Welcome to the ISLC course, Review of Diagnostic and Therapeutic Implications of EGFR Exxon 20 Insertions in Metastatic Non-Small Cell Lung Cancer. I'm Dr. Joshua Sabari, Thoracic Medical Oncology at NYU Langone Health Perlmutter Cancer Center, and I will be your presentation moderator today. We will start this activity with some brief housekeeping items. After the presentations have concluded, you may ask any questions by using the Q&A button feature located on your video screen. This activity is supported by an independent educational grant from Johnson & Johnson Innovative Medicine, Janssen. This activity has been planned and implemented in accordance with the accreditation requirements and policies of the Accreditation Council of Continuing Medical Education. The International Association for the Study of Lung Cancer is accredited by the ACME to provide continuing medical education for physicians. The International Association for the Study of Lung Cancer designates the live format for this educational activity for a maximum of 1.5 AMA PRA Category 1 credits. Physicians should only claim course credits commensurate with the extents of their participation in the activity. The International Association for the Study of Lung Cancer designates the live format for this educational activity for a maximum of 1.5 AMA PRA Category 1 credits. Physicians should only claim credit commensurate with the extent of their participation in the activity. All faculty planners and reviewers for the course today have disclosed their conflicts of interest. This information is provided on the following for the slide in your review. Our first presenter is Dr. Dipali Jain. Dr. Dipali Jain is a professor of All India Institute of Medical Sciences or AIMS in New Delhi, India. She is the editorial board member of the WHO 2021 Classification of the Thoracic Tumors and WHO IAC 2022 Lung Cytopathology Reporting System. She is a member of the Pathology Committee of International Association of Lung Cancer and Dr. Jain is a recipient of numerous distinguished national and international awards, including the IASLC Mary J. Matthews Pathology Translational Research Award. Dr. Jain, the time is yours. Thank you, Dr. Sabari for kind introduction. So I'm going to speak in this 20-25 minutes or so about conventional EGFR mutations such as L858R and exon 19 deletions and lung cancer, and then I'm going to talk about resistant mechanisms and conventional EGFR mutations, and then overview of EGFR exon 20 insertions, including its heterogeneous mutation sites and biological diversity. So I'll start with the basic structure of epidermal growth factor receptor protein. So this is an inactive EGFR when there is a ligand binding. Ligand may include epidermal growth factor, may include connective tissue growth factor, epiregulin, amphiregulin, anything which promotes cell growth. So these are ligands for EGFR, and when this ligand binds to the ligand binding domain of EGFR, it gets homodimerized or heterodimerized. So in this picture, it is homodimerizing with another EGFR, and upon dimerization of the receptor, what happens is that there is a phosphorylation. Phosphorylation of kinase domain as well as tyrosine residues, which you can see here, they also get phosphorylated, and ATP binds to the ATP binding pocket, which is present in between N and C lobe of the kinase pocket. And upon binding of ATP and phosphorylation, there is attachment of intracellular growth proteins, which kind of attach to these docking sites, which are phosphorylated tyrosine residues, and ultimately there is signaling pathway activation. So this is an active phase of the EGFR, this one is inactive phase. So what happens after activation of downstream signaling pathways, as I mentioned, these are few of them, the one is PIK3CA-AKT1 mTOR pathway, RAS-RAF-MAP kinase pathway, and then there are JAK-STAT pathways, etc. So these pathways are all growth-promoting pathways, and upon ligand binding and phosphorylation of tyrosine kinase domain, these pathways get activated. And then therefore, there will be cellular proliferation, angiogenesis, anti-apoptosis to promote cellular growth. So this is the story of normal EGFR, which I'm talking about. But what happens when there is a mutation in this tyrosine kinase domain, this doesn't need ligand binding at all. So without even ligand binding, this tyrosine kinase domain autophosphorylates, and it is constitutively activated, constitutively stimulated. So therefore, it is on all the time. So therefore, cellular proliferation goes on and on, and therefore, tumors develop because of this particular site mutation. This is the exonic distribution of entire EGFR, which has around 28 exons, and if we talk about tyrosine kinase domain itself, it spans from exon 18 to exon 24, and most of the mutations occur from exon 18 to 21. And this is the extracellular domain, which has alternating sites of ligand binding and cysteine-rich areas. Now coming to the type of EGFR alterations or aberrations, which we see in lung cancer, they are five types of mutations. One of them are common mutations, and other categories, uncommon or atypical mutations, exon 20 insertions, compound mutations, and resistance mutations. So I'll be talking about exon 20 insertions at last. First I'll talk about these common mutations, which are around 80 to 85% of all EGFR mutations in lung cancer. And most common ones are these two, in which in-frame deletions of exon 19 occurs, wherein amino acids residues from actually 746 to 750, that means five amino acids and 15 base pairs, because one amino acid is coded by three base pairs. So all in all, 15 base pairs deleted from exon 19. This is first of the mutations. And then second one is wherein leucine is substituted by arginine at the location 858. So that is a point mutation of exon 21. So if we see that panoramic view of chromosome 7 where EGFR sits here, and this is the zoom-in view of the tyrosine kinase domain, particularly exon 18 to 21, where all the mutations or more frequent mutations happen. So these two mutations, which I just described del 19 and L858 are, fortunately, these are tyrosine kinase inhibitor sensitive mutations. In contrast, there are tyrosine kinase inhibitor mutations also occur, and most of them occur in exon 20, as you can see here. But fortunately, they are lesser than TKI sensitive mutations. So common mutations respond well, as I said, to EGFR tyrosine kinase inhibitors, and they are actually a current standard of care for first-line treatment of advanced and metastatic lung cancer. So first-generation EGFR TKIs are gefitinib, arlotinib, second-generation TKIs are afatinib, etc. They show statistically significant longer progression-free survival as compared to platinum-based chemotherapy in the first-line setting. And if we talk about the mutation per se, what happens when there is a mutation in the tyrosine kinase domain, because of this mutation, what happens is there is an increased affinity of the ATP to ATP binding pocket. So therefore, the kinase domain is always activated because ATP is continuously binding to it. So therefore, there is cellular proliferation, and we see in clinics as it is in the form of lung cancer, which is oncogene-driven, because here EGFR is driving lung cancer. Now what happens when we give patients tyrosine kinase inhibitors? So this particular mutation, which we have talked about, they also increase affinity for tyrosine kinase inhibitors. So here, what happens, TKI displaces ATP, because it competitively binds or competitively inhibits ATP binding to the ATP binding pocket. So TKI, instead of ATP, binds to the kinase pocket, and therefore, there is cellular proliferation, which is checked by tyrosine kinase inhibitors. So this is precisely the mechanism of action of tyrosine kinase inhibitors. And if we talk about conformational changes or crystallographic structure of EGFR tyrosine kinase domain, what happens here in inactive phase or inactive form of wild-type EGFR is that the C helix, I showed you N and C lobes of tyrosine kinase, wherein alpha C helix is attached to the C lobe, and when EGFR is in inactive phase, the C helix is in out position. That means it is farther from the ATP binding pocket. So there is no ATP binding if EGFR is in inactive phase. So you can see here also alpha C helix is in out position. And if receptor is just, you know, without dimerization, this alpha C helix is in out position. So therefore, there is no kinase pocket activity. In comparison to active form of EGFR, even if it is wild-type, what you see here is that because of, you know, dimerization of the receptor, you know, upon ligand binding, there is dimerization. So therefore, this C helix loop goes inside the pocket. It actually goes towards ATP binding pocket. So therefore, there is ATP binding is happening, and therefore, cellular proliferation goes on. So this is inactive phase where C helix is in out position. This is active phase of kinase pocket where C helix is in in position. And what happens when there is a mutation? So this is one of the commonest mutation, exon 19 deletion. So this is a stretch of amino acid residues. I said five amino acid residues, excuse me, or 15 base pairs where this particular portion is kind of deleted, and this pulls this C helix into inside the pocket. So therefore, this is constitutively active all the time because of this particular