2nd Generation EGFR-TKI Research Articles

Next-generation sequencing based mutation profiling reveals heterogeneity of clinical response and resistance to osimertinib

ObjectivesThe 3rd generation epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR TKI) osimertinib has shown promising efficacy both in EGFR-mutant, T790M positive non-small cell lung cancer (NSCLC) patients who have become resistant to 1st or 2nd generation EGFR TKIs and patients with sensitizing EGFR mutations as the first line therapy. However, the degree and duration of response to osimertinib are heterogeneous. We hypothesized that the concurrent genomic landscape of these tumors could play a role in clinical outcomes and/or mechanisms of resistance. Materials and methodsWe conducted a retrospective multicenter study of lung cancer patients who had developed resistance to osimertinib. Genomic profiling was done for all the patients by using targeted next-generation sequencing encompassing 59–1021 cancer-related genes. Results and conclusionKnown EGFR-dependent resistant mutations and activation of alternative pathways were identified in 44 % of all the patients with great heterogeneity. Gain-of-function mutations of CTNNB1 were highly enriched in our cohort. Some other putative resistance mechanisms to osimertinib, such as the recurrent EGFR V834 L mutation, were also identified. Moreover, pathogenic mutations of TP53 were negatively related to the efficacy of osimertinib. To sum up, heterogeneity of resistance to osimertinib was not only manifested by inter-individual differences, but also embodied in its intra-individual diversity.

486P - Phase II study of low-dose afatinib maintenance treatment for patients with EGFR-mutated non-small cell lung cancer (NJLCG1601)

BackgroundAfatinib is a 2nd generation EGFR-TKI and has been a standard first line treatment for patients (pts) with advanced NSCLC with EGFR mutations. Afatinib has good efficiency, whereas afatinib has severe adverse events (AEs) and often lead to discontinuation of treatment or a dose reduction. NJLCG1601 was conducted to evaluate the efficacy of low-dose afatinib maintenance treatment. MethodsEGFR-TKI-naïve stage III / IV / recurrent NSCLC pts harboring EGFR mutations (exon 19 deletion or exon 21 L858R or minor mutation) were enrolled. Pts received afatinib 40mg orally once a day. When prescribed AEs (grade ≧ 2 in CACAE ver 4.0) or unacceptable AEs occurred, a dose of afatinib was reduced from 40mg to 30mg, 30mg to 20mg. The treatment was continued until disease progression, occurrence of intolerable severe AEs, or withdrawal of consent. The primary endpoint was 1-year progression free survival (PFS) rate. Secondary endpoints were PFS, overall response rate (ORR), the incidence of AEs, and the incidence of grade ≧ 3 AEs. Assuming a threshold 1-year PFS rate of 42% and an expected 1-year PFS rate of 63%, a total of 26 pts was required to have 90% power at a two-tailed alpha of 0.05. This is a subset analysis of patients with adenocarcinoma in NJLCG1601 study (Trial Identifier, UMIN000020688). ResultsBetween Apr 2016 and Sep 2017, 30 pts were enrolled. Among 30 patients, 28 patients (93%) were adenocarcinoma. Pts characteristics were as follows: median ages, 70 years (range 46-79 years); proportion of males, 43%; clinical stage, IV 61% and post-op 39%; EGFR mutation types, exon 19 deletion 54%, ex21 L858R 36%, and minor mutations 11%. 1-year PFS rate was 53.6% (95% CI, 33.8-69.8), median PFS was 13.0 months, and ORR was 78.6%. Dose reductions occurred in 86% (24/28). Incidence of all cause grade ≧ 3 was 60% which is smaller than the frequency of Japanese subset in LUX-Lung 3. ILD was occurred in 3 patients and 1 patients died. Treatment-related deaths were observed in 2 pts. ConclusionsLow-dose afatinib maintenance treatment reduced treatment-related adverse events without detracting from the therapeutic efficacy. Legal entity responsible for the studyUMIN000020688. FundingHas not received any funding. DisclosureA. Nakamura: Honoraria (self): MSD; Honoraria (self): Chugai; Honoraria (self): AstraZeneca. S. Inoue: Honoraria (self): AstraZeneca; Honoraria (self): Eli Lilly; Honoraria (self): MSD; Honoraria (self): Otsuka Pharmaceutical; Honoraria (self), research funding: Kyorin Pharmaceutical; Honoraria (self): Ono Pharmaceutical; Honoraria (self): GlaxoSmithKline; Honoraria (self): Daiichi Sankyo Company; Honoraria (self): Taiho Pharmaceutical; Honoraria (self): Chugai Pharmaceutical; Honoraria (self): Teijin Pharma; Honoraria (self), research funding: Novartis Pharma; Honoraria (self): Pfizer Inc; Honoraria (self): Boehringer Ingelheim; Honoraria (self): Meiji Seika Pharma. S. Sugawara: Honoraria (self): AstraZeneca; Honoraria (self): Chugai Pharma; Honoraria (self): Nippon Boehringer Ingelheim; Honoraria (self): Taiho Pharmaceutical; Honoraria (self): Pfizer; Honoraria (self): Eli Lilly; Honoraria (self): Novartis; Honoraria (self): Kyowa Hakko Kirin; Honoraria (self): Bristol-Myers Squibb; Honoraria (self): Ono Pharmaceutical; Honoraria (self): MSD. All other authors have declared no conflicts of interest.

