EGFR C797S Mutation Research Articles

Abstract 1170: NCI-H1975 L858R/T790M/C797S isogenic cell line for next-generation EGFR inhibitor screening

Abstract Non-small-cell lung cancer (NSCLC) is the most common cause of cancer death. Over-activation of EGFR occurs in 13%~23% of NSCLC. The identification of EGFR as a cancer driver gene had led to rapid development of NSCLC targeted therapy, but the mutation of EGFR during treatment can cause drug resistance and cancer relapse. For example, T790M mutation causes resistance to the first generation of EGFR inhibitor Erlotinib and C797S mutation contributes to the resistance to the 3rd EGFR inhibitor AZD9291. There are several human cell lines for T790M mutation research, but there is not yet any primary human cancer cell line with C797S mutation. In the current study, a human isogenic EGFR C797S mutation cell line was generated through introducing nucleotide mutation into the EGFR gene in NCI-H1975 cell line using CRISRP technology, which originally bears L858R and T790M mutations of EGFR. First, the sgRNA sequence around the exon-20 of EGFR gene was screened using CRIPSR plasmid. Second, the donor plasmid with the homology arm containing exon-20 of EGFR and selecting marker was constructed and co-transfected into the parental cell line. Then, the NCI-H1975 C797S isogenic cell line was obtained and confirmed after screening over 100 single clones using the PCR method. The NCI-H1975 C797S isogenic cell line was resistant to AZD9291 but sensitive with EAI045 (the potential 4th generation EGFR inhibitor) combined with cetuximab. These data indicate that our NCI-H1975 L858R/T790M/C797S isogenic line is a useful model for next generation EGFR inhibitor study. Citation Format: Feng Hao, Feng He, Wenna Zhang, Zhaoshuai Bai, Jinying Ning. NCI-H1975 L858R/T790M/C797S isogenic cell line for next-generation EGFR inhibitor screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1170.

Investigating Novel Resistance Mechanisms to Third-Generation EGFR Tyrosine Kinase Inhibitor Osimertinib in Non-Small Cell Lung Cancer Patients.

Purpose: The third-generation EGFR tyrosine kinase inhibitor osimertinib is approved to treat patients with EGFR T790M-positive non-small cell lung cancer (NSCLC) who have developed resistance to earlier-generation drugs. Acquired EGFR C797S mutation has been reported to mediate osimertinib resistance in some patients. However, the remaining resistance mechanisms are largely unknown.Experimental Design: We performed mutation profiling using targeted next-generation sequencing (NGS) for 416 cancer-relevant genes on 93 osimertinib-resistant lung cancer patients' samples, mainly cell-free DNAs (cfDNAs), and matched pretreatment samples of 12 patients. In vitro experiments were conducted to functionally study the secondary EGFR mutations identified.Results:EGFR G796/C797, L792, and L718/G719 mutations were identified in 24.7%, 10.8%, and 9.7% of the cases, respectively, with certain mutations coexisting in one patient with different prevalence. L792 and L718 mutants markedly increased the half inhibitory concentration (IC50) of osimertinib in vitro, among which the L718Q mutation conferred the greatest resistance to osimertinib, as well as gefitinib resistance when not coexisting with T790M. Further analysis of the 12 matched pretreatment samples confirmed that these EGFR mutations were acquired during osimertinib treatment. Alterations in parallel or downstream oncogenes such as MET, KRAS, and PIK3CA were also discovered, potentially contributing to the osimertinib-resistance in patients without EGFR secondary mutations.Conclusions: We present comprehensive mutation profiles of a large cohort of osimertinib-resistance lung cancer patients using mainly cfDNA. Besides C797 mutations, novel secondary mutations of EGFR L718 and L792 residues confer osimertinib resistance, both in vitro and in vivo, and are of great clinical and pharmaceutical relevance. Clin Cancer Res; 24(13); 3097-107. ©2018 AACR.

Abstract 3116: Developing engineered and primary cancer cell lines for oncology drug development