mutation. So this goes off from here and the C helix pulls inside the, it is pulled inside the kinase pocket. So these are the common mutations and coming to uncommon or atypical mutations, all mutations except del 19, L858R and T790M mutations are uncommon mutations. Most frequently identified ones are these, out of which exon 18G719X is more frequent out of all these uncommon mutations. So X denotes here that it may be more than one amino acid. It's not just, you know, always, for example, here it is I, that means isoleucine is replacing serine, but when X is, you know, present, that means any amino acid can come at this particular position. So that is what it means. Apart from all these three mutations, there are very rare kinase domain duplications occur as an uncommon aberration in EGFR mutation, but this is very, very uncommon in which what happens is tandem in-frame duplication, entire, you know, kinase domain gets duplicated from exon 18 to 25, but it is very rare. And then second is EGFR amplification, oncogenic fusions can occur as an uncommon mutation or uncommon aberration, but they are very rare. Then coming to compound mutations, they may also be called as double mutations, complex mutations or multiple mutations. They, as its name suggests, there are multiple independent mutations in the same sample accounting for 4 to 26% of all EGFR mutations. So any combination can occur in compound mutations, but the most common combination is to have common and uncommon mutations. So for example, del 19 along with some exon 18 mutation and so on. And the second combination which is common is to have common or uncommon mutation with a de novo T790M mutation, which is a exon 20 mutation, wherein methionine is substituting threonine at 790 locus. So this is another common combination. Then coming to the resistance mutations, and we know that one of the most common resistance mutation is T790M, which emerges under the pressure of first generation and second generation tyrosine kinase inhibitors. And the presence of this mutation confers sensitivity to osimaltinib, and this is one of the actually clinically validated EGFR second site mutation, which is the most common cause of TKI resistance. This is just a simplified diagram of showing various possible mechanisms of, you know, a disease progression at cellular level in patients who are taking EGFR TKI. And as one can see here, the ideal response is that death of all tumor cells, which is very, very uncommon, but the common ones are to have genetic resistant mutations. For example, de novo T790M, which is present and under the pressure of TKI, it kind of outgrows and goes out of proportion. So therefore tumor stop responding to tyrosine kinase inhibitors. So this mechanism can be on target, that means if it is EGFR T790M, that means it is including or it is involving EGFR gene per se. But then there are off target mechanisms, that means genes other than EGFR can be activated. Another mechanism is to have histologic transformation from, you know, from adenocarcinoma to begin with, it turns out to be small cell or squamous cell carcinoma under treatment of TKI. Sometimes they can show EMT, they just become sarcomatoid and they don't, you know, they don't behave like adenocarcinoma. So in conventional EGFR mutations, the first and foremost is T790M. So what it does is that it replaces or displaces first generation TKI. So here, instead of first generation TKI, now T790M is increasing affinity for ATP. So it is reduced, it is reducing binding of TKI to the ATP binding pocket. So therefore ATP goes and bind there, and therefore, you know, there is a stimulation of kinase domain. Apart from on target mechanisms, there are various bypass signaling mechanisms, for example, you know, activation of bypass signaling pathways, other tyrosine kinase receptor pathways, for example, R2, MED, KIT, these are other tyrosine kinase receptors, which also get activated. And then sometimes what happens is there is a co-dominant or co-oncurrent mutations which are present already at the baseline. And when EGFR is kind of taken care of by TKIs, they become drivers for the cancer, and they cause resistance to TKIs. So this is another, I mean, this is frequency wise, if you see after T790M in liquid biopsy as well as tissue biopsies, the other common mechanisms which are on target and off target include histologic transformation, as I said, PIK3CA mutations, MET amplification, and so on. But still in one third of cases, we don't know what is the exact cause of TKI resistance in first generation, after first generation, or second generation TKIs. But having said that, with the widespread use of osimertinib in first line setting nowadays, the importance of recognizing T790M has gone down, because that has been taken care of by osimertinib. But that is also not free of resistance. So patients develop resistance even after osimertinib, and these are the mechanisms of acquired resistance in which off target mechanisms are more common than on target mechanisms. Histologic transformation does occur after osimertinib. And frequency wise, on target mechanism C797X is the most common secondary EGFR mutation after osimertinib, and off target mechanisms, MET amplification is the most frequent kind of resistance mechanism after osimertinib. Then coming to the exon 20 insertion, which is an earliest EGFR mutation identified in NSCLC along with exon 19 deletion and L858R mutation, but it has not been much talked about because it has always been underdiagnosed. And this is, again, a larger subtype of atypical mutations, 4 to 10% of all EGFR mutations. It is similar to other EGFR mutations. It is also mutually exclusive for EL-ALC, ROS1, and KRAS, et cetera. This is an actionable mutation, but very heterogeneous. Some of the insertions are TKI sensitive, some of the insertions are TKI resistant. So from a biological and clinical and molecular point of view, it is a very heterogeneous entity. They don't respond well to conventional EGFR TKIs, as I said, except for some subtypes among which FQEA subtype is one of them. I'll just talk about in a minute. So they show, they confer some TKI sensitivity, but limited evidence of in vivo efficacy is currently available for second generation or third generation EGFR TKIs. Similar overall survival to EGFR mutation negative tumors and worse prognosis than classical TKI sensitive EGFR mutation positive tumors. They are characterized as in-frame insertions or duplications of between 3 and 21 base pairs that corresponds to 1 to 7 amino acids and clustered between amino acid position 762 and 774 of EGFR protein, I show you in a picture. And NGS is required to detect the full range of exon 20 insertion. As I said, they are commonly underdiagnosed because people use PCR based methods for EGFR protein detection and sole reliance on PCR based methods may miss approximately 50% of cases of exon 20 insertion. And if you see the picture again, the panoramic view of chromosome 7 EGFR sits here and this is entirely EGFR where this is tyrosine kinase domain from 18 to 24 or so. And this is particularly EGFR exon 20 amino acid sequence. And this is the location where most of the insertions take place. And so far, more than 122 EGFR exon 20 insertions have been identified. And if you can relate there, here it is P790M, which is on the same exon. But this is the area where 90% of insertions found between amino acid 766 and 775. And these particular insertions are TKI resistant. But if insertions occur proximal to the C helix, I'll show you these mutations or these insertions are TKI sensitive. So therefore, they are very heterogeneous, as I said. This is vertical view. I'll just show you picture wise so that you can make a mental picture that, okay, wherein the problem is. So this is a vertical picture of EGFR and here is a horizontal dimension. And this is exon 20, this is CT790M here. This is alpha C helix I showed you here. And this is a loop following C helix. And here are the most of the insertions take place. And this is the area where they are TKI resistant, whereas mutations or insertions occur in this area, they are relatively TKI sensitive. So if you see here, this is exon 20 insertion, which is present proximal to C helix and this kind of, you know, drag C helix inside the kinase pocket. And that's how the EGFR kinase domain gets constitutively activated. This is another picture to just show you how this insertion and this terminology, what does it mean? for example here d 761 e 762 so that means this is aspartic acid this is glutamic glutamine and in between these two amino acid there is insertion of some amino acids so they may be you know different kind of amino acids which are inserted and similarly here this is alanine and this is um i guess this is lysine and there are some amino acids which are inserted here so this is the terminology and this is this is the same picture which i have shown you and this is uh this is another picture where uh wherein you can see this is f u e a wherein f means phenylalanine this is uh i guess this is uh tryptophan glutamine yeah so this is glutamine this is glutamic acid this is alanine so these four amino acids are inserted at this particular site so as i said the mutations which are insertions which are present in the c helix they are relatively tki sensitive but most commonly mutations occur in this area where these are tki resistant