 Hello, sorry, Hussain. We've just started the recording feature earlier, but we will edit, well, of course, edit the presentation to start with Dr. Lakwa Van's presentation. We'll remove the upfront introductions. Okay. Okay, hearing nothing. Yeah, we stopped talking. Hi, sorry. It's Hussain Naraini from BCBSA. We're just gathering for the call. We should start in a few minutes. Thank you. Recording. I'm not hearing anything. Heather, sorry. Good afternoon. It's Hussain. Heather, we could begin with the introduction, please. Welcome. Good afternoon, everyone, to the February genetics webinar. Today, we are very pleased to have as our presenter, Dr. Felipates Lakwa Van, who is the Medical Director of Genetics at Quets Diagnostics Nichols Institute. Today, she will be presenting next-gen sequencing channel testing for hereditary cancer syndrome and for cancer targeted therapy. Before we begin, though, I would just like to remind everyone, please mute your lines during the presentation, and we will open up the conversation at the end of the presentation for questions, and as always, the webinar is being recorded. So, Dr. Lakwa Van, call yours. Thank you, Heather. Good afternoon, everyone. It's a pleasure to present to you this topic for today, and hopefully, it would be helpful in your individual companies or offices. So, in the application NNGS panel, Testing for Hereditary Cancer Syndrome and Cancer Targeted Therapy, we would try to cover as much in terms of the content of the NNGS panels, how they are put together, how they can be validated, what are the guidelines that are controlling or burning the use of this test panel, both in germline as well as in somatic cancers. Based on the 2015 American Cancer Society slide, there are areas in the United States where there are more than 100,000 cases per year, and that is actually in California, Florida, New York, and Texas. Ten most primary cancer sites are prostate, breast, female, cancer, lung cancer, colon, rectum, uterus, and pretty much some of this or the prostate, a lot of this are part of the hereditary cancer syndrome. By distribution, 80% of cancers are sporadic, meaning that there's no germline mutation or inherited gene that's causing the cancer. 10 to 15% are familial, meaning that they occur in families and they could be low penetrance and they need a gene environment interaction or both to cause cancer. The 5 to 10% though are inherited cancers and they arise from highly penetrant germline mutations. Inheriting a genetic mutation or pathogenic variant doesn't mean that the patient or the person who has the variant develops cancer, but it increases his or her risk. The most common hereditary cancers are breast, ovarian, colorectal, and prostate cancer. Understanding if cancer is due to an inherited pathogenic mutation can help clarify the risk of developing cancer and it also helps determine options for cancer prevention as governed by guidelines and possibly therapy. Some cancer risk for common cancers are more or less variable, but it actually is around the range of almost 40 to 80% cancer risk versus the general population. As an example, breast cancer, BRCA2 has 4 in 10 versus BRCA1, which is 6 in 10, chances of developing cancer by the age of 70. There are red flags of inherited cancer in a family or in an individual. Cancer in two or more closely related relatives, multiple generations affected by, depending on what type of cancer there is, an early age of diagnosis, multiple primary tumors, bilateral or rare cancers, as well as constellation of tumors consistent with specific cancer syndromes, and certain ethnic backgrounds like the Ashkenashi Jewish panel, I mean cancers, can be a clue that there is an inherited susceptibility gene. Family history is one good clue that something is happening in the family that could be inherited. With improvement or advances in technology, the cost per genome has drastically changed since 2007, and with that, the massive parallel sequencing or next generation sequencing had really propelled the use of this technology in a lot of donor-based sequencing, and that we can also utilize that technology to interrogate several genes at a time. Right now, when we talk about sequencing, we would be able to do several things. We can do risk management depending on the diagnostic test that actually is used for certain gene panels or certain genes, and we can use it also for screening when we apply it to high-risk patients and identify the disease early before the cancer occurs, and also for diagnosis when we would want to ascertain what kind of cancer that patient has, and staging, of course, as well as therapy selection, and I would expound on the therapy selection when I get to the solid tumors, and of course, monitoring for efficacy. For cancer gene panel, pretty much similar to any next generation sequencing, what varies is actually the input or the source, the DNA, or the RNA. So normally, for germline cancer panel testing, we use blood. There are some times when you would use FFP if you know that there is a somatic mutation that could be subtracted, you would have a germline mutation from removing the somatic mutations, and sometimes rarely, though, this is not yet established, but eventually, if there is a germline mutation that can be followed using circulating free DNA. But right now, most of the cancer germline panels are run using blood. So after DNA extraction or RNA extraction, the library prep is done, and then the target enrichment is something that is important to remember, because this is where different laboratories really refer, and depending