but did have a high LOH score, which in this trial actually was set at 14 percent, so a little bit lower. It was actually the randomized phase 3 trial that set it at 16 percent, which is the current standard based on the ARIEL trial data, demonstrated that while those patients who did not have a BRCA 1 or 2 mutation but did have a higher LOH score still had benefit from a PARP inhibitor. And that is compared to the green line where we see these are patients who were BRCA wild-type and had a low LOH score. So really the group that benefits may not just be the BRCA mutants, but also another, a kind of larger subset that have other alterations in the DNA that may ultimately cause that homologous recombination deficiency biology, and that's really what we're trying to get. So, the official companion diagnostic that is approved by the FDA is an HRD score, which includes the LOH, the TIA, and the LST. That is reported by the specific company that has been granted the companion diagnostic designation by the FDA. They actually report it as either negative or positive. I have an example to show you what these tests look like. However, the HRD cutoff that they validated was a score of 42, and it is the official companion diagnostic for the drug olaparib with bevacizumab in the first-line maintenance setting for ovarian cancer, and this was based on the PAOLA-1 trial. You can see the results here and kind of similar to the story that we saw. This is looking at HRD. So it's adding in, in addition to LOH, we're now looking at the large-scale state transitions and the telomic allele imbalance. So adding in additional measurements of homologous recombination deficiency, and it was the PAOLA-1 trial, which was a randomized double-blind placebo-controlled study, again, in the high-grade ovarian cancer patients who had a response after first-line platinum taxane and bevacizumab therapy. They were randomized two-to-one to either get the PARP inhibitor in combination with bevacizumab or placebo for up to 24 months. And what we saw when we look at the forest plot here is that, again, no surprise patients who did have a BRCA mutation did have benefit from the PARP inhibitors, but again, if we had a homologous recombination deficient tumor, that those patients also had benefit. Similar, obviously, overlap to what we're seeing with the BRCA mutation, but there were patients who did not have a BRCA mutation, who had the homologous recombination deficiency, who did still have benefit. And that's really what we're trying to do with these scores, is kind of moving away from just the genes, but looking at the underlying function of the cancer. And again, this trial utilized the cutoff of 42 for that HRD score, which is the FDA-approved standard. And so this is just an example of how the official companion diagnostic gets reported. That genomic instability score, again, is reported as positive if it is greater than or equal to 42, and then will also report if there is a BRCA alteration in the tissue as well. And this is a tissue-based assay. This same company also does have a germline assay, so it is important to know what tissue is being tested and kind of what the question being asked is so that we can interpret it correctly. So whenever we think of homologous recombination deficiency, we think of ovarian cancer, like our patient case that we introduced and that we'll come back to, I think on the slide or two, breast cancer, prostate cancer, and pancreatic cancer. But we know that we can see alterations in homologous recombination pathway in other types of cancers, and that's actually what we are looking at here with the changes in the allelic imbalance, the large-scale state transitions, and the loss of heterozygosity. So you can see most of them kind of clustering at the bottom. One that I'll point out that's very near and dear to my heart are our glioblastoma patients, and again, the majority of them are not going to have homologous recombination deficiency, but we do know that a subset of these patients do have homologous recombination deficiency, and whether or not we should utilize a PARP inhibitor is an active area of investigation. We have looked at that in a unselected population, but you can definitely see that there are outliers, obviously not just in glioblastoma, but in some of the other cancer types as well. And one of the essence of precision medicine is really delving into each individual patient, and while they may not cluster towards the norm, what is it about these patients and what treatment can we offer them to help them in the best way possible? And really the genetics and all the molecular investigation that we do are additional pieces that help us understand that. And so it's really exciting for me, I think, as we understand homologous recombination deficiency, and how it may predict response to PARP inhibition in the cancers that it is associated with, as well as cancers where it may be less commonly seen, so they're not enriched in these PARP inhibitor studies, but that it's still important to look at those extraordinary responders, and again, understand the biology, go back to the bench, and understand why a certain drug may have a benefit, and what we can do to understand that process better, so that we can help each individual patient. So let's come back to our patient case. So again, just a reminder that this is our patient with ovarian cancer who has a BRCA2 truncating alteration. I will tell you that that is occurring earlier in the gene, and so if we do look that one up, it will be pathogenic. Could it be germline? We're seeing it at 48%, so it definitely is suspicious for being germline. This patient does have ovarian cancer, and so this is a patient who I would recommend for to be seen by our genetic counselors to discuss whether or not the patient wishes to get germline testing. We cannot definitively say that it is germline based on these results, however. As we kind of alluded to already, these tumor-based tests are not tumor next-generation sequencing tests are not optimized for detecting germline alterations. The germline tests especially are better designed for detecting larger deletions, and they can miss some of the larger deletions. We've actually seen that in clinical practice. So while my ultimate interpretation would be that this is suspicious for being germline in nature, however, the patient should be referred to our genetic risk assessment service to discuss germline testing so that the next step can be done by our experts in that area. Is the TP53 germline? I kind of talked about that a little bit already that we see TP53 mutations very commonly that as well, and that this may be not germline even though we do have a high allele frequency, but ultimately germline testing would be needed to definitively determine that. We talked about the LOH score. In this case, the LOH score is high. It's greater than 16% at 22.4%, which would make sense given that we do see this pathogenic BRCA2 alteration, so kind of this causing this to be high. And then is there additional MGS testing that may be needed? I think that this gives us a lot of answers. I think the additional testing would be the germline testing. And then is there an FDA-approved targeted