Abstract Cancer cell lines are effective in vitro systems in oncology drug development. The success of many targeted cancer drugs benefited from the utilization of cancer cell lines such as ones from American Type Culture Collection (ATCC) and Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ). Recent multiplatform omics research revealed complex diversity of cancers, many of which were classified into subtypes that have distinct genomic signatures, show different prognosis patterns, and may respond differently to same drug treatment. For example, colorectal cancers were separated into four consensus molecular subtypes, and subtype 1 is marked by high microsatellite instability and has worse survival after relapse1. Cancer subtypes may not be well represented in existing cell line libraries. Immortal cell lines for some cancers, such as soft-tissue sarcoma2, may also be rare. It is therefore pertinent to expand current cell line collection, a common approach being to establish primary cell lines directly from cancer patients, preferably with complete clinical information. In addition, current library may also lack of cell lines with rare mutations that exist at low frequency in naïve patient tumors and frequently occur under drug treatment. For example, Erlotinib—a first generation of EGFR inhibitor—induces the EGFR T790M mutation, and Tagrisso—a third generation of EGFR inhibitor—induces the EGFR C797S mutation. It is desirable to create engineered lung cancer cell line with both mutations. An engineered cell line with both mutations can be more efficient in developing EGFR inhibitors overcome the resistance of both mutations. In this study, we report the establishment and characterization of over 120 engineered cell lines and several dozens of primary cell lines across multiple cancers. For the engineered cell lines, we used CRIPSR technology to create cell lines bearing specific mutation for well-studied target proteins such as EGFR, FLT3, ELM4-ALK. In addition, we also created cell lines that may be used for immunotherapy development targeting PD-1, PD-L1, TIM3, OX40, etc. We used Sanger sequencing, flow cytometry, cell viability assay, in vitro and in vivo efficacy studies to validate and characterize the cell lines. To make the resources easily accessible to researchers, we created a searchable database named InnopediaTM that presents detailed information of the engineered and primary cell lines. Access is open with registration. The database also provides comprehensive genomic and efficacy data, retrieved from published research, for over 1000 conventional cell lines.

EGFR T790M and C797S Mutations as Mechanisms of Acquired Resistance to Dacomitinib.

Dacomitinib was superior to gefitinib in terms of progression-free survival in patients with EGFR-mutant lung cancer in a recent ARCHER 1050 trial. However, despite a marked initial response, lung cancers eventually acquire resistance to these inhibitors. This study aimed to elucidate the mechanisms of acquired resistance to dacomitinib invitro. Dacomitinib-resistant clones were established by exposure to fixed concentrations of dacomitinib by using N-ethyl-N-nitrosourea (ENU) mutagenesis or by chronic exposure to increasing concentrations of dacomitinib without ENU. EGFR secondary mutations were analyzed by Sanger sequencing. Time to resistance in each clone was compared according to the mutational status. EGFR Del19, L858R, and G719A mutations were introduced into Ba/F3 cells by using retroviral vectors. Chronic exposure to dacomitinib without ENU induced T790M in Ba/F3 cells expressing Del19. ENU mutagenesis resulted in 171 dacomitinib-resistant clones. Among these clones, 90% acquired T790M. However, C797S occurred in 11% of L858R-mutant clones (four of 35) and in 24% of G719A-mutant clones (12 of 38) established by using low-dose dacomitinib. Time to resistance was not significantly different between T790M- and C797S-mutant clones in both of L858R clones (p= 0.93) and G719A clones (p= 0.86). Cells expressing Del19 that acquired T790M were sensitive to osimertinib, whereas cells with L858R plus C797S mutations were sensitive to gefitinib or erlotinib. These invitro data demonstrate that dacomitinib can directly induce T790M or C797S secondary mutations. Our data suggest the importance of analyzing these secondary mutations because appropriate selection of EGFR inhibitors could overcome acquired resistance to dacomitinib in a subset of lung cancers.

Combination Osimertinib and Gefitinib in C797S and T790M EGFR-Mutated Non–Small Cell Lung Cancer

IntroductionOsimertinib, a third-generation EGFR tyrosine kinase inhibitor has demonstrated efficacy in tumors harboring the EGFR T790M resistance mutation. Inevitably, resistance to third-generation inhibitors results in disease progression, with the EGFR C797S mutation being one of several resistance pathways identified to date. On the basis of preclinical data, we report what is the first known case of a patient harboring the T790M and C797S mutations in trans treated with combination gefitinib and osimertinib. MethodsOn development of progressive disease after multiple therapies, the patient’s plasma was sequenced using the Oncomine Lung cfDNA Assay (Thermo Fisher Scientific, Waltham, MA). Subsequent monitoring of circulating tumor DNA in plasma was performed by droplet digital polymerase chain reaction. ResultsSequencing showed that the T790M and C797S mutations were in trans. Within 2 weeks of commencement of combination therapy, rapid clinical improvement occurred. Accompanying this, a rapid decline in the C797S mutation subclone in plasma was detected. However, the levels of the EGFR exon 19 deletion driver mutation and the T790M resistance mutation in the circulating tumor DNA continued to rise and the patient died from progressive disease 6 weeks after commencement of combination therapy. There were no adverse events seen with the combination therapy. ConclusionThis is, to the best of our knowledge, the first reported case of combination EGFR tyrosine kinase inhibitor therapy tailored to the allelic conformation of T790M and C797S mutation that resulted in brief clinical improvement without toxicity.

