Epidermal Growth Factor Receptor TKIs Research Articles

Efficacy and safety of afatinib for non-small-cell lung cancer: state-of-the-art and future perspectives

ABSTRACT Introduction Afatinib is a second-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, acting as an irreversible and multitarget blocker of ErbB family members. Afatinib is currently approved for advanced non-small-cell lung cancer (NSCLC) harboring common and uncommon sensitizing EGFR mutations and for squamous NSCLC patients progressing after first-line platinum-based chemotherapy. Areas covered This review summarizes the efficacy and safety profile of afatinib compared with chemotherapy and other EGFR TKIs, in order to evaluate its characteristics and potential role in the increasingly complex treatment landscape of EGFR-mutant lung cancer. Future perspectives and innovative drug combinations are also discussed. Expert opinion Afatinib has been demonstrated to improve efficacy and quality of life compared with chemotherapy with a managed toxicity profile. However, in recent years, the increasing availability of different treatment options for advanced EGFR-mutant NSCLC has made the current treatment scenario more complicated, with an increasing need of new and deeper scientific data. In this light, the identification and validation of potential clinicopathological and/or molecular predictors of benefit, as well as the clarification of resistance mechanisms, may help to clarify the most appropriate treatment strategies and sequences for EGFR-mutant patients.

AXL/MET dual inhibitor, CB469, has activity in non-small cell lung cancer with acquired resistance to EGFR TKI with AXL or MET activation

ObjectivesIn non–small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) mutations, acquired resistance to EGFR-tyrosine kinase inhibitors (EGFR-TKIs) may occur via bypass signals such as AXL or MET activation. We investigated the antitumor activity of CB469, a newly developed drug that targets both AXL and MET, in EGFR TKI-resistant NSCLC cells. Materials and methodsWe generated EGFR TKI-resistant NSCLC cell lines with acquired resistance to erlotinib, gefitinib, and osimertinib (PC9/ER, HCC827/GR and HCC827/OR, respectively). We characterized the mechanisms of CB469 action in resistant cells and investigated the antitumor efficacy of CB469 both in vitro and in vivo. ResultsResistant cells showed activation of phosphorylated EGFR, as well as AXL and MET activation and phosphorylation. The combination of CB469 and EGFR TKIs synergistically inhibited cell proliferation and colony formation rates in resistant cell lines. The combination of CB469 and erlotinib induced apoptosis of PC9/ER cells. Mechanistically, resistant cells showed an interaction of AXL and MET. CB469 and EGFR TKI also demonstrated antitumor activity by reducing phosphorylated AXL and MET in mouse xenograft models with HCC827/GR cells. ConclusionThe combination of CB469 and EGFR TKI can overcome the acquired resistance to EGFR TKI mediated by AXL and MET activation. We anticipate that the dual inhibitory actions of CB469 will assist with the development of targeted therapy for EGFR-mutant NSCLC patients who fail initial EGFR TKI therapy.

CTONG1104: Adjuvant gefitinib versus chemotherapy for resected N1-N2 NSCLC with EGFR mutation—Final overall survival analysis of the randomized phase III trial 1 analysis of the randomized phase III trial.

9005 Background: ADJUVANT-CTONG1104, a randomized phase 3 trial showed adjuvant gefitinib treatment significantly improved disease-free survival (DFS) vs standard doublet chemotherapy in patients (pts) with epidermal growth factor receptor ( EGFR) mutation-positive resected stage II-IIIA (N1-N2) non-small-cell lung cancer (NSCLC). 5-year survival rate of N1N2 were 38%-50% in IASLC staging system. Here, we present the final overall survival (OS) results from the study. Methods: From Sep 2011 to April 2014, 222 patients, aged 18-75 years, with EGFR activating mutation through completely resection and diagnosed as stage II-IIIA (N1-N2) NSCLC pathologically from 27 sites were enrolled. The enrolled patients were 1:1 randomized to receive adjuvant gefitinib (250 mg once per day) for 24 months (G, n=111) or vinorelbine (25 mg/m2, d1 and d8) plus cisplatin (75 mg/m2, d1) every 3 weeks for 4 cycles (C, n=111). The primary endpoint was DFS in the ITT population. Secondary endpoints included OS, 3 and 5-year DFS rate, 5-year OS rate. The subsequent therapy data were collected, including crossover from C to G, re-challenge TKI and other treatment. Data cut-off date was Jan. 13, 2020. Results: A median follow-up was 76.9 months. The median OS (mOS) was 75.5 months based on 95 (42.8%) events in ITT whole population. The mOS was 75.5m in G arm and 79.2m in C arm (HR 0.96, 95%CI 0.64-1.43, p=0.823). The 3, 5-year OS rate were 68.6%, 53.8% in G and 67.5%, 52.4% in C respectively. DFS in 3, 5-y were 40.3%, 23.4% in G and 33.2%, 23.7% in C, respectively (P3-y=0.395, P5-y=891). All predefined subgroups including age, gender, lymph node, EGFR mutation type had no significant difference in statistics but in favor of G arm in trend. Subsequent treatment especially targeted therapy contributed most to OS (HR = 0.46, 95% CI 0.26 – 0.83). Median OS of patients receiving subsequent target therapy was75.5m (n=35), 36.4m in other treatment (n=33; (P<0.001). For G mOS were 75.5 (n=15; target therapy) and 35.0 (n=18; other, p<0.001), for C 62.8m (n=20) and 46.8m (n=15; p=0.251). The RR was 26.7%, DCR 66.7%, mPFS 14.1m and mOS 19.6m for patients with rechallenged EGFR TKI in G arm (n=15). No novel unexpected SAE was observed during follow up. Conclusion The DFS survival advantage did not translate to OS difference in ADJUVANT trial. The OS with 75.5m was the best one of survival in completely resected N1N2 NSCLC comparing with historical data and sequent TKI treatment contribute to overall survival. Clinical trial information: NCT01405079.