on how they capture the DNA or the RNA, pretty much that could give you the sensitivity, specificity as well as the depth of coverage and also the type of coverage within the whole gene, whether they include the promoter sites or the other genetic structure within the gene. So then after that, sequencing can be normally done depending on what platform is available in that laboratory, and of course, the other thing that differentiates its lab would be the informatics that's being used, meaning the informatics pipeline, because that would actually differ in each of the laboratories that do certain gene channels. The reporting is also something that it may differ within the lab. So just a review. The normal or the more common test that one can do is BRCA-1, BRCA-2, and therefore breast ovarian cancer and the most common high-risk breast cancer is just up to build the syndrome, because they occur in 1 in 300 to 1 in 800 individuals, more so in Ashkenasha, Jewish, where they have 1 in 40 individuals. So the cancer risk by age 70 for BRCA-1, BRCA-2 mutation carriers without personal history of cancer is reflected in this table. So for female breast cancer with BRCA-1 mutation, it's up to 65 percent, and for BRCA-2 it's up to 47 percent, and for ovarian cancer, 39 percent for BRCA-1, and 17 percent for BRCA-2. And for male breast cancer, more so on BRCA-2 it's 6.8 percent. So there are other hereditary breast cancer genes, and every year there are more than 200,000 women in the U.S. that are diagnosed with breast cancer. I mentioned earlier that it is mostly BRCA-1 and BRCA-2, but there are certain genes which are highlighted in this figure like TP53, P10, STK11, CDH1, and PAWB2 that are also responsible for breast cancer and around 4 percent of cases. So gene-speed highest increased risk for breast cancer, as I mentioned earlier, can be included with BRCA-1, BRCA-2 in the panel, and that increases the number or the presence of cases that can be detected with that panel. So why are they included in a panel? Pretty much one would understand that breast cancer tumor genesis, it actually affects DNA repair, that is, BRCA-1 and 2, and check 2, chromatin remodeling for BRCA-1, as well as protein ubiquitination. There is cell cycle regulation, it's regulated by P53, and apoptosis are cell death by P10, as well as cell proliferation. So these genes participate, or the products of these genes participate in tumor genesis in different ways, and that actually is the reason why they are put in certain panels for BRCA testing or for breast and avarian cancer testing. Can I just follow in the second? You need to go that way. Sorry for that, I have a competing lecture on the other side. Okay, so just to show that the NDAG, BRCA-1 and BRCA-2, they actually form complexes with the Phanconi anemia core complex, and they do repair DNA that are damaged, and they also promote chromosome stability. So I wouldn't belabor some of these components, but just showing the fact that there are certain genes that interact with BRCA-1 and BRCA-2, and these are the Phanconi genes, as well as the ATM and Pal-B-2. BRCA-1, BRCA-2, we know it's hereditary breast and avarian cancer syndrome. TP53 is actually responsible for leaf romani syndrome, P10 for the hammer-toma tumor syndrome, which includes cowden syndrome, and then CDH1 for hereditary diffuse cancer, gastric cancer, and Poich Degger syndrome for STK-11 and Pal-B-2 with the associated breast cancer. In terms of lifetime risk for breast cancer, TP53 has a relative risk of 6.4 times when you have a mutation, a pathogenic mutation, a likely pathogenic mutation for TP53, and then P10, we have for breast cancer, 85% approximately at the age of 70 years of age. CDH1, lobular breast cancer risk of 39% or 52% for age 80 years of age, and STK-11 breast cancer risk of 45% by age 70, and Pal-B-2 breast cancer of 35% by age 70. Then, as I mentioned earlier too, there are several other cancers that can be associated with different genes, and for TP53, bone, connective tissue, brain, pancreas, colon, and liver are also increased in patients who have TP53 mutations. For cowden, besides breast, one can have thyroid, endometrial, renal, colorectal, and melanoma. Of course, hereditary diffuse gastric cancer in males and females may differ, and then poitzvierger for gastrointestinal cancer, including pancreatic cancer, 11% by age 70. The NCCM guideline actually gives a very well-defined criteria for testing based on age, family history, personal history, and pretty much I wouldn't read through the whole criteria, but it does tell you who are to be tested, and that within the family, you can actually identify who needs to be tested after an individual in the family has been documented to have a mutation. Of course, within the NCCM guidelines, there are also management guidelines for women and men who have the mutation and ranging from graph examination, MRI, as well as other procedures that would prevent development of associated cancers within the family or within the patient. This table just summarizes what are the guidelines that are out there, and probably justifies why these NGS panels are really offered to individuals who really fulfill the genetic testing criteria. So for BRCA-1, BRCA-2, and TP-53, as well as PTEM, NCCM guidelines have the genetic familial high-risk assessment best variant, and then for CDH-1, there's an international gastric cancer linkage of consortium, transensors guidelines, and although STK-11 and POP-V2 don't have genetic testing criteria, NCCM has genetic familial high-risk assessment for colorectal in STK-11, as well as ACS