Abstract 4112: Overgrowth of competing resistance mechanisms, such as an acquired KRAS mutation, underlies a poor prognosis subtype of acquired resistance to osimertinib in T790M-positive NSCLC

Abstract Introduction: Osimertinib is a third-generation EGFR tyrosine kinase inhibitor (TKI) which is highly active in EGFR-mutant NSCLC with resistance to prior EGFR TKI. Acquired resistance to osimertinib had been observed clinically; an improved understanding of the molecular mechanisms of resistance is needed. Methods/Results: We initially studied an institutional cohort of 86 patients (pts) treated with osimertinib for advanced T790M-positive NSCLC. 50 pts had progressed on therapy, of whom 25 underwent a resistance biopsy and 17 had NGS results available. 6 pts maintained the T790M mutation at resistance, of whom 3 also acquired an EGFR C797S mutation. The remaining 11 pts had loss of T790M, of whom 5 had evidence of a competing resistance mechanism: 2 with histologic transformation to SCLC, one with BRAF V600E, one with an FGFR3-TACC fusion, and one with KRAS Q61K. For the final case, we confirmed the acquired KRAS Q61K on therapy using serial plasma genotyping. Time to treatment failure (TTF) on osimertinib was 3 months median in pts with loss of T790M and 14 months median in pts with maintained T790M. To test the hypothesis that loss of T790M is a poor prognosis subtype of resistance, we analyzed 127 pts treated for T790M-positive NSCLC on the phase I AURA trial of osimertinib. Plasma drawn after progression was submitted for genotyping using droplet digital PCR. 88 pts had a detectable EGFR driver mutation and were eligible for resistance analysis. 45 pts (51%) had detectable T790M at resistance, 17 (19%) of whom also acquired a C797S mutation; the remaining 43 pts (49%) had loss of T790M and no C797S. Median TTF on osimertinib was 6 months in pts with loss of T790M and 11 months in pts with maintained T790M; among pts with TTF <5 months, 72% had loss of T790M. To study whether baseline plasma genotyping could predict the eventual pattern of resistance on osimertinib, we studied 30 pts from the AURA trial with pretreatment plasma genotyping positive for T790M and an EGFR driver. The relative allelic fraction (AF) of T790M was calculated as the ratio of the T790M AF to the driver AF. In 9 pts with eventual loss of T790M, the relative T790M AF trended lower (25% median) than in 21 pts with maintained T790M at resistance (38% median, P = 0.086). Conclusion: Different molecular mechanisms of acquired resistance to osimertinib have different clinical presentations, with early resistance being associated with loss of T790M and the potential for overgrowth of a competing resistance mutation. These findings have implications in steering pts towards suitable trials for overcoming resistance - pts with early resistance to osimertinib could favor trials targeting a competing resistance mechanisms (e.g. MET inhibition) while pts with late resistance could favor trials targeting EGFR C797S. Citation Format: Geoffrey R. Oxnard, Yuebi Hu, Philip Tracy, Nora Feeney, Cloud P. Paweletz, Kenneth S. Thress, Pasi A. Janne. Overgrowth of competing resistance mechanisms, such as an acquired KRAS mutation, underlies a poor prognosis subtype of acquired resistance to osimertinib in T790M-positive NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4112. doi:10.1158/1538-7445.AM2017-4112

Abstract 3156: Multiple acquired resistance mechanisms against third generation EGFR-TKI osimeritinib in non-smal cell lung cancer cells

Abstract Background Osimertinib (AZD9291; Tagrisso) is a third generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) known to be effective against the EGFR-T790M variant, which is accounts for half of the acquired resistance mechanisms to the first generation EGFR-TKIs. However, resistance to osimertinib is likely to progress and the study of potential osimertinib-resistance mechanisms in advanced is necessary. In this study, we investigated the molecular and cellular profiles of the acquired resistance cells to osimertinib in EGFR-mutant non-small cell lung cancers. Materials and Methods Five EGFR-mutant cell lines were exposed to osimertinib by stepwise escalation or high-concentration exposure methods, and resistant sublines to osimertinib were established. The molecular profiles and cellular phenotypes of these resistant sublines were characterized. Results EGFR-C797S mutation which was reported to be a major mechanism of resistance to osimertinib in clinical samples was not detected in established resistance cells by using direct sequencing. Several osimertinib-resistance cell lines displayed MET amplification, and some of these cells were sensitive to the combination of osimertinib plus MET inhibitor crizotinib. However, one cell line that displayed MET amplification was not sensitive to this combination therapy. On the other hand, several osimertinib-resistance cell lines displayed epithelial-to-mesenchymal transition (EMT) features. The HCC827-derived subline established by the high-concentration exposure method exhibited not only EMT features but also cancer stem cell (CSC)-like properties, including aldehyde dehydrogenase isoform 1 (ALDH1A1) and ATP binding cassette subfamily B member 1 (ABCB1) overexpression. Conclusion We established osimertinib-resistant cells and found that MET amplification, EMT, and CSC -like features were observed. These findings may provide clues to overcome acquired resistance to EGFR-TKIs. In some cell lines, however, the mechanisms of acquired resistance to osimertinib were not revealed yet. Citation Format: Kei Namba, Kazuhiko Shien, Takahiro Yoshioka, Hidejiro Torigoe, Hiroki Sato, Hiromasa Yamamoto, Junichi Soh, Kazunori Tsukuda, Shinichi Toyooka. Multiple acquired resistance mechanisms against third generation EGFR-TKI osimeritinib in non-smal cell lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3156. doi:10.1158/1538-7445.AM2017-3156

Characterization of EGFR T790M, L792F, and C797S Mutations as Mechanisms of Acquired Resistance to Afatinib in Lung Cancer.