Inhibition of JAK1/2 can overcome EGFR-TKI resistance in human NSCLC

Non-small lung cancer (NSCLC) is the most common cancer in the world. The epidermal growth factor receptor (EGFR) gene is mutated in approximately 10% of lung cancer cases in the US and 50% of lung cancer in Asia. The representative target therapeutic agent, erlotinib (EGFR tyrosine kinase inhibitor; EGFR TKI), is effective in inactivating EGFR in lung cancer patients. However, approximately 50–60% of patients are resistant to EGFR TKI. These populations are associated with the EGFR mutation. To overcome resistance to EGFR TKI, we discovered a JAK1 inhibitor, CJ14939. We investigated the efficacy of CJ14939 in human NSCLC cell lines in vitro and in vivo. Our results showed that CJ14939 induced the inhibition of cell growth. Moreover, we demonstrated that combination treatment with erlotinib and CJ14939 induced cell death in vitro and inhibited tumor growth in vivo. In addition, we confirmed the suppression of phosphorylated EGFR, JAK1, and Stat3 expression in erlotinib and CJ14939-treated human NSCLC cell lines. Our results provide evidence that JAK inhibition overcomes resistance to EGFR TKI in human NSCLCs.

Serum CRIPTO does not confer drug resistance against osimertinib but is an indicator of tumor burden in non-small cell lung cancer

BackgroundAdenocarcinoma is the most common subtype of non-small cell lung cancer (NSCLC) and often harbors oncogenic driver mutations in the epidermal growth factor receptor (EGFR). Osimertinib (AZD9291), a third generation EGFR TKI, has replaced earlier generation EGFR TKIs for first line treatment of EGFR mutant lung cancer due to its improved overall survival, longer progression free survival, and better tolerability compared to earlier generation inhibitors. However, like earlier generation EGFR TKIs, only about two thirds of patients respond, indicating an unknown mechanism of intrinsic resistance for the non-responders. We previously identified overexpression of CRIPTO as a potential mechanism of intrinsic resistance to EGFR TKIs of first and second generation. ObjectiveTo determine if CRIPTO could promote drug resistance against the third generation EGFR-TKIs osimertinib. We also wanted to investigate whether this resistance was conferred by both membrane bound and secreted CRIPTO. Finally, we wanted to explore the potential of secreted CRIPTO as a non-invasive biomarker for EGFR-TKI resistance. Materials and methodsHCC827 and H1975, EGFR mutant non-small cell lung carcinoma (NSCLC) cell lines, were transfected with wildtype CRIPTO, two secreted variants of CRIPTO, a membrane only version of CRIPTO, and the mock backbone vector as the control. Western blotting, immunoprecipitation, and in vitro viability experiments were performed.In vivo work was carried out in athymic nude mice; 2 × 106 CRIPTO overexpressing HCC827 cells were implanted per mouse.EGFR mutant NSCLC patient blood samples were collected before treatment with and EGFR-TKI, during response while on treatment, and at progression while on treatment. ResultsAlthough both membrane bound and secreted CRIPTO forms were able to activate downstream pathways such as SRC, CRIPTO was unable to elicit resistance towards osimertinib in vitro or in vivo. CRIPTO serum levels in mice were higher in larger xenograft tumors. Furthermore, CRIPTO serum levels were higher in patients with progressing lung cancer when compared to their CRIPTO serum levels during EGFR-TKI response. ConclusionsCRIPTO does not cause resistance against third generation EGFR-TKI osimertinib. CRIPTO levels in serum might be a potentially useful biomarker for tumor burden in NSCLC patients.