has recommended guidelines for POP-V2. Expanded menu. Remember I mentioned that BRCA-1, BRCA-2 explains 15 to 20 percent of hereditary breast cancer cases, and with the additional GNS, which are TP-53, PTEM, CDH-1, STK-11, and POP-V2, which are mostly probably not just high penetrance but moderate to high penetrance, breast cancer susceptibility GNS can explain up to 4.5 percent of hereditary breast cancers. So that in itself justifies the fact that the seven GNS can be put together as initial screen for patients with breast cancer that would fulfill genetic testing criteria. So I mentioned that POP-V2 is an emerging GN and pretty much right now there are more reports of POP-V2 positive breast cancer patients. So it looks like that should be enough, but not really because there are other GNS that are actually responsible for breast cancer risk also. And just summarizing the fact that when will you use a panel versus just BRCA-1, BRCA-2? Depending on the family history, one can actually prioritize BRCA-1, BRCA-2 if it's mostly hereditary breast cancer variant. But if there's a mixture within the same family of certain other cancers, one can opt to take the seven GNS or even a 34 GNS which actually I can explain. Okay, so right now in certain laboratories there is just BRCA-1, BRCA-2 as complete coding exon sequencing and that can be just a comprehensive BRCA-1, BRCA-2. There are times when a family member has been tested with the single mutation or variant within the family, especially in Ashkenazi Jewish families where they have specifically any one of these common mutations, then they can just be screened for that type of mutation. The other thing that can happen is that if the family is Ashkenazi Jewish but then the Ashkenazi Jewish screen is negative, one can order a reflex for the comprehensive so that the whole exon of BRCA-1 and BRCA-2 can be tested. Single site means that within the family there's a known pathogenic mutation and so when ordering a test, the clinician can just order that specific mutation so that the lab doesn't have to sequence the whole BRCA-1 or BRCA-2 GNS. And then there are times in the early days where rearrangements were not tested. One can also do adjust the rearrangement if the BRCA-1, BRCA-2 were sequenced earlier and not the rearrangement. Then I mentioned that the 17 panel, we can call expanded panel and who are the cases that would need this panel. It depends. Sometimes when the family history is not very specific and not focusing or directing the test to just BRCA-1, BRCA-2, then you can order a seven-gene panel including BRCA-1, BRCA-2, TP53, P10, CDH-11, STK-11, and Pulse B2. You can do a reflex also. You can just do BRCA-1, BRCA-2 then later on if it's negative and the patient has some other family members involved. You can do a reflex of the five genes or just the five genes if the BRCA-1, BRCA-2 was earlier tested and there were no point mutations, deletions, or duplications. Then comes the bigger panel. This has been up and coming in different laboratories and I'm just showing you what we have, but pretty much the reason why they are put together is because they can have only breast cancer. Here we have the seven-gene panel that I discussed earlier, but then within the other genes which are actually, chances are they're low-to-moderate risks and low-penitence. They can also cause breast cancer and some other cancers like ovarian cancer and for this other genes, which are actually Lien syndrome genes, they can also cause breast cancer. I'll discuss a paper that was just published and just to show you that there is some reason why cancer predisposition panel is bigger compared to the more high-penitence genes because of the different conditions that can be tested for the 34-gene panel. Here's a paper by Desmond and it was just published recently at the JAMA Oncology 2015. Pretty much you have different labs here. There are 34 genes in two laboratories and 25 genes in one laboratory and here's what the gene panel that this study did. They actually included 1,054 cases which are BRCA 1 and 2 negative and they went through all the testing and found that there are 63 of those 10,24 cases that are positive for other genes other than BRCA 1 and BRCA 2 and significantly actually affected management as well as familial testing. It does support the need or the use for multiple gene panels and pretty much the advantage of having this multiple gene panel is you would have a lower turnaround time and you cover several genes at a time. However, just to iterate some of the differences in the different panels that are being offered out there, one would need to understand that they were validated in different ways depending on the platform that they were ran and at the same time there should be an accuracy sensitivity specificity limits of detection for each of the lab that had validated this test. One very important thing to consider too is that not only the panels, the contents of the panel itself but also the assay design and the genes that are included in the panel because there are certain genes that are actually very difficult to do massive parallel sequencing because they can have pseudogenes and I just gave one, two genes here, the CHEC2 and the PMS2 pseudogenes can complicate sequencing and so if the laboratory is offering these two genes pretty much one can ask them what are the ways that they had improved on so that they are sure that they're not sequencing the pseudogenes but rather the real gene. And then, of course, the third one here where laboratories do optimize their conditions in terms of capturing the real