Lung cancers harboring common EGFR mutations respond to EGFR tyrosine kinase inhibitors (TKI). We previously reported that tumors with exon 18 mutations are particularly sensitive to irreversible second-generation (2G) afatinib compared with first-generation TKIs (1G-TKI). However, data on the mechanisms of acquired resistance to afatinib are limited. We established afatinib-resistant cells by transfecting Ba/F3 cells with common or exon 18 (G719A and Del18) mutations and subjecting them to chronic exposure to increasing concentrations of afatinib. Afatinib-resistant clones were separately established through N-ethyl-N-nitrosourea (ENU) mutagenesis and exposure to fixed concentrations of afatinib. Rebiopsy samples from patients whose tumors acquired resistance to afatinib were analyzed. Afatinib-resistant cells with Del19, L858R, or G719A developed T790M, whereas those with Del18 acquired novel L792F mutation. ENU mutagenesis screening established 84 afatinib-resistant clones. All Del19 clones and most of the other clones acquired only T790M. However, C797S occurred in subsets of L858R, G719A, and Del18 clones. In addition, subsets of Del18 clones acquired L792F. C797S-acquired cells were sensitive to 1G erlotinib. L792F demonstrated intermediate resistance between T790M and C797S to both 1G- and 3G-TKIs, whereas L792F was the least resistant to 2G-TKIs, particularly dacomitinib. Chronic exposure of Del18 + L792F cells to dacomitinib induced additional T790M. T790M was detected in one of four clinical samples. In conclusion, L792F and C797S, in addition to the major T790M, can develop in afatinib-resistant cells particularly using a low dose of afatinib, and these minor mutations appear to exhibit sensitivity to dacomitinib and erlotinib, respectively. These secondary mutations should be tested in clinical practice. Mol Cancer Ther; 16(2); 357-64. ©2016 AACRSee related article by Talbert et al., p. 344.

SC04.03 Sequencing of EGFR Tyrosine Kinase Inhibitors

Treatment of EGFR-mutant (EGFRm) lung cancer with specific EGFR TKIs, such as gefitinib, erlotinib or afatinib, has opened the door to the precision medicine in the management of advanced non-small cell lung cancer with remarkable tumor shrinkage and improvement in progression-free survival (PFS) and quality of life compared to standard chemotherapy. Despite such a remarkable initial clinical response with EGFR TKIs in patients with EGFR+ NSCLC, however, the disease eventually comes back with the emergence of acquired resistance and median PFS is ∼ 1 year. The most common mechanism of resistance is acquisition of the T790M gatekeeper mutation and the 3rd-generation EGFR TKIs irreversibly inhi bit mutant EGFR, esp. T790M, with sparing wild-type(WT) EGFR. There are several EGFR mutant specific inhibitors (EMSIs) under development including AZD9291, CO-1686, BI1482694 /HM61713, ASP8273, etc. All these 3rd-generation EGFR TKIs have shown a promising early clinical efficacy in T790M(+) EGFRm NSCLC patients with ORR of ca. 60% and PFS of 9.6 – 10.3 months and appear to be well tolerated. Based upon these encouraging early results many confirmatory phase 3 trials(e.g., NCT02151981, NCT02322281) comparing to the standard chemotherapy in the 2nd-line setting are underway. It is very tempting that one might like to move the 3rd-generation EGFR TKI to 1st-line setting. The development of the 3rd-generation agents as the first-line therapy for patients with EGFRm disease has already started. Recently AZD9291 demonstrated an encouraging clinical activity and a manageable tolerability profile in 1st-line: confirmed objective response rate of 77% (95% CI 64, 87) and mPFS of 19.3 months (investigator-assessed). Currently it is being compared with the 1st/2nd-generation EGFR TKI in the 1st-line setting. The Phase III FLAURA study (NCT02296125), comparing AZD9291 80 mg once daily versus current standard of care EGFR-TKIs for treatment-naïve patients, is enrolling. Though the preliminary result in the 1L setting is quite provocative, extreme caution needs to be exerted since the currently available data are not mature enough to determine which agent is the best in its class and only from a small subset of patients. Though it is hoped that the T790M-mediated resistance can be delayed or prevented by using the EMSIs in the TKI-naïve setting, it is also possible that other less well known escape mechanisms might emerge. Given that EMSI works well after failing 1st/2nd-generation EGFR TKI I believe it seems to be a more reasonable approach to investigate if EMSI in the TKI-naïve setting is more effective than 1st/2nd-generation EGFR TKI followed by EMSI when failing 1st/2nd-generation EGFR TKI with acquired resistance. One of the biggest questions to emerge in the era of next-generation inhibitors that have activity against the basic driver oncogene is whether it makes sense to use this approach before the development of acquired resistance to prevent it from occurring in the first place. Can its use in the 1st-line(TKI-naïve) setting prevent the development of acquired resistance and lead to a longterm control of the disease? Considering the well-known genomic heterogeneity with its possible association with resistance to EGFR TKIs we need better understanding of the biology and resistance mechanisms to this class of new generation EGFR TKIs in order to develop better strategies for subsequent therapies to overcome the resistance including how to best sequence the available EGFR TKIs in the clinic as well as combination therapies. It is fair to say that during the past few years we’ve clearly made another progress in the management of NSCLC patients with EGFRm, including those who failed previous EGFR TKIs. However, the currently available data are not mature enough to determine which agent is the best in its class, with the notable differences primarily related to toxicity and we’re not there yet and still lots of unanswered questions remain and further researches are warranted. 1. DR Camidge, et al. Acquired resistance to TKIs in solid tumors: learning from lung cancer. Nat Rev Clin Oncol 2014;11: 473-481. 2. SS Ramalingam, et al. The Next Generation of Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in the Treatment of Lung Cancer. Cancer 2015;121:E1-E6. 3. GR Oxnard et al. Acquired EGFR C797S mutation mediates resistance to AZD9291 in non–small cell lung cancer harboring EGFR T790M. Nature Med 2015;21:560-564. 4. LV Sequist et al. Heterogeneity Underlies the Emergence of EGFR T790 Wild-Type Clones Following Treatment of T790M-Positive Cancers with a Third-Generation EGFR Inhibitor. Cancer Discov 2015;5(7): 713-22. 5. CM Lovly et al. Shades of T790M: Intratumor Heterogeneity in EGFR -Mutant Lung Cancer. Cancer Discov 2015;5(7): 694-6. 6. S Ramalingam, et al. ELCC 2016; Abstract LBA1_PR. EGFR TKI, Resistance, Sequence