type of insertions so most common mutations or insertions are in this area these two are the more most common exon 20 insertions which are present in approximately 20 percent of all egf exon 20 insertion mutations and this i have already talked about more than 100 mutations are identified clinical associates are same lung adenocarcinoma female non-smokers and asian ethnicity tk resistance as i said similar to t790m it increases the affinity of egfr for atp and prevent binding of first generation and second generation egfr tkis and pre-clinical evidences indicate insertions between 769 and 775 that is a loop following alpha c helix i showed you this might convert egfr tki resistance but insertions which are more proximal they kind of resemble classical sensitizing egfr mutations and thus potentially predicting egfr tki sensitivity this has been dealt by another speaker in this same webinar and these are the two agents which are kind of fda approved in the form of monoclonal antibodies and small molecule kinase inhibitors and we got accelerated approval for these these targeted agents these are exon 20 insertion tkis they act on exon 20 insertion and this is a monoclonal antibody which is not only for egfr but also targeted against met or hepatocyte growth factor so this will be discussed in detail in next section of this webinar so in conclusions i would say it's on 19 deletion l858r mutations are the most frequently occurring mutations in tki domain of egfr while these mutations are sensitive to tki but resistance invariably occurs due to various on target and off target mechanisms and one of the de novo tki resistant egfr mutation mechanisms is exon 20 insertions which as explained in the talk that they are clinically and biologically diverse they may or may not be showing sensitivity to tki because they are pretty heterogeneous in terms of insertions so with that i i thank you all for your attention thank you dr jane our next presenter is dr pilar gorito who will be talking about treatment approaches in egfr dr grito is an associate professor of oncology at the universidad de alcala and serves as the head of medical oncology department at the hospital universitario ramon y cajal in madrid spain she is the co-director of the cancer research group at the instituto ramon y cajal de investigaciones in santeria her main areas of research and clinical interests are in thoracic malignancies so dr grito welcome and and the floor is yours so thank you again for giving me the opportunity to participate in this very interesting talk to speak about treatment approaches in patient with egfr mutation as i has already been mentioned egfr mutation are common particularly in asian population only 10 to 40 percent of non-small cell lung cancer tumor in western population and usually we classify patient in three different subgroups depending on the type of location because they have different prognosis and different treatment approaches so the most common subgroup is the common egfr mutation representing about 85 percent of cases the second most common is the subgroup of patient with tumor with egfr exome 20 insertion mutation and the last one is the uncommon egfr mutation subgroup that is williatherogen as has been already mentioned the key point is that for patient with advanced disease the the recommendation about frontline treatment are different depending on the type of mutation according to the ESMO guideline and also according to the MCCN guidelines so for those patients with common common sensitizing mutation meaning the the most common subgroup of mutation the standard first line approach is to use oral tki and egfr tki there are different generation first second and third generation this is the standard first line approach while for the subset of patient with egfr exome 20 insertion mutation the standard first line approach at least until the last ESMO meeting and i will talk about that later on the standard approach was or is chemotherapy and only at the moment of disease progression targeted agents are an option so starting with the review the review of the management of patient with common egfr mutation we know that we have different oral egfr tki is first second and third generation but usually at least in my country the the goal standard is the use of osimertinib because based on the result of the flaura trial osimertinib demonstrated advantage in terms of median pfs and also intensive overall survival when compared to as a first generation so when thinking about the design of clinical trial and considering that osimertinib is the standard frontline the standard option of the standard arm the numbers to take into account are median pfs 18 months and median overall survival 30 39 months or 20 months so we know that we need better option we need to improve progression for survival and we need to improve overall survival and there are different strategies some of them are focused on preventing or delaying the progression and this can be done by adding some additional treatment to our standard egfr tki different options could be the the addition to chemotherapy to egfr tki the addition to avimantamab to egfr tki because as i has already mentioned avimantamab is a monoclonal antibody with two target egfr plus also met and met is one of the most common mechanism of resistance at the moment of disease progression and the third theoretical option to prevent or delay progression is by adding anti-angiogenic therapy apart from modifying the standard upfront management in order to have better results that the other possibility is modifying the second line option in order to have a better survival at home so i will review briefly these different strategies starting by the the results of clinical trial focus on demonstrating the potential value of adding chemotherapy in this slide we can see a trial named again presented the year during the ASCO meeting and as you can see in this particular trial the standard arm was egfr tki alone at the beginning gefitinib and then they moved to oxymertinib and the experimental arm was complies by oxymertinib followed by chemotherapy and then oxymertinib or gefitinib at the beginning the primary endpoint of the trial was overall survival and as you can see the study was negative for this primary endpoint however during the last wclc meeting in singapore we learned about this trial the flaura2 study that was focused on the combination of oxymertinib plus chemotherapy in this particular trial patients went randomized one-to-one to receive oxymertinib alone standard treatment versus oxymertinib plus chemotherapy platinum based chemotherapy platinum plus pemetrexet followed by maintenance with oxymertinib plus pemetrexet the the primary endpoint was progression-free survival by investigator assessment and the stratification factor were race egfr testing modality and performance status and as you can see here the study was clearly positive demonstrating a benefit in terms of progression-free survival that was not only statistically significant but also clinically relevant improving nine months a pfs apart from this primary endpoint what that was pfs per investigator the the other outcomes focus on the efr subtype the cns metastasis and the different subgroup for all this subgroup the all the as you can see in the different picture the figure were in favor of the combination strategy including also the tumor response but this benefit in terms of longer pfs as i mentioned a clinically relevant comes at the cost of higher toxicity and this is important and is part of the divide we are currently involved in terms of grade three or superior toxicity as you can see here the combo had a grade three adverse event 64 percent versus 27 of course more hematological toxicity as expected and the percentage of adverse event leading to this continuation 11 versus 6 interaction 43 versus 19 that reduction 10 to two three percent apart from the scenario of exploring the role of adding chemotherapy as i mentioned another potential option is to use in addition to the efr tki tki monoclonal antibody also targeting a cement because it's a very common mechanism of resistance so this is the rationale for this study lasertini plus avimantamap was explored in a phase one trial with multiple cohorts as you can see here exploring monotherapy with avimantamap on the combination and one of the cohort on this phase one will provide the rationale for the phase three trial the mariposa study presented the year during the small meeting in terms of the the the standard arm as expected osimertinib they include two experimental arm avimantamap plus lasertinib randomization 222 and also lasertinib monotherapy as to assess the contribution of the compound although these are the randomization was one to do the the study was positive again the primary endpoint was a progression-free survival in this particular study was by blended blinded investigator assessment is external assessment and in this particular trial all patients required serial brain mri so all the information about my metastasis was really a value the stratification factors were different when compared to flaura 2 efr mutation subtype asian race and history of brain metastasis and the study as i mentioned was positive for the primary for the primary endpoint pfs improving median pfs by seven months and similar way that the flaura 2 the key results were consistent