exonic sequence as well as the flanking sequence using RNA dates or some other ways of capturing the sequence of interest because there are times when there are some entronic sequences that are commonly affected in some genes and those need to be... I'm sorry. I think I'm disconnected. I don't see any slides on my... Sorry. Okay. I got it. So with all these technicalities within the assay design one should understand that there's also a difference between using tissue and blood and the mechanism for capturing low copy number variants as well as addressing mosaic system and the other one is also the type of mutation or rearrangement that one actually can see using that assay because some assays would not be detecting the CNVs as well as large rearrangement, more so for massive parallel sequencing, triplet repeats are difficult to actually identify, although they don't occur in some of the... in most of the genes that we have anyway for the NGS panels for cancer. And again, sequencing performance and quality metrics need to be understood as well as I mentioned earlier, the bioinformatics pipeline is also important because some bioinformatics pipeline actually do not detect small deletions and they can be missed. Okay. So at any rate, once you have a pipeline that is working and annotation and classification is pretty much standard, however, in different laboratories also multiple database sources are something to highlight. There are publicly available mutation databases and some of them are reported or recorded here in the slide. So everybody can use those but there are certain private databases that others can query and consortia and other companies that are actually in participation or in collaboration with other foreign laboratories. So those are the things that can happen in terms of annotation and variations in annotation, but hopefully with more public databases and more publications, pretty much every... or most of the variants can be annotated similarly. There are multiple reviews for VUS and pathogenic cases in most laboratories and anything that can be reclassified within a certain period of time can be done by the lab and contact the clinician who had ordered the test. Co-segregation family studies can help in VUS reclassification and I think some labs are doing those and of course, pretty much most of the labs or all of the labs are doing final interpretation by board certified directors for experience in interpretation. So in a graph cancer report besides the fact that the turnaround time is important, one can reflect the options within 21 days, at least within our lab and the interpretation summary depending on which lab you're sending your test, it's pretty much there categorized and highlighted and all of the ACMG guidelines are utilized. So that actually is the germline side of cancer sequencing. I'm shifting to solid tumors and if you have any questions, we'll take it at the end of the talk. So solid tumors, many of you probably have, if you remember in medical or at least the theory that you have to have a double hit to have tumors or you have an environmental factor that affects a previous mutation within the tumor producing genes before you can have tumor and that becomes a little more of a challenge in terms of testing and that we have solid tumors with multiple genes and pathways being altered, suppressor genes as well as oncogenes being affected and with that a lot of therapies have been developed targeting specific genes as well as other molecules within the cells that could help prevent cell proliferation or induce cell death. And of course clinical annotation as well as clinical utility must be established before testing can be offered. So the clinical view of cancer, one could see the different stages or the different cell activity that can be affected during tumor formation and that basically you're either inducing proliferation, preventing death of the cell or pretty much there is a driver mutation within that type of cell that actually causes the proliferation of that cell. So here it's just giving you a broad view of cancer and what are the possible inhibitors or therapy that can be developed to target specifically those activities within the cell that produce cancer. Cancer pathways and targeted treatments. Most of you must have heard of a lot of receptor antagonists, tyrosine kinase inhibitors and some of these other gene products that can be inhibited within the intracellular part of the cells and any part or any protein within these pathways can be inhibited or depending on what the mutation or variants that can occur they can actually have a constitutional activation of that receptor leading to cell proliferation or some inhibition of apoptosis and this actually just summarizes the fact that there are several receptors, cell receptors that can be shared by different cancers and that certain drugs or emerging therapeutic agents can be used to target certain cancers and they can also be used in some other non-specific cancers depending on the target. So just to give you an example, Lank cancer or Lutinib is one of the drugs that can be used for treatment of Lank cancer and it is pretty much inhibiting the EGFR mutated receptor action. Again, you can probably look at the slides again but I don't need to belabor the point that several genes responsible for tuberogenesis are shared by a lot of solid tumors and there are specific solid tumors where they are very much more related to a specific tumor but again, since they share different genes and they can have driver mutations in different solid tumors then one can use a