MTE23.01 Biomarker Characterization: Challenges and Perspectives

The treatment of lung adenocarcinomas has improved with the identification of driver gene mutations and the development of drugs tackling the altered driver gene proteins.1 The interrogation of EGFR mutations as well as ALK and ROS1 translocations is nowadays part of the routine diagnostic workup of lung adenocarcinomas. Further therapies aiming at mutations in driver genes like RET, HER2, BRAF and MET are emerging. The main techniques currently employed in molecular pathology laboratories for mutation detection are mutation specific PCR, conventional capillary (Sanger) sequencing and next generation sequencing (NGS).2 The basic principles of these methods, advantages and disadvantages with a special emphasis on NGS and its potency for cancer diagnostics will be discussed. Recently the detection of driver gene mutations in DNA that is derived from cancer cells and released into the blood has become feasible (so-called “liquid biopsy“).3 It has already entered routine diagnostics with the detection of the T790M mutation in circulating tumor DNA of primary EGFR mutated lung cancers, that developed resistance to first or second generation tyrosine kinase inhibitors.4 The verification of a T790M mutation either by liquid biopsy or the analysis of tumor tissue is a prerequisite for therapy of lung adenocarcinomas with third generation tyrosine kinase inhibitors like osimertinib. The principles of liquid biopsy, employed techniques and potential applications will be introduced. The recent advent of cancer immunotherapy that interferes with immune checkpoint molecules like programmed death 1 (PD-1) offers a new treatment for subgroups of lung cancer patients. A biomarker that would predict responsiveness to this expensive therapy is greatly needed. The expression of the PD-1 ligand (PD-L1) by tumor or immune/stromal cells is a potential biomarker, however its utility is heavily debated.5 The confusion on PD-L1 as a biomarker is partly caused by technical difficulties in the determination of expression, such as the antibody used for immunohistochemistry and the determination of a threshold of expression that correlates with response.6 A further biomarker that is determined by immunohistochemistry is the expression of EGFR in lung squamous cell carcinoma. A therapy with the recently approved anti-EGFR antibody necitumumab requires the demonstration of EGFR expression by the cancer cells.7 The methods and pitfalls in PD-1 and EGFR immunohistochemistry will be presented. The requirement for biomarkers increases. This poses a challenge for diagnostics in respect to availability of techniques, infrastructure and budget. Particularly in lung cancer often very little tissue is available, but different assays should be run. To fulfill the increasing demand for a plethora of biomarker analysis multiplexing techniques that simultaneously interrogate a large number of gene mutations, gene fusions, gene amplification and deletions, as well as RNA and protein expression will be needed. An outlook on available and emerging multiplexing techniques will be provided. 1. The Cancer Genome Atlas Research N (2014) Comprehensive molecular profiling of lung adenocarcinoma. Nature 511 (7511):543-550. http://dx.doi.org/10.1038/nature13385 2. Buermans HP, den Dunnen JT (2014) Next generation sequencing technology: Advances and applications. Biochimica et biophysica acta 1842 (10):1932-1941. http://dx.doi.org/10.1016/j.bbadis.2014.06.015 3. Diaz LA, Jr., Bardelli A (2014) Liquid biopsies: genotyping circulating tumor DNA. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 32 (6):579-586. http://dx.doi.org/10.1200/JCO.2012.45.2011 4. Thress KS, Paweletz CP, Felip E, Cho BC, Stetson D, Dougherty B, Lai Z, Markovets A, Vivancos A, Kuang Y, Ercan D, Matthews SE, Cantarini M, Barrett JC, Janne PA, Oxnard GR (2015) Acquired EGFR C797S mutation mediates resistance to AZD9291 in non-small cell lung cancer harboring EGFR T790M. Nat Med 21 (6):560-562. http://dx.doi.org/10.1038/nm.3854 http://www.nature.com/nm/journal/v21/n6/abs/nm.3854.html - supplementary-information 5. Sunshine J, Taube JM (2015) PD-1/PD-L1 inhibitors. Current opinion in pharmacology 23:32-38. http://dx.doi.org/10.1016/j.coph.2015.05.011 6. Garon EB, Rizvi NA, Hui R, Leighl N, Balmanoukian AS, Eder JP, Patnaik A, Aggarwal C, Gubens M, Horn L, Carcereny E, Ahn M-J, Felip E, Lee J-S, Hellmann MD, Hamid O, Goldman JW, Soria J-C, Dolled-Filhart M, Rutledge RZ, Zhang J, Lunceford JK, Rangwala R, Lubiniecki GM, Roach C, Emancipator K, Gandhi L (2015) Pembrolizumab for the Treatment of Non–Small-Cell Lung Cancer. New England Journal of Medicine 372 (21):2018-2028. http://dx.doi.org/10.1056/NEJMoa1501824 7. Thatcher N, Hirsch FR, Luft AV, Szczesna A, Ciuleanu TE, Dediu M, Ramlau R, Galiulin RK, Balint B, Losonczy G, Kazarnowicz A, Park K, Schumann C, Reck M, Depenbrock H, Nanda S, Kruljac-Letunic A, Kurek R, Paz-Ares L, Socinski MA (2015) Necitumumab plus gemcitabine and cisplatin versus gemcitabine and cisplatin alone as first-line therapy in patients with stage IV squamous non-small-cell lung cancer (SQUIRE): an open-label, randomised, controlled phase 3 trial. The Lancet Oncology 16 (7):763-774. http://dx.doi.org/10.1016/s1470-2045(15)00021-2