Triple Mutation Epidermal Growth Factor Receptor (EGFR) Exon 18 (G719S), 20 (T790M) and 21 (L858R) in a Male Patient with Lung Adenocarcinoma: A Case Report

Background: Lung cancer is one of the deadliest cancers in the world. The percentage of non small cell lung cancer (NSCLC) is about 80% of the incidence of lung cancer. The type of NSCLC, adenocarcinoma, is usually found in the presence of epidermal growth factor receptor (EGFR) mutations.Case. A male patient aged 70 years, an active smoker, works as a farmer. He has complained of shortness of breath, and chest pain for three months. There was no family history of suffering from malignancy. The cytology result of the right pleural fluid indicated adenocarcinoma. He was diagnosed with pulmonary adenocarcinoma (D) stage IV positive mutation of EGFR exon 18 (G719S), 20 (T790M) and 21 (L858R) carnofsky score 70. He could survive for more than 11 months with the treatment of EGFR TKI, and received a good therapeutic response. Initially, for the first six months it was such a progressive disease, and for the next eleven months it became stable.Conclusion: In addition to the exon mutations found in this case, the cells in the tumor will continue to grow and develop into new mutants that are immune such drugs and rapidly split themselves into new, different forms. The therapy for complex mutations is still being developed. EGFR TKI therapy in this patient had a relatively good response. Further understanding of the molecular biology of lung cancer is seriously required.

Nazartinib in EGFR Thr790Met-mutant non-small-cell lung cancer

Mutations in the epidermal growth factor receptor (EGFR) gene are the most common actionable alterations in non-small-cell lung cancer (NSCLC). These abnormalities occur almost exclusively in lung adenocarcinoma, with the prevalence ranging from 10–20% among white patients to 40–55% among patients from the Asia-Pacific region. The development of EGFR tyrosine kinase inhibitors (EGFR TKIs), small molecules that specifically inhibit the downstream EGFR signalling pathway, have launched a new era of personalised therapy for lung cancer, with EGFR TKIs largely replacing chemotherapy for first-line and second-line treatment of EGFR-mutant advanced NSCLC. Subsequently, the family of EGFR TKIs has been supplemented by TKIs blocking other molecular targets, such as ALK, ROS1, RET, and BRAF, with a large potential for further developments. Safety and efficacy of nazartinib (EGF816) in adults with EGFR-mutant non-small-cell lung carcinoma: a multicentre, open-label, phase 1 studyNazartinib has a favourable safety profile, with low-grade skin toxicity characterised by a predominantly maculopapular rash that required minimal dose reductions. Full-Text PDF

T790M mutations identified by circulating tumor DNA test in lung adenocarcinoma patients who progressed on first-line epidermal growth factor receptor-tyrosine kinase inhibitors.

Background:Plasma circulating tumor deoxyribonucleic acid (ctDNA) test is an alternative method to detect the T790M mutation. Compared to conventional tumor rebiopsy, ctDNA possesses several advantages including less invasive, faster, lower costs, and having minimal risk of complications for patients.Objective:The main objective of the study is to identify the prevalence of T790M mutations in lung adenocarcinoma patients who progressed after tyrosine kinase inhibitors (TKIs) therapy using ctDNA examination.Materials and Methods:This was a retrospective cohort study based on medical records of lung adenocarcinoma patients in the Oncology Outpatient Clinic of Dr. Soetomo General Hospital within the period of January 2017–June 2018. Patients who progressed after receiving first-line epidermal growth factor receptor-TKI (EGFR-TKI) undergone plasma ctDNA examination and genotyping using digital platforms (Droplet Digital™ PCR) method.Results:In total, there were 39 patients who met the criteria for ctDNA testing. Thirty-three patients (84.6%) received first-line gefitinib, while the other six (15.4%) received erlotinib. The T790M mutations were detected in 46.2% of patients. In addition, EGFR common mutation in exon 19 and exon 21 were detected in 87.2% of patients. Median progression-free survival of patients receiving first-line gefitinib or erlotinib were both around 9 months and did not differ significantly.Conclusions:CtDNA examination successfully detected T790M mutation in a certain proportion of lung adenocarcinoma patients who progressed after first-line EGFR-TKI without the need for difficult and invasive rebiopsy.

PLGA Porous Microspheres Dry Powders for Codelivery of Afatinib-Loaded Solid Lipid Nanoparticles and Paclitaxel: Novel Therapy for EGFR Tyrosine Kinase Inhibitors Resistant Nonsmall Cell Lung Cancer.

Combination therapy of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFR TKIs) with other chemotherapeutic agents is a feasible strategy to overcome resistance that often occurs after 9-13 months of EGFR TKIs administration in nonsmall cell lung cancer (NSCLC). In this study, a pulmonary microspheres system that codelivers afatinib and paclitaxel (PTX) is developed for treatment of EGFR TKIs resistant NSCLC. In this system, afatinib is loaded in stearic acid-based solid lipid nanoparticles, then these nanoparticles and PTX are loaded in poly-lactide-co-glycolide-based porous microspheres. These inhaled microspheres systems are characterized including geometric particle size, drug encapsulation efficiency, morphology by scanning electron microscopy, specific surface area, in vitro drug release, and aerodynamic particle size. Cell experiments indicate that afatinib and PTX have a synergistic effect and the codelivery system shows a superior treatment effect in drug-resistant NSCLC cells. The biocompatibility, pharmacokinetic, and tissue distribution experiments in Sprague-Dawley rats show that afatinib and PTX in the system can maintain 96 h of high lung concentration but low concentration in other tissues, with acceptable safety. These results demonstrate that this system may be a prospective delivery strategy for drug combination treatment in cancers developing resistance, especially drug-resistant lung cancer.