targeted mutation for that specific gene product. For Lank cancer, pretty much in some solid cancers, they have at least a dozen of shared genes that one can target and that's the rationale in why some of the hotspots that are commercially available are being used in different laboratories and of course for Lank, melanoma, breast and colorectal, here are all the genes that are actually being targeted by some commercially available and some IVG diagnostic kits and that itself, pretty much because of the hotspots one can easily direct or target that particular cancer based on the cancer profile. In most next-generation sequencing there are several targeted actionable genes. First of all, we have a 34GN panel and it is applicable to all solid tumors and it's annotated directed at FDA approved drugs and sometimes with the less common genes we have clinical trials that are available and most of the other labs as well as us we actually can suggest which of these clinical trials can be used depending on the variants that are actually identified after the sequencing. So for next-generation sequencing for solid tumor one can use FFPE tissues, small biases, FNAs and sometimes there are tests where one can use some other types of cells but for the most part it's FFPE. For example, lung cancer. So there are treatment options based on the molecular profile as I alluded to earlier and specifically for lung cancer if you have an EGFRX in 18, 19, 21 on mutation you could use Erlutinib and for the other mutations in EGFR as well as some of the positive KRAS mutations for 12, 13 and 61. This drug cannot be used because it's non-responsive. So this table just gives you a flavor of different antibodies or the different drugs that can actually antagonize the driver mutations and here are the different genes here based on the mutations and then here are all the different drugs used for each of the different gene-specific mutations that can occur in lung cancer specifically the non-small cell lung cancer. So in most cases these are the ones that are targeted for diagnostic or profile. So for lung cancer essential is EGFR, BRAF and ALC. Some have used Los Juan red-met fish as well as HER2 mutation detection and for colorectal KRAS, NRAS, HRAS, PIC3CA and BRAF and for melanoma, BRAF and KIT. So for the ones that are not commonly associated with hotspots and are very negative in this more hotspot directed and less number of genes that are commonly tested, one can use a gene panel that can encompass more genes that are actually causing driver mutations that are associated with cancer. So again this is level one association between genes and FDA approved therapies and ranging from BRAF, EGFR, HRAS, KIT, WET, SM, SMUTAN, KRAS and NRAS and here are all the two word types here ranging from melanoma to colorectal cancer and then the association of the different mutations that can occur, whether they are sensitive or resistant to the specific drugs on column two. This can be the table where we refer the more common changes that are associated with the different cancer types as well as the genes that are related to targeted therapy. And here's a larger actually listing of gene targeting and again it could be that any of these genes on the far left, there are actually more than two dozens of genes on the far left, any of these genes can confer mutations and any of these genes can confer resistance or sensitivity depending on what is the mutation and the treatment can be identified or dictated depending on which gene is affected and what the mutation is. Okay, so what are the clinical applications of NGS multi-gene cancer panel? It's primarily for cancer patients with few or no standard treatment options remaining and this is sometimes after identifying the hotspots or the ones that are available out there and that if the patient doesn't respond to treatment given that initial stratification the oncologist can be assisted in deciding on potential effective drugs or clinical trials that can be utilized by the patient. So most of the multi-gene cancer panels are actually for solid tumor, all solid tumor types and pretty much it could be either a metastatic or a locally advanced disease on presentation and when no actionable mutation is based on guidelines then you can use this gene panel to actually get some of the other drugs that are pretty much under clinical trial or emerging into the market. They can be used for small specimens and they can be used for both recurrent and metastatic disease as well as tumor of unknown origin or primary origin and some rare tumors with no specific standard of care can also be analyzed in this platform. But of course results need to be guided in terms of how they could actually be used and the evolving concept now varies with patients, clinicians, guideline committees as well as payers and pretty much within the contextual stage of the disease whether it's primary or metastatic within the tumor type. The guidelines and FDA approved drug labels as well as inclusions for clinical trials and anticipation of additional genes and mutants in the near future needs to be there and it is not a binary action, it is actually a continuum of evidence because the tumor can evolve especially when there's metastasis and in that some of the drugs are quite new and there's just emerging and I think one of the best things about whether you target the primary tumor and knowing the genetic profile of that primary tumor one can also understand what the next treatment would be if there's resistance to no drugs. So this is pretty much like any other NGS counterpanels. There are