consistent benefit in patient with or without brain metastasis and it's important to take into account that this is a combo without chemotherapy and also benefit across all predefined subtype but again this benefit is associated with higher toxicity and as i mentioned this is part of our current debate but we are using a combo with efr and met activity this the adverse event are linked to this so the most common adverse event were related to efr inhibition paronychia ras diarrhea stomatitis but also met inhibition hyperbulinemia or peripheral edema in terms of number as you can see here the percentage of patient that required that presented adverse leading to to that was similar serious adverse event 49 versus 33 percent and the third strategy when thinking about how to delay the progression is by adding anti-bgf drugs and this strategy is based on a preclinical clear rational but also based on some clinical data particularly coming from the combination of adlotinib plus bevacizumab or ramutirumab but most of us were waiting for the result of oxymertinib in combination with anti-bgf therapy unfortunately this study published last year is exploring the role of oxymertinib plus bevacizumab at front was negative in terms of the primary endpoint pfs and also a due to the high toxicity profile with a almost 56 percent of patient presenting adverse event leading to treatment discontinuation the year during the ASCO meeting two studies were presented focus on oxymertinib plus ramutirumab with different designs and with different results the american study is randomized patient to receive oxymertinib alone or oxymertinib plus ramutirumab every three week randomization two to one different follow-up depending of the arm and pfs by investigator at primary endpoint however the japanese study the oxygram one study randomized one-to-one oxymertinib monotherapy standard oxymertinib plus ramutirumab the same dose but administered every two weeks a primary endpoint pfs by a external a committee evaluation the north american study was positive the japanese study was negative so i don't think that this phase two trial are going to change our standard of care so moving to the first line scenario in the subset of patient with common mutation to the scenario of pre-treated patient according to the guidelines we have chemotherapy as best second option after progressing to oxymertinib in spite of knowing that there are different mechanism of resistance some of them as have been already mentioned by my colleague a called efr on target mutation resistant or the mechanism of resistance are off target but currently we don't have targeted agents to treat differently this kind of patient so in this scenario during the the small meeting we learn about the mariposa two trial that was focused on improving the result after receiving oxymertinib in this particular trial the standard arm was chemotherapy according to the guidelines and two exploratory arm were included adimantama plus chemotherapy and avimantama plus lasertinib plus chemotherapy randomization two to two to one and stratification factor the oxymertinib line of therapy most of patients receive oxymertinib but for those patients who receive jefitinib or erlotinib and at the moment of disease progression have a t7 at the end mutation and receive oxymertinib this subset of patients were also possible to be enrolled in the clinical trial in terms of the primary endpoint the study was positive because as you can see here chemotherapy alone the results were inferior in comparison to any of the arm having avimantama the problem as always is that the toxicity is increased by combos particularly in the in the in the setting of patient receiving the four drugs and in fact while the study was a enrolling patient according to the idmc they identify higher hematological toxicity in the four drugs arm and they modify the regimen in terms of saying that a lasertinib has to be a start after completion of carboplatin so it seems that this is a good strategy but maybe four drugs in the in this particular scenario is too too high apart from the potential role of avimantama that maybe is will be moved to will be moved to the first line scenario we have results of two a disease in this scenario particular patient one is based on two clinical trial presented this year one is the tropion o5 that is a study phase two study exploring the role of that or the rustican in patient with genomic alteration actionable genomic alteration and as you can see more than 50 percent in particular 57 percent of patient had efr mutation and in this subgroup of efr mutation patient already treated the response rate was really interesting 43 percent of cases the median duration of response seven months the problem as always is the toxicity the toxicity particularly with this drug is stomatitis 10 severe stomatitis 20 percent of grade two stomatitis that from the clinical point of view could be also a problem also ocular toxicity and of course infusion related reaction that are also very common when when using avimantama and the last drug to be mentioned in the setting of common mutation is this drug the patitumab de rustican another adc explored in the setting of efr mutated already treated patient within the lung to trial and as you can see here the response rate almost 30 percent the median pfs 5.5 months so there are many changes potential changes many new drugs and many new option in this scenario and now i would like to talk about the management of patient with advanced non-small cancer efr exon 20 insertion mutation as i mentioned is the third most common subtype of patient with efr mutation and currently is an additional subgroup of patients suitable for targeted agent although it's true that this is very recent because according to guidelines the standard first line approach is chemotherapy but the results are very poor the response rate is less than 30 percent the median pfs is really short when using chemotherapy alone so based on different study we had different option at the moment of disease progression one of the options were a oral tki's efr tki such as mobile 13 mobile 13 was initially approved based on the result of phase one trial more effectively is an irreversible tki designed to selectively target efr and head to exon 20 insertion the first set of data based on 28 patients demonstrated in this scenario and this very difficult disease at that time a response rate of 43 percent pfs of seven months and some interesting data also in patient with brain metastasis so based on that the exclaim was the expansion cohort or that finally enrolled 114 patients to validate the result of phase one. Based on these results, FDA granted an initial approval. It's true that the result of this claim trial in terms of overall response rate was only 28% in comparison to the initial set of 43%, but the median progression-free survival was similar. The problem with this drug was the toxicity, particularly the diarrhea, 21% of brain death. It's really diarrhea. Anyway, the company with this result decided to go to the first line with a design exploring mobotherapy versus chemotherapy. And unfortunately, although we don't have the results, the company confirmed the voluntary withdrawal of the indication in the FDA based on the outcome of these results. So we don't have currently the possibility to use mobocertinib for our patient in this setting. Another oral TKI is sumbocertinib, a Chinese drug, two-pivotal second-line trial. Again, interesting results in this subgroup of patient. Again, a problem with the toxicity, particularly diarrhea, but based on the results, overall response rate, 45%. Interesting result. FDA granted approval. FDA is now running the phase one in the first-line setting comparing to chemotherapy. Another drug that I wanted to mention is GLU459, because this is very interesting, although the results are very preliminary based on the phase one, in which 75% of patients had already received a targeted AEN. And this is important because targeted AEN in EGFRX on twin insertion is going to be our standard of care, is and will be our standard of care. So we need drug in this scenario. And the key point of this drug is not only the response rate, but the toxicity, because no grade three diarrhea or risk has been seen. So moving to the scenario from oral TKIs to monoclonal antibody, we have avimantamab, albile-able, in post-platinum exon twin insertion population, based on the approval of FDA and also based on the approval of EMA. This approval is coming from the results of the arm of the CRISALIS study focused on this specific population. The initial results were published in GCO in 2021, demonstrated that overall response rate of 40% and a median duration of response of 11 months, as I mentioned in this very difficult clinical scenario. The long-term results were presented at ALCC this year, in April this year, demonstrated a median overall survival on 23 months and two-year overall survival of 47%. So no new data in terms of toxicity, no new concern. So we have currently the option to use avimantamab at targeted agents after chemotherapy in this patient. But probably this is going to change because the third key trial we learned in the recent meetings was about the role of avimantamab in combination with chemotherapy in a chemo-naïve patient in the first line. This is the Papillon study that compare our