P1.01-50 Impact of Concomitant HER2 Alterations in Mediating Clinical Outcomes of EGFR-Mutant Patients to Different Generation of EGFR-TKIs

The 2nd generation EGFR-TKI are selective and potent irreversible pan-HER inhibitors. Given their effect on HER2 besides EGFR, we investigated the role of HER2 co-alterations in mediating the clinical outcomes of EGFR-mutant non-small cell lung cancers to 2nd generation or other generation EGFR-TKIs. Plasma/tissue samples from advanced NSCLC before and after 1L EGFR-TKI treatment were sequenced using 168-/520-cancer-related gene panels, respectively. Clinical records of 94 EGFR-mutant pts (92 adenocarcinomas and 2 adenosquamous cell carcinomas) after 1st/3rd (n=73) or 2nd generation (afatinib, n=16; dacomitinib, n=5) EGFR-TKI treatment were collected for clinical outcomes evaluation. Event-time distributions were estimated using Kaplan-Meier and compared with long-rank test. Among the 94 pts identified as EGFR-positive at baseline, 8 (8.5%) had concurrent HER2 gain-of-function alterations (amplification or active mutation). Survival analysis showed that for those with concomitant EGFR and HER2 alterations at baseline, pts achieved favorable PFS (23.2 vs 5.6 months, p=0.04) to 2nd generation (n=2) than to 1st/3rd generation EGFR-TKI (n=6) as 1L treatment. And for those receiving 1L 1st/3rd generation EGFR-TKI (n=73), the presence of baseline HER2 alterations (n=6) was associated with shorter PFS (5.6 vs 9.8 months, p=0.16) and OS (15.7 vs 21.0 months, p=0.06) than absence of HER2 (n=67), although the difference was not significant due to a small sample size. In addition, we found all the samples (n=21) obtained from pts after 2nd generation EGFR-TKI resistance were HER2-negative, but pts progressed on 1st or 3rd generation EGFR-TKI with HER2 as the only resistance mechanism were observed in our study cohort. Our findings suggested that, for EGFR-mutant pts combined with HER2 alterations at baseline or those resistant to 1L treatment of 1st/3rd EGFR-TKI due to HER2 alterations only, 2nd EGFR-TKI might be a better choice. Our findings suggested that, for EGFR-mutant pts combined with HER2 alterations at baseline or those resistant to 1L treatment of 1st/3rd EGFR-TKI due to HER2 alterations only, 2nd EGFR-TKI might be a better choice.

P2.14-57 Efficacy of 3rd Generation EGFR-TKIs After Failing First or Second Generation EGFR-TKIs in EGFR Mutation-Positive NSCLC (ROOT-EGFR)