some that have all the 400 genes there is but pretty much they are the more common genes and as I mentioned earlier interrogating them simultaneously gives you an advantage of knowing some of the other driver mutations that are not very common and it's actually enhanced compared to some of the other platforms that are readily available including thyroid sequencing. With NGS one can also multiplex patient samples and that reduces cost of testing and also hopefully it actually reduces reimbursement and patients cost out of packets and that you could also do sequencing targeted sequencing and modify the content after verification of the panel that was previously developed. So most of the panels are because of the technical considerations and they can use some other sequencing like sequencers like the proton or the PGM and they can use as I mentioned earlier alternate specimens like FNA and other cells. Cement flow after surgery in the OR or after FNA one can use any of the tissue types and transported to the pathology department so it could be that the tissue is prepared and formally fixed and it could be that they prepare the tissue blocks and then from the tissue block sections one can extract DNA and then quantify and proceed to DNA capture and enrichment and pretty much DNA sequencing. So just to give you a flavor mutation distributions in common cancer types pretty much from melanoma, colorectal, lung and breath they're not really that viable but just to show that they can have ranging from no mutation to actually four variants within the same tissue and one can actually prioritize which driver mutations can be targeted and in that also just to mention that germline versus somatic testing there's a little bit of a challenge in terms of using tissue and I will sort of give that part next after this slide. So how do we do annotation for tissue? Similarly there are actually databases that are publicly available but a lot of cancer centers, MSK, Masset and some of the other cancer centers have their own mutation databases and also there are available mutation databases that are publicly available but they're not curated very well so one should know and this is common knowledge in most labs that some of the publicly curated databases are not as good but one needs to understand that this is a very dynamic field and it could be that that mutation or particular mutation is not available in terms of classification at the moment but pretty much with all the other data sharing one can identify exactly or at least classify a mutation based on information from the different databases as well as different publications that are available. Mutations are identified and clinical relevance are given based on what is out there in terms of literature. As I mentioned earlier there are national and international guidelines that actually give us more information on how to treat or manage patients and that the tumor type and additional tumor type can also be tested and gives us more treatment options depending on what drug has been used previously and of course the ones that are up and coming and there are no FDA approved drugs that are available for that patient the patient can be identified to join clinical trials and mentioned earlier is the evidence are all based on publication as well as available mutation databases for solid tumors but it's not as simple as the germline pretty much primary tumors are heterogeneous and depending on how much tumor or how much of that particular tumor you had on your sample that would be the mutation or the variant that one can detect and so therefore at times sampling would be a good thing to do. Metastases also can differ from primaries and I mentioned earlier that tumors can evolve and they become resistant and that depending on what clone is present if one predominant clone is present and the primary tumor and that has been targeted by a drug early on then that could have been really wiped out but a secondary clone can be more resistant and be present or detected within the next testing and again as I probably would allude to and more importantly with solid tumors the copy number is very the low copy number mutations are very critical also to detect and so sensitivity is also a requirement for the validation of this kind of test. So again individual with cancer can have multiple tests and that of course reimbursement is also an issue in terms of using next generation sequencing based test. So there are other approaches out there they're commercially available in some laboratories they have larger panels ranging from 100 genes to 400 genes and it could be that you could use whole exome sequencing or genome sequencing with or without comparison to germline and that could be probably something that some labs would eventually be able to offer the more evolving or the more the hot one now is actually liquid biopsy or circulating free tumor DNA and it has its own pros and cons but it could be used for monitoring as well as for drug selection if it is validated properly. So in summary with all this advances in the past years we have access to genetic testing for cancer predisposition as well as solid tumor genetic profiling. There are important technical advances but there are also differences in different laboratories so they may vary in terms of performance and that one should be worried about how these tests were designed and the platforms that are used and pretty much they can be probably gathered from the laboratories that are actually offering this test and that the other distinguishing or the different shading points for the different