standard of care chemotherapy versus chemotherapy plus avimantamab. The stratification factor, ECOG-PFS, brain metastasis and prior use of EFR-TKI. So here we can see the toxicity unexpected. This detoxicity is the combination of chemotherapy toxicity plus avimantamab toxicity related to EFR inhibition or MET inhibition apart from the infusion-related reaction. It's important to take into account that the duration of treatment was longer in patients receiving the combination. So it's important when thinking about toxicity that also the median treatment duration was almost 10 months. In terms of the most common adverse event, as I mentioned, infusion-related reaction, 42%. It's easy to manage this kind because most of them are grade two or grade one or two in the first cycle. The most common adverse event in the combo pionic and RAS in chemotherapy, of course, anemia, neutropenia, nausea, similar rate of serious adverse event, similar rate of discontinuation, treatment-related discontinuation due to avimantamab, only 7%. In terms of the efficacy, the study was clearly positive. Primary endpoint progression-free survival by blinded investigator committee has a ratio of 0.39. So really not only statistically relevant but clinically relevant. This is why I said that probably we are going to change our standard of care and the interim overall survival median outreach in the combined versus 24 months in the chemotherapy alone. And benefit across a predefined subgroup and response rate. So my last slide, different EFR mutation require different approaches. For patient with common sensitizing EFR mutation, there are different option for systemic first line at front. And we need to debate about what is best and what are the subset of patient to receive chemo plus osimertinib, flaura trial, the mariposa avimantamab plus lasertinib. We also have new option in the second line setting and we have option for patient with EFR exon twin insertion including potential first line new standard of care. So thank you. Thank you, Dr. Garrido. It's amazing how many opportunities and options are for patients now in the EGFR mutant setting. I wanna now turn to Dr. Natasha Leal who's going to be discussing next generation testing in metastatic non-small cell lung cancer. Dr. Leal leads the thoracic medical oncology group at the Princess Margaret Cancer Center. She's a professor in the department of medicine and an adjunct professor at the Institute of Health Policy Management and Evaluation at the University of Toronto. She holds the OSI Pharmaceuticals Foundation chair in cancer, new drug development through Princess Margaret Cancer Foundation. Dr. Leal, the floor is yours. Thank you so much, Dr. Sabari. And I wanna thank the other speakers as well and the whole team. I'm delighted to round off the session talking a little bit from a clinical perspective about NGS and biomarker testing in our patients with advanced non-small cell lung cancer. So I'm gonna talk about the current list which of course is growing rapidly of targetable genetic alterations or genomic drivers in lung cancer, the importance of next generation sequencing as part of what we do in our routine practice and talk a little bit about the challenges globally. So it's been great, this is an old slide now but I think it's really illustrative of how far we've come. Just before I started fellowship, we used a toposide and platinum for everything but things have really changed, better treatments by pathologic subtype then of course genotyping and now with PD-L1 testing and immunotherapy, we've just made such tremendous progress and really lung cancer is now considered one of the leading sites for precision oncology. And it's really been thanks to Dr. Jain and our many colleagues in pathology and genomics. So today, there's a long list of alterations that we test our patients for, as we've heard from Dr. Jain, EGFR, ALCROS1, BRAF, TRAC, MET and RET and that's for first line treatment decisions if you're in a jurisdiction where these are available and of course, second line KRAS, we heard a bit about exon 20 insertions and the potential to move treatment to the first line from Dr. Garrido and of course her too. And for everyone else, there's immunotherapy or chemoimmunotherapy depending on where you live and what the PD-L1 status is, but it really is amazing that there's this incredible wealth of options for our patients just through identifying biomarkers. So these are some of the current recommendations for biomarker testing and as you can imagine, the more up to date these lists are or the guidelines are, the more orange you'll see on the slide in particular for things like EGFR mutations, we now know that we also need to test in early stage disease, so patients with tumors that are three centimeters or larger, fusions we heard about in ESMO now, very relevant in early stage, PD-L1, I think something for discussion in early stage as we just talk about perioperative therapy and immunotherapy and then of course, the remainder of this list and certainly where there's any blue or gray, this is really just a function of the time of the update and I'm sure that the next version of the ISLC biomarker testing guidelines will include all of these and perhaps even more. So what do the guidelines say about how to test these at least nine different genes plus PD-L1? And I think that by and large multiplex panels have been recommended for more than five years now compared to single gene testing and so you can see at the top right of this slide, this is for a multi-gene targeted panel where you get all of these tests simultaneously versus one by one. Of course, the benefits of going sequentially or excluding is that you can stop testing once you get your marker, but the trade-off of course, is that each of these take time and patients often run out of tissue. The NCCN recommends broad-based panel testing and ESMO has gone a step further to strongly recommend NGS testing for level one or targetable alterations either in tissue or plasma with patients with non-squamous, non-small cell lung cancer, preferably using RNA-based techniques that allow us to better capture fusion events. But of course, what you find depends a little bit on where you live and where your patients are from. We know that in East Asians, the proportion of patients with EGFR mutant lung cancer is much higher, anywhere from 40 to almost 60%. Other alterations, fairly similar, I would say, perhaps KRAS alterations or G12C, a little bit less common in patients from East Asia as opposed to patients from Europe. And certainly in that group of patients, we see EGFR alterations, for example, at anywhere from 15 to 25%, but KRAS alterations, including KRAS G12C are more common and the rest of these are fairly stable around the world in terms of frequency. And because of these variances in molecular epidemiology, this has really promoted different testing strategies. So, if you're extremely likely or you've got a 60% chance of your patient having an EGFR mutation, you're much more likely to really focus on that as a single test before reflexing to other tests as opposed to other parts of the world, for example, Europe and North America and perhaps even Latin America where getting that broad base upfront really does lead to benefit. And so this is an interesting consensus recommendation that was recently published in the Journal of Thoracic Oncology by a number of our colleagues from Asia. And they recommended either single gene testing with reflex to a multi-gene panel, acknowledging the issues of cost with NGS versus upfront testing for everyone, really endorsing both and certainly in these recommendations, you want to adapt this to your local context and I'll talk about some of the local relevance. So, what are some of the challenges of NGS? We all talk about it and yet when you go to World Lung or other meetings, not everybody is doing it and even in places where it may be funded or widely available, even then not all of our patients are getting it. And so this was a great survey conducted by the ISLC about six years ago now and I'm delighted that Dr. Smeltzer and colleagues are actually repeating this survey to look at molecular testing and the barriers around the world. Cost in the gray bars is the most frequently identified barrier. Tissue or in particular the lack of tissue in orange is another important barrier, certainly something that we have challenges with in Canada. And of course, who orders the testing and who knows what to order? These are key issues that we really need to work out, particularly in systems where testing is centralized and time of course, very important. So many of our lung cancer patients are diagnosed late in their journey and so really a challenge to get NGS testing and getting patients to that first precision therapy in time is a major issue in almost all countries around the world. So we've learned that even in the United States, less than half of patients with advanced lung cancer receive comprehensive biomarker testing either at any point in their journey or in particular before starting first line therapy. And this is a really nice study from the US Oncology Network and I was actually meeting with them a few weeks ago and they talked about how much scrutiny they came under for publishing these results. They showed that just under 40% of their patients had NGS testing. This is between 2018 