Over the past decade, treatment of EGFR mutation –positive NSCLC has been revolutionized with the development of next generation EGFR TKIs. Four epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), erlotinib, gefitinib, afatinib and osimertinib, are currently available in Korea. Recent trials have compared the available and emerging EGFR TKIs head to head. The highly anticipated findings of the phase III FLAURA trial showed a nearly doubling of PFS with frontline osimertinib, a third-generation TKI, versus erlotinib or gefitinib. The optimal sequence of EGFR TKIs is still controversial. And many countries 3rd generation EGFR TKI is not readily available. The purpose of this study is to analyze the efficacy in patients who received first-line gefitinib, erlotinib, or afatinib and followed by 3rd generation EGFR-TKIs. This non-interventional observational study through big data analysis will retrospectively collect de-identified patient data from clinical data warehouse (CDW) using a unique algorithm with Standard Query Language (SQL) called ROOT project. Over 10 years, 2,358 patients were diagnosed recurrent or metastatic non-small cell lung cancer and received 1st or 2nd generation EGFR TKIs at Samsung Medical Center. 72.9% (1720/2358) of EGFR mutation-positive NSCLC patients received 1st or 2nd generation EGFR-TKIs as first line palliative chemotherapy. In total population, 30.8% (727/2358) patients received 3rd generation EGFR TKIs. Among them, patients who received 3rd generation EGFR TKI after 1st or 2nd generation EGF TKIs showed better overall survival (45.0 months (95% CI, 41.8-48.2). Among 1720 patients who received 1st or 2nd generation EGFR-TKIs as first line palliative chemotherapy, 313 patients received 3rd generation EGFR-TKIs as 2nd line chemotherapy and 200 patients received as more than 3rd line chemotherapy. However, there was no difference of overall survival between them (44.0 months vs 47.0months, p-value=0.104). This study was meaningful as a study of what can be the best sequence in EGFR-TKI treatment. Updated and detail clinical and exploratory biomarker outcome will be presented at the meeting.

P2.03-42 Highly Sensitive Sel-CapTM Lung Cancer Panel NGS for Detection of Tumor-Derived EGFR Mutations in Plasma Cell-Free DNA in Lung Cancer

Next-generation sequencing is a genetic analysis platform for multiple analysis and large-scale analysis. Sensitivity is limited by methods for detecting cancer mutations that are contained at low rates in blood. With two highly sensitive detection techniques for genetic alterations, we evaluated their diagnostic value for tumor-derived EGFR mutations in plasma cell-free DNA (cfDNA) and measured concordance rate of activating EGFR mutations between cfDNA and tumor tissue. We conducted this study using FFPE tissue and plasma samples which were donated from NSCLC patients (N=316) between 2011 and 2013 and stored in the Human-derived Biomaterials Bank in Seoul St. Mary’s Hospital. Meanwhile, we prospectively collected plasma samples simultaneously when tumor tissue was taken out by surgery. We also collected peripheral blood just before administration of EGFR TKIs and each time imaging study was done during the administration of EGFR TKIs. We analyzed activating EGFR mutations in both cfDNA and tumor tissues using PNAclamping, Cancer panel and Sel-CapTM Lung Cancer Panel. The sensitivity and specificity of three detection techniques, PNAclamp, cancer panel and Sel-CapTM Lung Cancer Panel NGS for EGFR mutations in tumor tissues were more than 90% in all. In particular, EGFR T790M was found not only in the patients acquiring drug resistance approximately 10-12 months following the administration of 1st or 2nd generation EGFR TKIs, but also in the patients in whom EGFR TKI was continuously maintained over 30 months. The sensitivity of Sel-CapTM Lung Cancer Panel NGS for EGFR exon19 deletion and exon21 L858R in cfDNA was 76.2% and 61.1%, respectively. In addition, the sensitivity for double mutation of 19del and T790M was about 30% higher compared to that of cancer panel sold domestically (2/20, 10.0% vs. 9/20, 45.0%), suggesting that Sel-CapTM Lung Cancer Panel NGS enables to detect EGFR mutation up to 0.1% in plasma cfDNA. As a result of tracking of outcomes of EGFR TKIs treatment over time for patients with low frequency of EGFR mutations, we found that 1st or 2nd generation EGFR TKIs were switched into 3rd generation EGFR TKIs on the basis of disease progression on radiologic imaging, when an average of 6.8 months (0.2-25.4 months) elapsed from the first detection time of T790M in cfDNA. The presence of T790M in peripheral blood harvested periodically from patients receiving EGFR TKIs was initially identified about 3-5 months ahead of disease progress assessed by imaging study. With regard to the detection of EGFR mutations in lung cancer patients, Sel-CapTM Lung Cancer Panel NGS platform showed superior sensitivity to conventional NGS-based cancer panels as well as higher concordance rate between tumor tissues and plasma cfDNA. In longitudinal plasma cfDNA samples isolated from periodic liquid biopsies, quantitative changes of EGFR mutation were found to be significantly correlated to tumor response by EGFR TKIs. Our data suggest that Sel-CapTM Lung Cancer Panel NGS is a highly sensitive diagnostic platform for detection of EGFR T790M induced by long-term administration of EGFR TKIs and for determining when 3rd generation EGFR TKI be begun.