labs that offer solid tumor as well as germline mutation analysis is the databases that they use at the same time there are a lot of recent publications on clinical utility of multiple gene panels and I alluded to one of the more recent ones and that overall the field of genetic testing for predisposition to cancer is becoming fundamentally important and providing clinical validity and utility and it does give hope to some of the patients who don't have the FDA drug-sensitive cancers and in that nowadays since we can do genetic profiling of tissue one can be guided on which drug can be used and that the targeted therapy will be better used than a shotgun therapy. So with that I think I have 10 minutes for question and answer. Thank you very much. Thank you Dr. Lechwell and is there anyone that has a question? Hi, this is Dr. Bob Wilden from HGRI that was a fantastic presentation I came in about 15 minutes late but I really enjoyed that. I have a question about the cancer predisposition genes and sort of the historical part of that so we think of BRCA-1 and BRCA-2 as very well established and clinically useful and do you think that that's because they are more common and more because they are... it was sort of first to discovery and first to market or is it because it is fundamentally more powerful than the other genes in the panel that you talked about? Okay, at least for breast cancer in our experience it's pretty much, as you mentioned, it is the more common ones in terms of... I'm talking about experience in terms of the seven gene panel and it is the more common... the BRCA-1, BRCA-2 are still the most common mutations that we find and in that of course there are some emerging or low-to-moderate penetrant genes like CHEC-2 and POP-B2 that we are actually getting more cases on but I guess because... the other thing too is because the BRCA-1 and BRCA-2 have been tested longer and who knows if there's... I mean, most probably it would stay as the most common one and the more dominant gene associated for breast cancer and a variant. But it's not fundamentally different than the others. It's just sort of more common and has a longer track record. I would say that is true for now. I guess the more testing or the more individuals are getting tested with the larger panels, we can understand more probably because the other... I'm a clinical geneticist too but the cancer is like to me, well, you're not testing a lot of other possible cancer syndromes. We are just testing mostly as you have alluded to because BRCA-1, BRCA-2 are the most associated one and it's more commonly tested. So again, will that landscape change? Maybe. And if you know more, probably you can share more. No, no, it just struck me that we think a lot about BRCA-1 and BRCA-2 and there's a lot of publicity and there was myriad genetics put a lot of publicity into it and that's not true of most of the other genes that are sort of coming to light now. So it kind of got this head start. It is a fairly high penetrance area which also helps it too. And I guess what I'm trying to point out is that we, I think, unconsciously make a division between BRCA-1 and BRCA-2 and then all the rest and I'm not sure that from a clinical standpoint that that's fair. And I'm with you in that. It's, I think, because the other thing too is that because we have more experience and we have more families that have been tested, we get all this referral most, I mean, not just from programs within a family but actually the ones that have been, I mean, had relatives who had been tested before. Which is another interesting point about that category of testing is that it has knock-on effects so even though you may use a panel in the pro-band, once you find, you know, associated mutations, then you can do targeted testing and the relatives are much lower cost. That's right, precisely. Hi, this is Dan Halibia-Horizon. Thanks for the presentation. That's been very interesting. I have a question about your comments regarding the recent studies talking about clinical utility for the tumor panels. And I think that one of the challenges we face in developing medical policy is using the right outcomes when it comes to demonstrating clinical utility. I think concern I had with these studies is that they really didn't look at any kind of hard outcomes. And I'm using this in comparison to what we've seen. I think that was called the SHIVA trial, which was several months ago. And that was, I think, the closer to the right kind of study design we'd be looking for, where the result of the panel would guide therapy and then we'd look at hard outcomes. Whereas these, in gem oncology, one of them looked at perhaps increasing the yield of abnormalities, which is relevant, but certainly doesn't demonstrate clinical utility when it comes to changing outcomes. And then the other trial was looking at, actually, I think they looked for targeted mutations, but when they actually looked at what happened, it was observational, and they found that very few patients actually did get the recommended targeted therapy. So are there other trials that, I mean, are my misunderstanding these recent trials? And the second question is, are there other trials which you think would be more convincing, consistent with looking for hard outcomes? Okay, so let's, I think, I don't know, the clinical actionability paper by Desmond and Company are the coworkers. It is a multi-center trial, and it's actually, they actually have demonstrated that 4% of their cases have significantly changed in terms of management. So I think that's like one of the more, up to this date.