P2.03-05 PHLPP1 Expression Through AKT and ERK Dual Signaling Pathways May Slow Down the Resistance to TKI in EGFR-Mutated Lung Adenocarcinoma

The epidermal growth factor receptor (EGFR) kinase inhibitors are effective treatments for lung cancers with EGFR activating mutations, but the magnitude of tumor regression varies and drug resistance is unavoidable. Multiple mechanisms of resistance to EGFR-TKIs have been identified, including the occurrence of secondary mutations in the EGFR gene, MET amplification, acquired BRAF rearrangements and activation of bypass pathways. Central to these mechanisms of resistance is the re-activation of AKT and ERK signaling, which enables escape of tumor cells from EGFR inhibitor treatment. However, the mechanisms of reactivation of PI3K-AKT and ERK/MAPK pathway are unclear. Pleckstrin homology (PH) domain leucine-rich repeat protein phosphatase (PHLPP) acts as tumor suppressors in various types of human cancer by suppressing cell survival pathways and promoting apoptosis through inhibiting AKT and ERK pathway activation. Here, we hypothesize that PHLPP is a key regulator of EGFR-TKI resistance in lung cancer and a potential treatment target for overcoming resistance to EGFR-TKI treatment. A transcriptomic of PHLPP1 in non-small lung cancer cell according to gefitinib sensitivity obtained from Gene Expression Omnibus (GEO) database under accession number GSE4342 were analysis. The lentivirus-mediated delivery of shRNA was used to generated stable knockdown of PHLPP1 expression lung cancer cells, and retrovirus-mediated delivery was used to generated stable overexpression of PHLPP1 lung cancer cells. Western blotting, real-time PCR (RT-PCR) and immunofluorescence were used to determined PHLPP expression in vitro. PHLPP1 expression in clinical sample was determined by Immunohistochemical (IHC) staining. MTT assay was conducted to determine the cell proliferation. Xenografts bearing PHLPP overexpression and control were evaluated EGFR-TKI induced tumor regression. PHLPP1 gene expression was higher in gefitinib-sensitive NSCLC cell lines than gefitinib-resistant NSCLC cell lines from a GEO public database. In vitro, EGFR mutated NSCLC cell line HCC827 continuously exposing to gefitinib exhibited dramatically reduced expression of PHLPP1 and increased phosphorylation AKT and ERK. Knockdown of PHLPP1 decreased cell death induced by the EGFR-TKI in EGFR-mutant lung cancer cells, overexpression of PHLPP1 enhanced gefitinib-induced apoptosis in gefitinib-resistance EGFR-mutant lung cancer cells. In xenograft model, overexpression of PHLPP showed significantly more tumor regression after gefitinib treatment at 1-week time point compared to control group. In patients, PHLPP1 were highly expressed in tumors with EGFR common mutations pre- and post-development of resistance to EGFR TKIs. PHLPP1 expression were down regulated in the post-relapse tumor samples compared to that of pre-treatment, and patients with higher PHLPP1 expression in pre-treatment had significantly longer progression-free survival (PFS). PHLPP loss may be a key molecule contributing to the resistance of EGFR-TKI by activating PI3K-AKT and ERK/MAPK signaling pathway. PHLPP may serve as a potential predictor of EGFR-TKI treatment response in these patients. Up-regulating PHLPP expression may prevent or /and delay the emergence of EGFR-TKI resistance. Further study is warranted to prove PHLPP as an effective strategy.

Study of epidermal growth factor receptor gene mutation in liquid biopsy of cerebrospinal fluid supernatant from patients with lung adenocarcinoma

Objective To study the detection protocol of epidermal growth factor receptor(EGFR)gene mutations in cerebrospinal fluid(CSF)specimens of lung adenocarcinoma by establishing a liquid biopsy technique, and to analyze clinical value of the detection findings for clinical therapy. Methods In the retrospective study, a total of 26 CSF specimens from lung adenocarcinoma patients who harbored EGFR gene mutations(L858R and EGFR exon 19 deletion)were collected.The included patients were treated with first-generation EGFR TKIs, and had brain metastasis detected by imaging examinations.Five CSF specimens from non-tumor patients confirmed by clinical findings and cytological and pathological diagnosis were collected during the same period.EGFR gene mutations in cell-free CSF supernatant fluid were detected by using the protocol recommended in this study. Results EGFR mutations were detected in 21/26(80.8%)of cell-free supernatants from CSF specimens of lung adenocarcinoma patients, including 13 cases(61.9%)with EGFR L858R mutations and 8 cases(38.1%)with EGFR exon 19 deletion.No T790M mutation was detected in cell-free supernatants from all CSF specimens.No EGFR mutation was detected in cell-free supernatants from all CSF specimens from non-tumor patients. Conclusions Our study-established protocol could be used to detect EGFR mutations in cell-free supernatants from CSF specimens with high sensitivity and specificity.Perhaps due to the characteristics of first-generation EGFR TKIs, the detection rate of T790M mutation is low.The detection of T790M mutation in cell-free supernatants from CSF specimen may have a limited clinical value for choice of 3rd generation EGFR TKIs. Key words: Lung neoplasms; Neoplasm metastasis; Receptor, epidermal growth factor; DNA mutational analysis