1535P - HER3 ligand heregulin expression and clinical implication in patients with epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer treated with EGFR-tyrosine kinase inhibitors

BackgroundEpidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) improve the prognosis of EGFR-mutant non-small cell lung cancer (NSCLC). However, other human epidermal growth factor receptor (HER) families contribute to EGFR-TKI resistance. The HER3 ligand heregulin is aberrantly expressed in NSCLC. Previously, heregulin genomic induction in an EGFR-mutant NSCLC cell line caused EGFR-TKI resistance, except against 2nd-generation EGFR-TKIs, which uniquely blocked the pan-HER family. However, the clinical relevance of heregulin is unclear in EGFR-mutant NSCLC. Here, we aimed to explore the implication of heregulin in patients with EGFR-mutant NSCLC treated with EGFR-TKIs. MethodsSoluble heregulin was immunologically measured in the plasma of patients with EGFR-mutant NSCLC. Cut-off values were determined via 1-year progression-free survival (PFS) receiver operating characteristic curve. Relationship between soluble heregulin and PFS, after EGFR-TKI therapy, was analyzed using a Cox proportional hazards model. ResultsSeventy-six patients were enrolled, of which 44 were treated with 1st-generation, 29 with 2nd-generation, and 3 with 3rd-generation EGFR-TKIs. Soluble heregulin levels were found to vary (range: 274–7,138pg/mL, median: 741.5pg/mL). Among patients treated with 1st- and 3rd-generation EGFR-TKIs, those with high heregulin (n=22, > 800pg/mL) had a shorter PFS than those with low heregulin (n=25, < 800pg/mL) levels; median PFS of 322 and 667 days were, respectively, observed. Cox proportional hazards model indicated a trend toward resistance (HR: 1.797, 95% CI: 0.833–3.877), except with 2nd-generation EGFR-TKIs (HR: 0.879, 95% CI: 0.325–2.376). ConclusionsResults showed soluble heregulin to potentially correlate with EGFR-TKI resistance, though not so for 2nd-generation EGFR-TKIs, in patients with EGFR-mutant NSCLC. Therefore, 2nd-generation EGFR-TKIs warrant comparative clinical examination regarding their anti-cancer efficacy in heregulin-expressing NSCLC. Legal entity responsible for the studyThe authors. FundingBoehringer Ingelheim. DisclosureK. Yonesaka: Honoraria (self), Research grant / Funding (institution): Boehringer ingelheim. E. Iwama: Research grant / Funding (institution): Boehringer Ingelheim. H. Hayashi: Honoraria (self), Research grant / Funding (institution): Boehringer Ingelheim. S. Suzuki: Research grant / Funding (institution): Boehringer Ingelheim. R. Kato: Research grant / Funding (institution): Boehringer Ingelheim. S. Watanabe: Research grant / Funding (institution): Boehringer Ingelheim. T. Takahama: Honoraria (self), Research grant / Funding (institution): Boehringer Ingelheim. J. Tanizaki: Honoraria (self), Research grant / Funding (institution): Boehringer Ingelheim. K. Tanaka: Research grant / Funding (institution): Boehringer Ingelheim. M. Takeda: Honoraria (self), Research grant / Funding (institution): Boehringer Ingelheim. K. Sakai: Research grant / Funding (institution): Boehringer Ingelheim. K. Azuma: Honoraria (self), Research grant / Funding (institution): Boehringer Ingelheim. Y. Chiba: Research grant / Funding (institution): Boehringer Ingelheim. S. Atagi: Honoraria (self): Boehringer Ingelheim. K. Nishio: Research grant / Funding (self), Research grant / Funding (institution): Boehringer Ingelheim. I. Okamoto: Honoraria (self), Research grant / Funding (self), Research grant / Funding (institution): Boehringer Ingelheim; Honoraria (self), Research grant / Funding (self), Research grant / Funding (institution): Bristol-Myers Squibb; Honoraria (self), Research grant / Funding (self), Research grant / Funding (institution): AstraZeneca. K. Nakagawa: Honoraria (self), Research grant / Funding (self), Research grant / Funding (institution): Boehringer ingelheim; Honoraria (self), Research grant / Funding (self), Research grant / Funding (institution): AstraZeneca; Honoraria (self), Research grant / Funding (self), Research grant / Funding (institution): Bristol-Myers Squibb.