P3.02b-120 EGFR T790M, L792F, and C797S Mutations as Mechanisms of Acquired Resistance to Afatinib: Topic: EGFR RES

P3.02b-120 EGFR T790M, L792F, and C797S Mutations as Mechanisms of Acquired Resistance to Afatinib: Topic: EGFR RES

Acquired BRAF V600E Mutation as Resistant Mechanism after Treatment with Osimertinib

IntroductionAZD9291 (osimertinib) is designed for acquired T790M mutation after first- and second-generation EGFR) tyrosine kinase inhibitors have been used. Some of the resistance mechanisms that present after osimertinib treatment, including a newly acquired EGFR C797S mutation, have been identified. It is unclear, however, whether the bypass pathway is also a mechanism of resistance in patients after osimertinib treatment. MethodsCells from malignant pleural effusion were collected and cultured at the time of progression in a patient being treated with osimertinib. Tumor genotyping was done by matrix-assisted laser desorption ionization–time of flight mass spectrometry. EGFR, AKT, MEK, and ERK phosphorylation were determined. An anchorage-dependent colony formation assay was used for drug sensitivity. ResultsAn acquired mutation, BRAF V600E, was found in the patient at the time of progression while being treated with osimertinib. Cells grown from malignant pleural effusion were sensitive to BRAF V600E inhibitor and were more vulnerable to a combination treatment with osimertinib. ConclusionsA potential mechanism of acquired resistance to osimertinib in patients with T790M is through the BRAF pathway. Simultaneous blockade of the BRAF and EGFR had a significant inhibitory effect.

Case series of EGFR C797S mutations in non-small cell lung cancer identified with cell-free circulating tumor DNA next generation sequencing.

e23021Background: In non-small cell lung cancer (NSCLC), targeted treatment with first line tyrosine kinase inhibitors (TKIs) selects for the evolution of resistance at EGFR T790M in 50% of cases. ...