and 2020, but you can see that these numbers are rising on the left. They also looked at how many patients had their results available to that first initial treatment decision. This was really focused on five biomarkers. You can see it's only 35%. Another 11% had biomarkers ready later after starting first line therapy. So still that potential to personalize the journey but more than half never got their results at all. And so I think it's a really great lesson that if you don't know where you are, you really can't improve. And I'm really impressed that their new target is to make sure that more than 85% of their patients have comprehensive testing before first line therapy. And again, I think it's really valuable for people to look at their own experience. And so what we learned from this in Canada was that to really maximize benefit for patients, we really need to get that going routinely for all patients and as early as possible to avoid missing key subgroups. And so when we looked at this in Canada, we've had single gene testing up until about 2020 at our center. And we had a program where we offered patients free NGS testing. They just had to send us tissue and enroll. And what we found was that after single gene testing, almost 27% of our patients didn't have enough tissue to actually have NGS. So the first lesson we learned was best test, first test. And so important to really prioritize those very small diagnostic lung cancer samples for the best possible test that you have. Interestingly, even in this group of patients, we found 35% had alterations that were not identified with routine single gene testing, EGFR Alkylmeros-1. 70% were able to consider clinical trial options that they hadn't had before. And 15% switched to new treatments as soon as they got their results done. And this increased over time to almost a full 35%. We also found that it wasn't a large incremental cost, although of course this varies. There are some countries where the drugs aren't affordable. So of course, testing is much less of a priority and other places where this type of incremental cost per case can be quite challenging. The good news of course, is that the cost of testing is coming down. So as we integrate other options into lung cancer diagnosis, we're learning that liquid biopsy can play a key role. We've learned both in studies from the US and also studies from Europe, that up to 43% of our patients may not have adequate testing for upfront NGS. This is a very nice study on the right from Dr. Shara Agarwal, where when she looked at their experience between 2019 and 2020, only a quarter of their patients with advanced non-squamous, non-small cell lung cancer were able to get comprehensive molecular testing in tissue. So those are the blue bars at any point in time, 20% prior to the first line treatment decisions. And when she added plasma testing or liquid biopsy into this, that number increased significantly, 76% overall and 80% prior to first line testing. So what are some of the benefits? And so of course, obviously you have the potential to access all of these great new therapies. You know, we established a pilot program where we built the business case for doing routine NGS at our hospital in Canada. And, you know, we thought we might find alterations in 30, 40% of patients. And we actually found alterations in 54% of our patients when we routinely did comprehensive NGS testing in our non-squamous, non-small cell lung cancer patients. Now, one of the challenges of course is turnaround time. For those who do not use systems like GeneXus, it is harder to get NGS test results and can often take anywhere from two to four weeks, which is longer than ideal, but there's a lot of work ongoing to try and improve this. So one of the other great things about NGS testing is we've learned that it can be very sensitive. And so on the topic of each of our Exxon 20 insertions, we've seen some very nice data from several databases now that show that if we look at PCR, you can identify about four, maybe five common variants of each of our Exxon 20 insertions. But if you identify NGS, you can double the number of alterations that you find. And all of these patients have gone into clinical trials that Dr. Garridos talked about and derived benefits. So again, so important for so many of these alterations that we remember that using PCR can miss up to half of the population where we want to offer targeted therapy. So very, very important for NGS. And of course, this is true in many other types of alterations as well, as well as common EGFR mutations, where we know that NGS is more sensitive and can pick up more alterations. We do know that targeted therapy does outperform chemotherapy. We saw some nice data looking at patients with RET-positive fusions from ESMO this year. And you can see I've got duration of response across the X-axis here and response rate on the Y-axis. And whether you look at chemotherapy or even chemoimmunotherapy, which I would say would probably come up to about the 45 to 50% range and up to about nine months here, you can see that patients with targeted therapies tend to have higher response rates, less toxicity, and longer durations of response, even when we don't have randomized trials for all of these. And of course, we're working to continuously do better. So how does testing and treatment impact on patient survival? And there's some nice retrospective data that show that even simple things like doing EGFR and ALK testing may associate with longer survival. Patients in blue from Italy, Spain, Australia, Korea, Taiwan, Japan, and Brazil had longer survival times if they were able to have testing and that option to access treatment compared to patients in red. Of course, there is some selection bias here and some prognostic things that we count on control for. But there's some other studies now. This is a nice study from Japan where they looked at patients who were able to access first-line targeted therapy on the left, those who had complete biomarker testing and actual alterations found versus those who could not, shown in red. And in particular, even in the second-line setting, when they looked at patients who had complete genotyping versus patients who didn't, the patients who had molecular testing had much better outcomes, particularly when compared to patients who were later discovered to have targetable alterations and did not receive targeted therapy. So we've learned that things like liquid biopsy can really help increase the odds of complete genotyping prior to making first-line treatment decisions. And we've also seen some recent published data, this was just published in JCL Precision Oncology, that suggests that this can also associate with survival. Now, I think some of this is certainly access to better treatments and targeted therapy, although some of it, again, will be selection bias. Clearly, those patients who couldn't wait for molecular testing or couldn't even get a liquid biopsy probably have a poor prognosis, although in this analysis, they did control for multiple factors such as performance status. So again, you know, really very interesting to see the potential for the impact of targeted therapy and complete molecular testing and the association with the outcome. I know there are a lot of drug studies where we'd like to see the curves separate like this. The other challenge of course, is when we don't find the target initially and patients go on to get chemo immunotherapy or immunotherapy and then later switch, we've shown really across different targetable subgroups and across different compounds that patients do worse. They have a higher risk of adverse events. For example, osomertinib after checkpoint inhibitors, much higher risk in about a quarter of patients of severe pneumonitis, colitis, and hepatitis. Similar data have been presented with sotavacib, chrysotinib, and metinhibitors where very consistently, if we fail to identify that marker upfront, patients get a checkpoint inhibitor or chemo immunotherapy and then switch to targeted therapy, they are at risk of higher side effects. And so, so important to really double down and make sure that patients have complete testing before we start treatment. If the patient needs to start and you don't have data yet, the common practice has become that people start chemotherapy without a checkpoint inhibitor, just to give you that extra week or two to get the molecular results and to avoid these types of toxicities. So, you know, I think certainly liquid biopsy has been very exciting as an addition to improving our ability to genotype patients easily, particularly with lung cancer. Of course, we need tissue. This is how we get our diagnosis, our pathologic subtype and PD-L1. Although, you know, certainly plasma has been very helpful, especially if you're going to look over time or in resistance. And so how do we put this together? Do you wait for tissue to fail and then look at plasma? Do we put the two together? And in Canada and some other countries, we're looking at even plasma first, just because it takes so long to get a biopsy as part of the diagnostic workup. And so this is a study that we did where we looked at patients that were light or never smokers. They had a diagnosis of lung adenocarcinoma and we did a liquid biopsy test in all of these patients. We found that more patients, when we added plasma testing to tissue