Abstract 4765: MERTK is a potential therapeutic target in osimertinib-resistant non-small cell lung cancer

Abstract Non-small cell lung cancer (NSCLC) remains a global problem causing more deaths in both men and women than any other cancer worldwide with an urgent need for more efficacious treatments. During the last decade the therapeutic landscape for NSCLC has been profoundly changed with the discovery of activating mutations in Epidermal Growth Factor Receptor (EGFR) (EGFRMT). Osimertinib, a 3rd generation EGFR TKI, was recently FDA-approved as a front-line agent for newly diagnosed EGFRMT NSCLCs due to its superior efficacy relative to earlier-generation EGFR TKIs in the international FLAURA trial. However, unmet clinical needs have arisen in conjunction with osimertinib use, including understanding mechanisms of osimertinib resistance and developing novel approaches to prevent or reverse resistance and/or enhance osimertinib efficacy in responsive patients. Our group previously identified MERTK receptor tyrosine kinase as a potential therapeutic target in NSCLC and developed MRX-2843, a novel small molecule inhibitor with dual MERTK and FLT3 activity, which is currently in Phase I clinical trials. Here, we report upregulation of MERTK and its ligand PROS1 in xenograft tumors derived from the EGFRMT H4006 NSCLC cell line and treated with osimertinib relative to vehicle-treated tumors. MERTK expression was also increased in osimertinib-resistant derivatives of the H4006 and H4011 cell lines generated by culture in escalating doses of osimertinib. However, overexpression of exogenous MERTK in H4006 cells was not sufficient to confer osimertinib resistance. In contrast, stimulation with TAM kinase ligands GAS6 or PROS1 protected H4006 cells from osimertinib treatment, as indicated by restoration of AKT, ERK, and ribosomal S6 phosphorylation in the presence of osimertinib. Together these data implicate MERTK as a mediator of resistance to osimertinib. Indeed, combined treatment with osimertinib and MRX-2843 effectively blocked PI3K-AKT and MAPK-ERK signaling and mediated synergistic inhibition of colony formation in osimertinib-resistant H4006 cells. Thus, MERTK inhibition may be an effective therapeutic strategy to re-sensitize osimertinib-resistant NSCLCs to EGFR TKI treatment. Citation Format: Dan Yan, Justus Huelse, Rebecca Parker, Xiaodong Wang, Stephen V. Frye, H. Shelton Earp, Deborah DeRyckere, Douglas Graham. MERTK is a potential therapeutic target in osimertinib-resistant non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4765.

Abstract 3019: Dynamic kinome profiling of EGFRvIII-driven murine astrocyte models of glioblastoma reveals targets for dual kinase inhibitor therapy

Abstract Glioblastoma (GBM) is an aggressive brain tumor with few effective treatments. Epidermal growth factor receptor (EGFR) is frequently amplified and mutated in GBM, leading to trials of several EGFR tyrosine kinase inhibitors, but none have proven successful. One potential reason for failure is acquired resistance, particularly acute, adaptive responses in the kinome. To study this adaptive resistance mechanism, we used RNA-seq and multiplex inhibitor bead/mass spectrometry (MIB-MS) to analyze transcriptomes and kinomes of genetically-engineered murine astrocytes with genotypes commonly seen in human GBM. We previously showed that 38% (86 of 228) of the expressed kinome varied among a panel of genetically diverse murine astrocytes harboring Cdkn2a deletion (C) plus Pten deletion (CP), wild-type human EGFR (CE) or EGFRvIII (CEv3) overexpression, or both overexpressed EGFRvIII and Pten deletion (CEv3P). Pairwise genotype comparisons revealed multiple differentially activated kinases, including Pdgfrb, Fgfr2, Lyn, Ddr1, and several Ephrin family members. We further investigated these potential targets for dual therapy with EGFR TKI by examining the transcriptional response of cultured astrocytes at 4, 24, and 48 hours after 3 μM afatinib. Afatinib induced no kinome changes in C and only 3 kinases (Fn3k, Prkg2, and Syk) were altered in CP astrocytes. Despite similar baseline gene expression profiles, CE astrocytes overexpressing wild-type EGFR responded significantly differently than C astrocytes without. Five kinases (Dclk1, Epha3, Epha7, Fgfr3, and Prkg1) were induced, while 14 were repressed. Six were similarly repressed in CEv3 (Bub1, Nek2, Pask, Plk4, Prkcb, and Vrk1). Whereas the kinase transcriptome response was blunted in C, CP, and CE astrocytes, afatinib induced altered expression of significantly more kinases in CEv3 (82) and CEv3P cells (49). One particularly attractive target in CEv3 astrocytes was Epha4, which afatinib induced >40-fold. Dual inhibition of EGFRvIII and Epha4 kinases may thus provide an opportunity for more effective targeted therapy. Citation Format: Erin Smithberger, Abigail K. Shelton, Madison K. Butler, Alex R. Flores, Ryan E. Bash, Steven P. Angus, Noah Sciaky, Harshil D. Dhruv, Gary L. Johnson, Michael E. Berens, Frank B. Furnari, C. Ryan Miller. Dynamic kinome profiling of EGFRvIII-driven murine astrocyte models of glioblastoma reveals targets for dual kinase inhibitor therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3019.