therapy in this case? Yes, a PARP inhibitor could be considered for maintenance. And just, I won't spend a lot of time on this, however, this is just a reminder of ovarian cancer, the PARP inhibitors that are approved. We have elaparib, recaparib, and noreparib. The approvals have changed over time, but these are the current ones, and we can see that in the maintenance setting, which is what is relevant for our patient, that we could utilize the elaparib in combination with the bevacizumab because of the HRD status being positive. And if we did not have that, but we still wanted to utilize a PARP inhibitor, the noreparib is also an option. And PARP inhibitors are approved in other cancer types. We have elaparib, noreparib, recaparib, and telozoparib. And I have their approvals listed here in different lines of therapy for ovarian cancer, breast cancer, pancreatic cancer, and prostate cancer. All right, so we wanted to end with kind of a case challenge, and I'm interested to hear some feedback, or we can discuss this in a little bit more detail. So our patient is a 67-year-old female with stage 3c ovarian primary peritoneal high-grade serous carcinoma, as before. Her past medical history is notable for triple negative breast cancer. And because of that, she had germline testing that was done that showed a pathogenic BRCA1 exon 15 deletion. She received a standard therapy with neoadjuvant carboplatin paclitaxel, and she underwent optimal debulking. Tissue from that debulking surgery was sent for outside next-generation sequencing, and you see the results there, where we have a TP53 mutation, we have an RB1 loss, we have this specific assay's version of their homologous recombination deficiency measurement, and this was an HRD signature that was positive. And then you see the tumor mutational burden and the microsatellite status listed there. So my question was, why was the BRCA1 alteration not seen on this tissue-based assay? And so if anyone wants to write in the chat what they think, this was actually a case we had I think about a month ago that we thought illustrated these concepts well, so if anyone wants to I hope that what you're thinking is that because this was a large deletion in Exxon-15 that this tissue-based assay was not optimized to detect it. Conversation with the company did verify that this alteration, which would not be detected by the assay because it's not an area that's baited by the assay. So communicating whenever we see findings like this that may be less intuitive, I think, are really important and, again, illustrate that these tests, somatic tests, are not optimized for detecting germline alterations. Thanks. So, yeah, there's your answer. And I think with that, we'll take questions and then also turn it back over to Dr. Bowie. Wonderful. I really enjoyed this. You two not only show us challenging cases and tell us what is the significance of this testing, how it's tested, and how to make decisions, so pathologists can really play a very important role in patient care by collaborating with molecular pathologists, PharmDs like yourself. So this is really great. And I start to see questions coming in. And I have this one question, it's asking, there are a lot of HDR testing out there in the pathology community. Is there an effort to harmonize how these are tested and reported? Yes, there's an effort by Friends of Cancer where they, I believe, have published. And also, I lead this Sequoia group, which is an NGS user group for the TSO500 assay. We have a Sequoia effort to standardize HRD reporting with this particular platform. And Chris might know about some other efforts. The Friends of Cancer one is the one that I'm most aware of. And I have found their resources incredibly helpful, as well as their passion for attacking this question, because patients live and die by these numbers. And they are continuous measurements. And so it's really, really important that, obviously, the information that we're giving is valid. But also, what do you do when you're in that gray area? And as this is kind of a newer and evolving area, it's through these collaborations that we can get the experience and different thoughts and, I think, examples so that we can understand it better. Very good. Well, we're waiting for people to type their additional questions into Q&A box. And we're going to go through the rest of some slides. All right. Next slide, please. All right. So this is the learning center that I was talking at the beginning. This is a historical for FSP. So this Precision Medicine Academy is hosted by this learning center. So we have a lot of great content there already. This lecture today will be recorded and posted. And you're going to receive a link and ask you to do your Q&A, your evaluation, and then receive CME. So there is a QR code for you to scan and go to this learning center. Next slide, please. And in addition, Florida Society of Pathologists has the best educational programs. We have it three times a year. The first one usually is February. So the one coming is February 14 to 16. So we have strong lectures promoted here. The Mayo Clinic, Jacksonville Pathology Department will also put on a webinar at the day one of the meeting. So that's a Friday. In addition, we give a challenge to our trainees. The program has the highest percentage of participation of this particular webinar. You'll receive a goodie bag at the FSP booth to congratulate them for their eagerness to learn and participate in FSP activities. The second lecture or conference is going to be in summer. That's in July. So those are the doctors who are our featured faculty. You can see their younger generation of pathologists. Some are very active on social media. So this is more targeted to our younger pathologists. We will also have a precision medicine multidisciplinary symposium. The date and time has not been decided, but likely it's going to be in September. And our education program also did a fantastic job, lined up the next FSP meeting for 2026 February. So these are the faculty. The topics are GI, breast and hematopathology. Next slide. Yeah. So please save the date. And our next Precision Medicine Academy webinar is going to be on February 27th at 12 p.m. Eastern Time. So the topic is application of molecular genomic biomarker testing in solid tumors. And Dr. Boyle will be returning, Dr. Kim Newsom from University of South Florida will join. So please complete the evaluation in the Online Learning Center to claim CME. So with that said, this is a very successful webinar. And I thank you for Dr. Boyle, Dr. Walco for your excellent presentation. Thank you for the organization. And I thank everybody who signed up and support us. And also, today is also a special day. So that's celebrating the Lunar New Year. So this is the year of snake on the Zodiac calendar. So that's in the year of snake, it's showing the wisdom, intuition and transformation. So with that note, I wish everybody have a very wise and transformative year. Thank you very much. All right. Bye.

Precision Medicine

Genetic Changes

Somatic Mutations

Oncology

Molecular Diagnostics

Personalized Treatment

Genetic Profiling

BRCA Mutations

Next-Generation Sequencing

Homologous Recombination Deficiency