EP1.14-12 KRAS Exon 2 Mutation - A Resistance Pathway to 1st and 2nd Generation EGFR TKIs

EP1.14-12 KRAS Exon 2 Mutation - A Resistance Pathway to 1st and 2nd Generation EGFR TKIs

Re-administration of EGFR-TKI to mutated EGFR NSCLC patients after remission of osimertinib induced ILD

Abstract Background Osimertinib is a key-drug to treat NSCLC harboring EGFR sensitizing mutation at 1st line setting as well as T790M resistant mutation at late line. Interstitial lung disease/pneumonitis (ILD) is a fatal adverse event induced by tyrosine kinase inhibitors including osimertinib especially in the Japanese patients. On the other hand, transient asymptomatic pulmonary opacities (TAPO) caused by osimertinib were reported as relatively frequent (10-20%) phenomenon and usually safe, permitting continuous or stop and go treatment with osimertinib. Purpose & Result We retrospectively analyzed consecutive 60 sonsequtive EGFR mutation positive NSCLC patients treated with osimertinib in our institution by Feb. 2019. Fifteen patients were TKI naive and the other 45 were pretreated with 1st or 2nd generation EGFR-TKI. ILD occurred in ten (16.7%) patients then administration of osimertinib was interrupted. Corticosteroid was administered to all patients for the ILD, resulting in fully remission of the lung adverse event without any related patient death. Three patients were re-administered with TKI (2 osimertinib for T790M positive, 1 gefitinib for 1st line setting) after remission of ILD. Re-challenged TKI was not successful for these patients resulting in ILD recurrence and permanent cease of TKI treatment. Conclusion We suggest that strategies other than re-administration of osimertinib should be considered even though osimertinib induced ILD is fully recovered by corticosteroid therapy.

EP1.01-72 Treatment Outcome of 2nd Generation EGFR-TKI for Non-Small Cell Lung Cancer

EP1.01-72 Treatment Outcome of 2nd Generation EGFR-TKI for Non-Small Cell Lung Cancer

P2.03-13 Acquired Resistance to Afatinib in Non-Small Cell Lung Cancer with EGFR G719X Mutation

The frequency of EGFR mutations is said to be relatively high within East Asian population. The most common EGFR mutations such as exon 19 deletions and exon 21 L858R mutation are strong predictors of good response to EGFR-TKIs in non-small-cell lung cancer. Exon 20 T790M mutation which accounts for a large part of 1st and 2nd generation EGFR-TKI resistance, is well known to be detected after failure of prior EGFR-TKI therapy. Furthermore, T790M mutation is a predictor of good response to osimertinib, 3rd generation EGFR-TKI. Meanwhile, other uncommon EGFR mutations are identified, such as exon 18 G719X mutation, exon 20 S768I mutation, and exon 21 L861Q mutation. LUX-Lung study reported afatinib may be effective for these uncommon EGFR mutations, however, their treatments are still controversial and their resistance-gaining mechanisms to EGFR-TKI are also unknown. We evaluated the characteristics of patients with G719X single mutation and their effectiveness of afatinib treatment. Furthermore, we analyzed the mechanism of acquired resistance to afatinib from their re-biopsied samples. Eighteen patients had G719X single mutation, which include fifteen patients with smoking history. Of all patients with G719X mutation, ten patients were treated with afatinib, which response rate was 40% (4/10). Eight patients of those treated with afatinib underwent re-biopsy after failure of the therapy, and their results revealed that G719X mutation was disappeared in six samples, and T790M mutation was not detected after afatinib treatment. As previously reported, the presence of G719X mutation might be used as a predictor of sensitivity to afanitib therapy. Our data suggest that the loss of cancer cells with G719X mutation could be the main mechanism of acquired resistance to afanitib. Furthermore, patients with smoking history were seen frequently in those with G719X mutation. Considering that G719X mutation negative cancer cells may coexist with G719X mutation positive cells in NSCLC patients, smoking might be associated with development of its heterogeneity.