testing, were actually able to have an alteration detected and go on targeted therapy, higher response rates, as you can imagine. And also when we added liquid biopsy, really as part of this concept of enhancing complete testing, patients actually had better quality of life, again, because they were able to access that treatment. And in terms of cost, you know, again, cost is really a very, the meaning of the different studies is very variable based on where you're from. Certainly if you do both, it doubles the cost. But in particular, what we found in Canada was that that's really a drop in the bucket. That first red arrow are the very small costs of testing. It's really the cost of treatment that outweighs everything else. And in the Canadian system, you know, giving patients with an actionable alteration that hasn't been discovered, immunotherapy is actually very expensive and very ineffective. And so we're actually able to save money on that, leading to something very cost-effective. So, you know, I think for many of us, the barrier really is cost. I think if NGS were highly affordable, we would be doing it much more routinely across a number of different settings in lung cancer. And these are some very nice data on the left from Nate Pennell and colleagues, where they actually looked at Medicare-insured and commercially-insured patients, and they showed that very consistently, upfront NGS was less expensive than all of the other possibilities. Sequential single gene testing, you know, testing for EGFR or KRAS, and then reflexing to NGS, or just looking at specific gene hotspots. And that included both the cost of re-biopsy, among other things. It was also faster. You know, you could get results faster and start your patients on treatment faster. And also they've updated their work with a recent cost-effectiveness study, showing that this is very highly cost-effective, $16,000 per life year gained. Just to give you an idea, things like antiemetics are several hundred thousand dollars per quality gained. There's also a nice study over here on the right. This is a Canadian study where they really looked at the downstream costs of delayed testing. So admissions, restaging, palliative care, people who really lost the opportunity to have targeted therapy. And what they found was that the most cost-effective or least costly approach was upfront NGS in green. I've got a green arrow there, compared to all of the others. Again, really bringing in this cost of delayed testing and lost opportunity. In Asia, it may still be very feasible to do upfront NGS. This is a nice study from Singapore, from Aaron Tan and Daniel Tan and the team there. And you know, what's very commonly done in Asia is you do EGFR testing. If it's negative, then you reflect to NGS. And so you will get EGFR results a little bit early, but for those patients that are EGFR negative, anywhere from 40 to 60%, that's quite delayed for them to get their results. So when they looked at upfront NGS for everyone, they found that people were able to get their NGS results faster, just a few days after or at the same time as the initial EGFR testing results in the EGFR first arm. And the cost was very similar, only about $100 difference. And so of course, in Singapore, this is very commonly used throughout. It's also a very nice study from Hong Kong, from Herbert Leung and his group, where they looked again at upfront NGS versus sequential or exclusionary testing. And what they found was cost-wise, exclusionary testing was best for the budget. You know, you do EGFR or KRAS first and then test from there, but upfront NGS was the best for patients. It really made sure that patients had their alteration identified as quickly as possible and got to treatment. And based on that, you know, despite the incremental budgetary cost, Hong Kong has now launched upfront NGS in most of their public hospitals, which is really terrific. So I do think that upfront NGS testing should be standard of care. Again, of course, you know, you need to do what is most relevant and most possible for where you live. But certainly if you can achieve NGS upfront, you are more likely to find actionable alterations faster than the other approaches. You need fewer repeat biopsies. You have fewer missed alterations, for example, half of EGFR XL20 alterations, and fewer missed opportunities for targeted therapy and better outcomes. The economic impact ranges anywhere from cost savings in the US and Canada to minimal added costs. And there's some studies from Europe as well that I didn't show and Asia, as we've talked about. And I do think that as sequencing costs fall and technologies improve, more countries will be able to adopt upfront NGS, both in advanced disease. And now as we're learning, this is also critical in our early stage patients as well to help more of our patients get access to precision oncology with much better outcomes. With that, I wanna thank you so much and I'm gonna turn it back to Dr. Sabari. Thank you, Dr. Leo, it was an excellent presentation. So I ask all the speakers to come back on camera. We have about 15 minutes here for question and answer. So Dr. Jane, I wanna start with you. You went through the different EGFR mutations. One question from the audience is, does mutated EGFR need dimerization to signal? And we'll build upon that with mechanism of action of some of the therapies, but what is unique about EGFR mutant disease? Yeah, the question was whether mutated EGFR needs dimerization. So the answer is that when I showed that picture, when the EGFR is wild type, of course it needs ligand binding and then ultimately dimerization of the receptor and then phosphorylation. But when it is mutated, it doesn't need ligand binding. So therefore there is no need for dimerization of the receptor to happen. And then phosphorylation and so on and so forth. So mutated EGFR does not need ligand binding and dimerization. Thank you. So when we think about therapeutics, TKI and how they function versus amivantamab, which you mentioned, Dr. DeGrito, we had two questions about the mechanism of action of amivantamab. Is it dependent on EGFR? Is it dependent on MET? How is this therapy working is the question from our audience. Yeah, it's a very good question. And I don't have the answer, no. I know that we have toxicity coming from the two mechanism of action, but what is the percentage? What is more relevant? We don't know yet, no. Yeah, I think this ME, whatever, monoclonal antibody, it actually causes degradation of the receptor, EGFR and MET both. So it kind of internalized those two receptors and then by antibody dependent cytotoxicity, it kind of degrades those two receptors. So therefore it provides cellular proliferation, inhibiting cellular proliferation by degrading both these two receptors, which are tyrosine kinase receptors. Yeah, that is a mechanism of action of this particular monoclonal antibody. Yeah, I understood that the question was more relevant, which one was more relevant? And I think we don't have the answer to see what is more relevant, the two of them. Yeah, I think the mechanism of action is not clear. There are three potential mechanisms, as you mentioned, ligand binding, right? There's a sort of immune activation per se. And then there's also this trogocytosis, as you mentioned, internalization. You know, one of the other questions was, you know, the real MET overexpression, is that necessary for response? And I'll bring this question to you, Dr. Leo. You know, in the second line setting, you know, post-osteomertin, let's say common EGFR mutations, what are your common therapeutic strategies? And do you look for MET expression in that patient population? One of the questions was, are you resequencing your patients at the time of progression? Thanks, Dr. Sabari. So we are resequencing, you know, wherever possible we get a tissue biopsy. And in the Canadian setting, the way we justify the payment for that is that there's a possibility of small cell transformation. So that's how we get tissue. But certainly we do, we are able to repeat NGS testing. MET, of course, very important and not uncommon, right, at least 15% of our patients have that. And then of course, you know, sometimes we do get very lucky. There are other emergent fusions. And so then can you combine, not entirely evidence-based, but there are a lot of case reports out there. And then of course, this whole other strategy of the untargeted, targeted therapies, you know, Patricia Mabb, you know, and the Trope 2 ADCs, and the role that they play. So I think what we try and do is we try and get the molecular testing and then stream them, whether it's to a MET, amnesia for a MET study, or that sort of targeted approach. Patients that have another sort of emergent fusion, and so sort of an off-label approach. And then for everybody else, you know, trials and, you know, some of these other opportunities like ADCs. Yes, I agree. I'm definitely repeating NGS testing in this patient population to look for these, you know, resistance alterations. Dr. Grito, if someone is treated with osimertinib in the frontline setting, third-generation EGFR TKI, at the time of progression, let's say we don't have any trial available, there is no actionable alteration. What are your standard second-line therapeutic options and how may that change in the coming years? Yeah, unfortunately, my standard second-line option, if