Abstract C80: Development of EGFR C797S mutation in serial liquid biopsy assessments in the clinical practice setting

Abstract Introduction: EGFR T790M has emerged as an important biomarker to predict response to 3rd-generation tyrosine kinase inhibitors (TKI). Therapies such as AZD 9291 and Rociletinib demonstrated impressive results in the clinical setting in patients with non-small cell lung cancer (NSCLC). However just as this resistance biomarker (T790M) has developed as a result of treatment with prior EGFR TKIs, EGFR C797S mutation is an acquired resistance mechanism to EGFR T790M inhibitors. This mutation was recently reported for the first time in clinical practice utilizing tissue-biopsy based next generation sequencing (NGS) and in the clinical trial setting in cell-free circulating DNA (cfDNA). Here we report the first case of a patient with NSCLC and emergence of C797S mutation utilizing a cfDNA biopsy-free NGS panel in serial assessments in the clinical setting. Methods: Patients with NSCLC were seen at Levine Cancer Institute and had serial blood draws to assess molecular aberrations in cfDNA over time for comparison with radiographic reviews. Blood samples were sent to Guardant360 for assessment by NGS with a targeted cfDNA NGS panel for 68 genes with complete exon sequencing for all 28 exons in EGFR, and other genes in the panel. Mutant allele fractions (MAF) are reported as% of mutant DNA molecules divided by total molecules (mostly leukocyte DNA-derived) overlapping the same mutated nucleotide base position. Results: A 50 year old African-American female never-smoker with lung adenocarcinoma developed a tissue-biopsy confirmed EGFR T790M mutation after 12 months on erlotinib for EGFR exon 19 deletion. She had significant clinical and radiographic responses to a 3rd generation TKI started May 2014. cfDNA NGS tests in March and April 2015 showed no genomic alterations detectable. In late May 2015, she developed clinical symptoms of cough, dyspnea and fatigue but no definitive radiographic evidence of progression on scans June 2015. She received steroids and antibiotics and continued treatment with the 3rd generation TKI. However, corresponding cfDNA NGS testing revealed emergence of multiple low level mutations and the original deletion in EGFR (EGFR L747_P753 DelInsS, T790M, C797S, and A755G variant of uncertain significance) and TP53 (all <1.1%). Repeat scans in July 2015 demonstrated clear progression of disease and a fourth cfDNA NGS test showed a rise in all low level aberrations previously detected in cfDNA: EGFR A755G 4.2%, TP53 P278R 3.6%, EGFR T790M 3.1%, EGFR L747_P753 DelInsS 2.9%, EGFR C797S 1.4%. NGS indicated that there may be 2 clones present for EGFR C797S but confirmation is required as disease progresses. Discussion: Complete exon sequencing of cfDNA upon clinical progression uncovered the simultaneous re-emergence of the original driver EGFR exon 19 del and resistance driver mutation EGFR T790M as well as the emergence of the next resistance driver mutation EGFR C797S. The latter mutation interferes with covalent binding of a 3rd generation TKI and under treatment pressure, the tumor appears to have simultaneously evolved two different EGFR C797S clones at low MAFs. cfDNA can be a dynamic measure of tumor response, easily obtained from a patient on any visit with potential for detection before macroscopic/radiographic evidence of progression, as demonstrated in this case with tumor evolution of the EGFR T790M and C797S alterations. Citation Format: Kathryn F. Mileham, Qing Zhang, Carol J. Farhangfar, Daniel E. Haggstrom, Stephen Fairclough, Oliver A. Zill, Daniel R. Carrizosa, Richard B. Lanman, Edward S. Kim. Development of EGFR C797S mutation in serial liquid biopsy assessments in the clinical practice setting. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C80.

Abstract LB-123: Analysis of cell-free plasma DNA (cfDNA) identifies 3 molecular subtypes of acquired resistance to AZD9291, a novel EGFR tyrosine kinase inhibitor (TKI), in patients (pts) with advanced lung cancer