Abstract 2113: KRAS amplification mediates resistance to osimertinib in acquired afatinib-resistant NSCLC harboring exon 19 deletion/T790M in EGFR

Abstract Background: The second mutation of T790M in epidermal growth factor receptor (EGFR) exon 20 mediates resistance to first- and second-generation EGFR tyrosine kinase inhibitors (TKIs; gefitinib, erlotinib, and afatinib). The critical T790M mutation in EGFR has facilitated the development of third-generation mutation-selective EGFR TKIs (rociletinib and osimertinib). We previously reported heterogeneous afatinib resistance in lung adenocarcinoma PC-9 cells, harboring 15bp deletions in EGFR exon 19. Among the afatinib-resistant PC-9 cells, we noted the emergence of T790M in EGFR exon 20 and responses to third-generation EGFR-TKIs. Here, we used afatinib-resistant lung adenocarcinoma PC-9 cells, called AfaR, harboring the exon 19 deletion/T790M in EGFR. Purpose: To identify novel resistance mechanisms in post-afatinib treatment, two osimertinib-resistant cell lines, OsiR1 and OsiR2, were established using increasing concentrations of osimertinib by stepwise dose escalation up to 1 μmol/L over 10 to 12 months. Results: A linear and reversible KRAS amplification and overexpression with increased osimertinib concentrations in OsiR1 and OsiR2 cells, respectively, was detected. OsiR1 cells maintained osimertinib resistance with KRAS amplification after osimertinib withdrawal for 2 months. After osimertinib withdrawal for 2 months, OsiR2 cells exhibited KRAS attenuation and osimertinib sensitivity was entirely recovered in vitro and in vivo. Phospho-EGFR (Y1068) and growth factor receptor-bound protein 2 (GRB2)/son of sevenless homolog 1 (SOS1) complex was found to mediate osimertinib resistance in OsiR1 cells with sustained KRAS activation. The association of phosphorylated EGFR with the GRB2/SOS1 complex leads to KRAS activation. After 2 months of osimertinib withdrawal, the GRB2/SOS1 complex was dissociated, and the EGFR signal but not the GRB2/SOS1 signal was activated. Concomitant inhibition of mitogen-activated protein kinase kinase and EGFR could overcome osimertinib-resistance in OsiR1 cells. Conclusion: We characterized novel acquired resistance mechanisms for third-generation EGFR-TKI, osimertinib, providing insights into the development of novel treatment strategies. Citation Format: Toshimitsu Yamaoka, Ohba Motoi, Yasunari Kishino, Sojiro Kusumoto, Junji Tsurutani, Tohru Ohmori. KRAS amplification mediates resistance to osimertinib in acquired afatinib-resistant NSCLC harboring exon 19 deletion/T790M in EGFR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2113.

Abstract 1055: Evaluation of immune checkpoint inhibitor efficacy in EGFR mutant tumors

Abstract Tyrosine Kinase Inhibitor (TKI) of Epidermal Growth Factor Receptor (EGFR) is a first-line therapy for non-small cell carcinoma lung cancer patients with EGFR mutations, but patients often develop resistance to such a target therapy. Recently several PD-1 and PD-L1 inhibitors were approved for treating NSCLC, however, it remains unclear whether EGFR mutant NSCLC would benefit from PD-1/PD-L1 treatment. PD-1 response in cancer patients is closely associated with oncogenic mutation status, tumor microenvironment, PD-L1 expression and tumor mutation burden (TMB); however, the lack of pre-clinical models limited the investigation of the checkpoint inhibitors in EGFR mutant tumors. Here we use human PBMC reconstitution system for immune-humanization, and established a series of IO-Xenograft models (IO-CDX and IO-PDX) with cancer cell lines (IO-CDX) or patient-derived tissues (IO-PDX) that contain various EGFR mutations, including EGFR 19 del, L858R, T790M, and EGFR over-expression, amplification and fusion. We treated these models with either EGFR TKI (erlotinib, afatinib or osimertinib) or nivolumab, a PD-1 inhibitor. The predictive biomarkers including PD-L1 expression and TMB were analyzed by whole exom sequencing (WES) and FACS for tumor infiltrating lymphocytes (TILs), respectively. We found that 3 TKI-resistant PDX models bearing EGFR T790M mutation exhibited lower TILs than those bearing EGFR wild type or exon 19del mutation. Of the 8 IO-CDX or IO-PDX humanized models we have examined, those exhibiting higher TILs achieved better responses to nivolumab treatment. The 3 most sensitive models are EGFR amplification (wt), EGFR over-expression (wt) and EGFR exon 19del. In contrast, the EGFR T790M model from H1975 cell line was observed with hyper-progressive disease after nivolumab treatment. We will also discuss the contribution of PD-L1 expression and TMB to nivolumab in other models. In summary, these well-established IO-CDX and IO-PDX models can provide a human-resembling immune system for of immune therapeutics and combination with targeted therapies, and help to facilitate the understanding of relationship among tumor microenvironment, driver oncogenic mutations and drug response. Citation Format: Xuzhen Tang, Li Yang, Hui Qi, Xianzhi Zhai, Fuyang Wang, Xiangnan Qiang, Jie Xu, Xiaoran Qin, Qingyang Gu, Shaoyu Yan, Qunsheng Ji. Evaluation of immune checkpoint inhibitor efficacy in EGFR mutant tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1055.