I don't have the option to offer a clinical trial is chemotherapy in this setting, no? Because at least in Spain, we need reimbursement. So we don't have data coming from clinical trial already approved by EMA and then reimbursement in Spain. But I would say that I think this is going to change because as you mentioned, and I talk about that, there are many different options, but I think that my second-line option will depend on my first option, no? Because avimantamab in the second-line option is a possibility, but if we move avimantamab up front, that could be possible based on the Mariposa trial. This is not going to be a second option. So I think we are going to have a lot of different possibilities, but currently, based on clinical practice, apart from clinical trials, I have to say that my standard option currently is only chemotherapy. Dr. Leal, any difference in your practice? Are you continuing osimertinib in the second-line setting without much data, or are you doing chemotherapy alone outside clearly of clinical trials? So it's a great question. Our standard, the Canadian standard is you stop and you switch to chemotherapy. If there are a few extra boxes of osimertinib lying around and patients continue, nobody says anything, but it is a challenging candidate. It's a challenge to justify the funding. So we're really looking forward to the COMPEL trial and other trials like that that compare continuing versus stopping. So of course, patients all want to continue, but who's going to pay? Yeah, I agree. And I think many therapeutic strategies are emerging in this setting. So an exciting time for our patients. Another question was, again, based on MET in the second-line, therapeutics like sabalitinib targeting MET, AMP or MET overexpression. Dr. Jain, can you sort of explain the difference there between MET overexpression and MET amplification? Is one more actionable than the others? And then, Dr. Garita, I'll turn to you to run us through that chrysalis data where we did look at MET overexpression. So Dr. Jain, to you first. Yeah, so MET expression or MET amplification, that is the commonest mechanism after osimertinib resistance. So for that testing, the recommendation is, I mean, they're not set standards. But it has been recommended that if you see more than five signals and more than 15% of tumor cells by fish, or there are antibodies available, you can detect it by IHC plus minus fish. And then you can say that this is MET amplified. And in those cases, MET inhibitors can be given. So that's one mechanism. And then, of course, exon 14 skipping is another ball of game. And that's entirely different thing, but that can be tested by NGS. But for MET amplification, of course, IHC and fish cytogenetic testing. Yeah, so really important points that overexpression is not the same as amplification. They can co-occur, as you mentioned. Dr. Garido, for the chrysalis two study where we initially combined amivantamib and lasertinib, we interestingly did see a signal for the MET overexpressed population. The MET high response rate about 61%. The MET negative or low, I think it was about 14, 15%. Is that important moving forward? And how do you then translate that potentially to the frontline setting? Are we testing for MET upfront? Yeah, yeah, you are right. And really it's amazing area, but we don't have the answer yet because the retrospective data are useful to think about the design of clinical trials. So I think we shouldn't make decision based on that, but for sure we need to identify different subset of patient and one option. And it's very clear the rationale is to select a combo based on MET inhibition in those patients who have MET signal, no? But we don't have the data yet. And the design of clinical trial we currently have are not prepared to answer this question because all of the data are coming from the retrospective analysis. So we are always moving in the tricky area of making decision without the right kind of clinical trials, no? I couldn't agree. It's interesting, we've moving away somewhat from biomarker driven strategies in the EGFR mutant patient population. So I wanna shift to EGFR exon 20 in the last couple of minutes. And this is to you, Dr. Leo. You mentioned this concept of PCR only identifying about half of the exon 20 alterations. Dr. Jane, we heard a comment or we saw a comment from one of the participants that this is such a diverse range of alterations. How do you identify all of these? So Natasha, how many are there? And are we identifying new ones and are all of these actionable? I mean, how do you think about this in your practice? Thanks, Dr. Sabari. And I think this is something we could all chat about because I know you have a lot of experience as well. So I think the last paper I read there are at least 150 different alterations and I'm sure that number is now much more. I think new alterations are being discovered every day because of course these insertions are complex, right? Once you develop that level of genomic heterogeneity, anything can happen. So far, I think many of these patients have been included in trials. I don't think that this class of agents, and again, the other speakers kind of feel differently. I don't think that they've yet gone the way of, for example, trastuzumab druxacan where they have a very specified list of who was on the trial and who was not. So I think for us, it's more sort of near loop and far loop. Certainly far loop, much more challenging, I think for all the agents, patients with near loop alterations. They're much more enthusiastic about kinase inhibitors, but of course, drugs like imbantamab work incredibly well across and then sort of the mutations in the helical domain. So I mean, in GS, you learn all the time and I really, I phone my genomics people if I don't really understand, is this an exon 20 insertion or isn't it? So I phone them, reach out to colleagues, but there's this huge growing area of novel alterations. Dr. Jain, what's your experience been? What advice would you give to clinicians when they get a report and they've got something novel? Yeah, so in our institute, we are actually doing all PCR based for patient care. So we are not doing NGS in routine, but as a research mode, yes. So I guess we are under diagnosing and we are not getting, I just get 10% of exon 20 by PCR based methods. So NGS, since NGS is not currently a standard of care here in our institute, so that is how I don't get so many questions related to EGF exon 20 insertion. So maybe in future, yeah, definitely I'll get those queries and challenges, but since we are doing the PCR based methods, we are kind of under diagnosing these insertions. Yeah, I get an email or two a week and I also send many emails a week asking colleagues like you, Natasha, that have you seen this? Is this actionable? There is one exon 20 insertion mutation or a few of the FQEA, for example, that is responsive to osimertin of a third generation EGFR TKI. So not all exon 20 insertion mutations are the same. And it's interesting how we may be moving away from an exon classification more into a functional classification here. So really terrific discussion today, exciting how many therapeutic options we are starting to have for our patients. I'll end with you, Dr. Leal here, if we don't test, we can't identify alterations, if we don't test, we can't identify alterations. So I'm curious, what are your thoughts on this? I mean, I think there's a lot of convergence across the country and the world, right? Thinking about just North America and globally, we don't have access for NGS. So what do you tell your colleagues around the world? I mean, if you can have access or don't have access to NGS, what are you recommending at this time to care for our patients? So, I mean, I think you have to use what you have, obviously. I mean, I think there's a lot of people who are using NGS in Asia, and then I think Dr. Jain can speak a bit to the experience in India, but EGFR is very powerful, it's often affordable. Things like ALK-IHC and ROS1, IHC to screen for negative, and then of course you need to fish later. But these are relatively inexpensive and possible to do even with small amounts of tissue. So I do recommend that you use what you can. I also think it's really important to build a team. That we might not normally align with. So for example, for our NGS project, we actually had 10 partners that came from the drug industry and they put together the money that we needed and then we established the base case and then the province funded it. So I think there's a lot that we could do even locally to build that experience, build the funding, prove to government that this is worthwhile. Very nice paper that's just come out from Germany showing the real value of NGS testing. And I think the more we do around the world and the cheaper NGS gets, the more progress we'll make. Excellent points. And also a plug for clinical trials. If you have them available, it's a very cheap and easy way to get next generation sequencing testing done for your patients. So with that, I want to thank everybody for joining today. Have a great day.

EGFR exon 20 insertions

metastatic non-small cell lung cancer

diagnostic implications

therapeutic implications

conventional treatments

mutation detection

mutation classification

amivantamab

Papillon study

chemotherapy

Next-generation sequencing

liquid biopsy