Abstract Introduction: EGFR T790M is the most common mechanism of acquired resistance to EGFR TKIs in pts with EGFR-mutant lung cancer. AZD9291 is an irreversible, mutant-selective EGFR TKI developed to have potency against both sensiziting EGFR mutations and T790M. In the ongoing phase I study of AZD9291 (AURA, NCT01802632), the response rate in pts with T790M-positive lung cancer was >60%. The molecular mechanism underlying acquired resistance to AZD9291 is not known. Methods & Results: To explore for mechanisms of resistance to AZD9291, we studied cfDNA extracted from pretreatment and post-progression plasma collected on AURA.Next-generation sequencing (NGS) of cfDNA was first performed on an exploratory cohort of 7 pts. All exons of a 20 gene panel (including EGFR) underwent PCR amplification and NGS using an Illumina HiSeq. In 1 pt, NGS of progression plasma identified a new EGFR C797S mutation in exon 20, not present in pretreatment plasma. Stable expression of C797S in Ba/F3 cells induced a >100-fold increase in IC50 to AZD9291 compared to EGFR activating and T790M mutations alone. To validate the plasma NGS, digital droplet PCR (ddPCR) assays were developed to detect key EGFR mutations including C797S. 15 T790M-positive cases were identified with progression plasma available for analysis. Serial plasma ddPCR showed that both the EGFR activating and T790M mutation levels decreased with AZD9291 treament and increased at progression, with 3 molecular subtypes of resistance apparent. In 6 pts (40%), C797S was detected in addition to T790M; NGS of resistance biopsies from 2 of these pts confirmed presence of acquired C797S. In 5 pts (33%), T790M was detected without evidence of C797S. Intriguingly, in 4 pts (27%), the T790M levels became undetectable with treatment despite high levels of the EGFR activating mutation at progression, suggesting overgrowth of a competing non-T790M resistance mechanism. Further NGS of progression plasma revealed additional evidence of the genomic heterogeneity of resistance. Individual sequencing reads indicate that C797S and T790M can occur either in cis or in trans (i.e. on competing resistant alleles). In the 2 pts with tumor NGS demonstrating C797S, plasma NGS identified both the DNA alteration seen in tumor as well as a second DNA alteration encoding for C797S. Conclusion: Using complementary assays for genomic analyses of cfDNA, we identified 3 molecular subtypes of acquired resistance to AZD9291, including an EGFR C797S mutation never before reported in pts. Due to the key role of the C797 residue in drug binding, C797S is expected to induce resistance to all irreversible EGFR TKIs currently in clinical development. Plasma NGS revealed substantial genomic heterogeneity and highlights the need for combination therapies to effectively prevent or treat drug resistance in cancer. Citation Format: Geoffrey R. Oxnard, Kenneth S. Thress, Cloud P. Paweletz, Enriqueta Felip, Byoung Chul Cho, Daniel Stetson, Brian Dougherty, Zhongwu Lai, Aleksandra Morkovets, Ana Vivancos, Yanan Kuang, Dalia Ercan, Mireille Cantarini, J Carl Barrett, Pasi A. Janne. Analysis of cell-free plasma DNA (cfDNA) identifies 3 molecular subtypes of acquired resistance to AZD9291, a novel EGFR tyrosine kinase inhibitor (TKI), in patients (pts) with advanced lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-123. doi:10.1158/1538-7445.AM2015-LB-123

Abstract 4832: Novel EGFR mutations that cause drug resistance to irreversible pyrimidine but not quinazoline based EGFR inhibitors

Abstract Background: Oncogenic EGFR T790M causes drug resistance to quinazoline based EGFR kinase inhibitors by increasing ATP affinity (Yun PNAS 2008). Mutant selective irreversible pyrimidine EGFR kinase inhibitor, WZ4002, is effective in non-small cell lung cancer (NSCLC) models harboring EGFR T790M (Zhou Nature 2009). We aimed to determine potential mechanisms of resistance to WZ4002 and explore alternative strategies to overcome acquired resistance to pyrimidine based EGFR inhibitors. Methods and Results: We performed an ENU mutagenesis screen in Ba/F3 cells expressing EGFR L858R, L858R/T790M, Del E746_A750 and Del E746_A750/T790M followed by culture in the presence of WZ4002 (100 nM or 1 μM). Using RT-PCR, we sequenced resistant clones for secondary EGFR mutations. No EGFR T790M mutations were identified. We detected novel secondary EGFR L718Q (9/27; 33%) or L844V (1/27; 3%) mutations in the drug resistant cells. We also recovered the EGFR C797S (1/27; 3%) mutation previously known to prevent covalent binding and decrease potency of WZ4002. Unlike EGFR T790M, EGFR L718Q and EGFR L844V did not lead to constitutive EGFR phosphorylation, were not transforming in Ba/F3 cells and required EGF for proliferation and survival. The EGFR L858R/L844V Ba/F3 cells were resistant to WZ4002 (IC50 0.7 μM) but sensitive to irreversible quinazoline EGFR inhibitors CL-387,785, HKI-272 (neratinib) and BIBW2992 (afatinib) (IC50 values all < 10 nM). Similar findings were observed with the EGFR L858R/L718Q cells although the IC50 values were slightly higher (100 nM or less) and with the Del E746_A750/L718Q and Del E746_A750/L844V cells. All triple mutants harboring EGFR T790M (e.g. L858R/T790M/L844V) were resistant to WZ4002 and irreversible quinazoline EGFR inhibitors. EGFR L858R/L844V and L858R/L718Q Ba/F3 cells were growth inhibited by clinical concentrations (1 μM) of gefitinib and the combination of 1 μM gefitinib and 100 nM WZ4002 completely prevented the emergence of resistant clones in our ENU assay. Using structural modeling, both the L718Q and L844V mutations likely lead to steric hindrance and could impact WZ4002 binding. To verify this hypothesis, we developed a biotinylated-WZ4002 compound and used it to assay binding to different EGFR mutant proteins. This agent effectively bound to EGFR L858R and DelE746_A750 (with or without T790M). However, in the presence of a concurrent L718Q or L844V mutation, protein binding was significantly reduced, consistent with the reduced in vitro efficacy in the Ba/F3 cells. Conclusions: We identify novel EGFR mutations that confer drug resistance to irreversible pyrimidine but not quinazoline EGFR kinase inhibitors. Our findings have implications for understanding drug resistance mechanisms and for the development of combinations of EGFR kinase inhibitors as therapies for cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4832. doi:1538-7445.AM2012-4832