Structure-based inhibitory peptide design targeting peptide-substrate binding site in EGFR tyrosine kinase.

EGFR (epidermal growth factor receptor) plays the critical roles in the vital cell activities, proliferation, differentiation, migration and survival in response to polypeptide growth factor ligands. Aberrant activation of this receptor has been demonstrated in many human cancers, particularly in non-small cell lung carcinoma (NSCLC). L858R point mutation is the most common oncogenic mutation in EGFR tyrosine kinase domain in patients with EGFR-mutated NSCLC. A feedback inhibitor of EGFR is MIG6 molecule which binds peptide-substrate binding site of the receptor and leads to degradation of activated EGFR. In this in silico study, the peptide-substrate binding site of EGFRL858R mutant has been targeted to inhibit it using molecular docking, MD simulation and MM-PBSA method. Finally, physicochemical properties of the designed peptides have been evaluated. A peptide library was provided composed of 31 peptides which were designed based on the MIG6 structure. The results indicated that, two peptides were able to inhibit EGFRL858R mutant selectively. This computational study could be helpful in designing novel inhibitory peptides to inhibit oncogenic EGFR mutants which do not respond to available EGFR TKIs.

Impact of KEAP1/NFE2L2/CUL3 mutations on duration of response to EGFR tyrosine kinase inhibitors in EGFR mutated non-small cell lung cancer

ObjectivesFor patients with Epidermal Growth Factor Receptor (EGFR)-mutated non-small cell lung cancer (NSCLC), frontline EGFR-tyrosine kinase inhibitor (TKI) therapy compared to chemotherapy improves outcomes. However, resistance to these agents uniformly develops. Recently, mutations in the KEAP1-NFE2L2 pathway have been implicated as a potential mechanism of acquired EGFR TKI resistance. Materials and methodsWe examined all patients with metastatic NSCLC with mutations in both EGFR and KEAP1/NFE2L2/CUL3 identified on next generation sequencing from 2015 - 2018. These patients were compared to a NSCLC control cohort with mutations in EGFR and wild type in KEAP1/NFE2L2/CUL3 matched on the basis of sex, smoking status, age and race. Time to treatment failure on EGFR TKI therapy and overall survival were examined. ResultsAmong 228 EGFR mutant NSCLCs, 17 (7%) also carried mutations in KEAP1, NFE2L2, or CUL3. The most common co-mutation in both the KEAP1/NFE2L2/CUL3 mutant and wild-type cohort was TP53. Patients with KEAP1/NFE2L2/CUL3 mutations had a shorter median time to treatment failure on EGFR TKI (4.7 months) compared with the wild-type matched cohort (13.0 months), p= 0.0014. There was no difference in overall survival. ConclusionFor NSCLC patients with mutations in EGFR, co-mutations in KEAP1/NFE2L2/CUL3 are associated with significantly decreased time to treatment failure. Our results suggest that these mutations represent a mechanism of intrinsic resistance to TKI treatment.

Standard dose osimertinib for erlotinib refractory T790M-negative EGFR-mutant non-small cell lung cancer with leptomeningeal disease.

Leptomeningeal spread in non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutations who experience disease progression on TKIs portends a poor prognosis. Mutation profiling of tumour DNA in cerebrospinal fluid (CSF) samples can be used to determine the presence of the EGFR T790M resistance mutation, indicating that osimertinib, a CNS-penetrating 3rd generation TKI may be efficacious. Eight patients on EGFR TKIs who progressed with cytology-proven leptomeningeal disease at our institution were studied. EGFR mutations were profiled in CSF using droplet digital PCR (ddPCR) and compared to matched plasma samples. Clinical characteristics and survival outcomes on subsequent therapies tailored to ddPCR analysis were reported. None of the four patients who developed leptomeningeal disease while receiving 1st generation EGFR TKIs developed the EGFR T790M mutation in CSF. One patient who did not have extra-cranial disease and was EGFR T790M-negative in both plasma and CSF was nevertheless treated with standard-dose osimertinib, and achieved a rapid and durable response lasting 9 months to date. Three patients developed leptomeningeal disease on osimertinib, with one patient developing the EGFR C797S mutation in a cis-allelic conformation with the EGFR T790M mutation in plasma. Standard-dose osimertinib resulted in a clinically meaningful response in a patient with EGFR T790M-negative 1st generation EGFR TKI refractory leptomeningeal disease. Next generation sequencing and ddPCR has a role at identifying the C797S mutation and its allelic conformation with the T790M mutation with clinical implications.

Diagnosis of lung biopsy employing the 2015 WHO criteria and detection of related oncogenic driver mutations

Objective: The diagnostic criteria of lung biopsy specimens by 2015 WHO lung tumor classification were used to evaluate lung biopsy specimens along with detection of genetic alterations of major tumor driving genes including epidermal growth factor receptor (EGFR). Methods: The clinical data, histological slides, immunohistochemical stains and special stains of 806 lung biopsy specimens at Beijing Hospital from July 2015 to July 2018 were retrospectively analyzed. Diagnosis of lung cancer was reclassified according to the 2015 WHO lung tumor classification and related gene mutation data were analyzed. Results: During a three-year period, the total number of lung cancer diagnosis was 483 cases, including 221 female and 262 male patients with age ranging from 37 to 85 years (median age of 65 years). There were 40 cases(8.28%) of small cell carcinoma,11 cases (2.28%) of large cell neuroendocrine carcinoma, 3 cases (0.62%) of combined neuroendocrine carcinoma, 2 cases(0.41%) of atypical carcinoid, 208 cases (43.06%) of adenocarcinoma, 92 cases(19.05%) of non-small cell carcinoma, favor adenocarcinoma, 66 cases (13.66%) of squamous cell carcinoma, 42 cases(8.70%) of non-small cell carcinoma, favor squamous cell carcinoma, 16 cases(3.31%) of non-small cell carcinoma, not otherwise specified, and 3 cases (0.62%) of non-small cell carcinoma, possible adenosquamous carcinoma. Among 202 cases tested, 107 cases (52.97%) showed EGFR mutations, including 86 of 133 cases (64.66%) of adenocarcinoma and 18 of 52 cases (34.62%) of non-small cell carcinoma, favor adenocarcinoma. Twenty two cases were found to have T790M mutation among 27 patients after EGFR TKI targeted drug therapy. Immunohistochemical staining of ALK (D5F3) was positive in 3 of 354 cases of non-small cell lung cancer, confirmed by EML4-ALK fusion gene fluorescence PCR. ROS1 gene fusion was found in 1 of 38 cases. Splicing mutations in exon 14 of MET gene were seen in one case of non-small cell carcinoma with spindle cell differentiation. Conclusion: The new diagnostic criteria by the 2015 WHO lung tumor classification is better suited for diagnosing lung biopsy specimens and providing accurate treatment guidance and improving the patient outcome.

HSR19-084: Real-World Treatment Patterns and Clinical Outcomes in Non-Small Cell Lung Cancer Patients with EGFR Exon 20 Insertion Mutations

Background: Mutations in the epidermal growth factor receptor (EGFR) gene have been identified in 10%–50% of patients with non-small cell lung cancer (NSCLC). Exon 20 insertion mutations represent about 2%–10% of this group. Reports of real world experience of NSCLC patients with EGFR exon 20 insertion mutations are limited. This study describes patient characteristics, treatment patterns, and survival outcomes of NSCLC patients with EGFR exon 20 insertions based on real world data. Methods: Flatiron Health electronic health record data, largely from community oncology practices, from January 2011–April 2018 were used for this retrospective observational study. Treatment-naïve (TN) and relapsed/refractory (RR) second-line patients diagnosed with NSCLC with EGFR exon 20 insertion mutation aged ≥18 years at treatment initiation were included. Patient characteristics were described, and Kaplan-Meier analyses were used to assess real world overall survival (rwOS) separately for TN (starting at first-line therapy) and RR (starting at second-line therapy) patients. Results: There were 128 TN and 71 RR patients identified. Median age was 66.5 and 65.0 years for TN and RR patients, respectively, and over half were female (TN: 59.4%, RR: 53.5%). Among 83 TN and 47 RR patients with known ECOG score at advanced diagnosis, most had score 0–1 (TN: 56.3%; RR: 62.0%). Central nervous system metastases were observed in 35.2% of TN and 33.8% of RR patients. While 45.3% of TN patients received any chemotherapy, approximately 20% of both TN and RR patient groups had exposure to various EGFR TKIs. Overall, median rwOS was low at 14.6 months for TN patients, and 10.1 months for RR patients. Conclusion: Real world survival of patients with EGFR exon 20 NSCLC remains poor. Treatment with any chemotherapy regimen was most commonly used followed by EGFR TKIs in TN patients, while the proportion treated with chemotherapy and EGFR TKIs was similar in RR patients. Despite limited evidence in this population, over a fifth of TN and RR patients received EGFR TKI monotherapy. This study demonstrated unmet need for improved therapeutic options in TN and RR patients with NSCLC with EGFR exon 20 insertion mutation.