Abstract 3999: Advanced lung adenocarcinoma cell bank (ALACB) : A comprehensive preclinical platform

Abstract Purpose: Patients with advanced lung adenocarcinoma often lack large clinical specimens required for molecular testing which limits next therapeutic options. Patient-derived cells (PDC) from malignant effusions can predict patient drug responses and provide a valuable tool for studying drug resistance mechanisms. In this study, we created an Advanced Lung Adenocarcinoma Cell Bank (ALACB) consisting of 28 unique PDCs established from patients who progressed on various tyrosine kinase inhibitor TKIs including 3rd generation EGFR TKI osimertinib. Experimental design: Patient malignant effusion samples were tested for malignancy, processed, observed by light microscopy, and cultured with appropriate media and supplements. The criteria for established PDCs include the following: sharing the same driver mutation as the patient; free of stromal fibroblasts confirmed by FACS staining; recapitulating patient’s drug response; and can be cryopreserved and re-grown. Established PDCs were further authenticated by STR profiling and microplasma testing to maintain cell quality. We were able to perform various in vitro assays and next generation sequencing for detailed analysis of individual PDC. Results: We were able to successfully establish 28 PDCs incorporating unique patient characteristics. Among the total 146 samples, we observed a success rate of 30% only accounting 95 malignancy positive samples. Established PDCs consists of 20 EGFR mutation positive cell lines, 3 with ALK rearrangements, 6 with ROS1 rearrangements, 1 with BRAF K601E mutation and 1 with KRAS G12D mutation. Seven PDCs were established from patients who progressed on osimertinib, two from olmutinib (HM61713), and others from 1st and 2nd generation EGFR TKI such as gefitininb and erlotinib. We were able to newly generate 3 PDCs harboring a T790M mutation with an activating EGFR mutation. Moreover, we are one of the few labs to successfully establish an EGFR del19/T790M/C797S triple mutant PDC showing resistance to osimertinib treatment in vitro. Other established PDCs harbored rare EGFR mutations including exon 20 insertion, L861Q and G719X/S768I. PDC with EGFR G719X/S768I compound mutation showed high sensitivity to afatinib but was resistant to gefitininb and osimertinib treatment. Three ALK-positive PDCs were derived from crizotinib-resistant, alectinib-resistant and ceritinib-resistant tumors. Furthermore, of 6 ROS1 fusion positive PDCs, 3 were derived from TKI-naïve tumors, and 3 from crizotinib-resistant tumors. Conclusions: Successfully established PDCs reflecting patient genomic profiles and drug response is a valuable resource for biological assays, generating in vitro drug-resistant models, and evaluating novel drugs and therapeutic targets. Further advances in drug development combined with a library of cell bank will facilitate lung cancer translational research. Citation Format: Hyeong-Seok Joo, Dong Hwi Kim, Seok-Young Kim, Ji-Yeon Lee, Mi-Ran Yun, Han-Na Kang, Byoung Chul Cho, Hye Ryun Kim. Advanced lung adenocarcinoma cell bank (ALACB) : A comprehensive preclinical platform [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 3999.

Mechanisms of acquired resistance to afatinib clarified with liquid biopsy.

Although mechanisms of acquired resistance to 1st and 3rd generation EGFR-TKI continue to be elucidated, there have been few clinical investigations into the mechanisms of acquired resistance to the 2nd generation EGFR-TKI afatinib. We analyzed data from 20 patients with advanced lung adenocarcinoma who acquired resistance to afatinib, including resistance during EGFR-TKI re-challenge. We examined EGFR T790M and C797S mutations, BRAF V600E mutation, and MET amplification with the MBP-QP method and with droplet digital PCR using ctDNA and re-biopsy samples obtained before and after afatinib treatment. Just before afatinib treatment, 15 of the 20 patients were T790M negative and five were positive. Among the T790M negative patients, 40.0% (6/15) became positive at the time of PD under afatinib. In patients positive for T790M, changes in T790M allele frequency were correlated with afatinib treatment efficacy. C797S was not detected in any patients just before afatinib treatment, but it appeared after treatment in three patients, although with very low allele frequency. Two of these three patients, although positive for both C797S and T790M, achieved PR to osimertinib. However, PFS of these patients was somewhat shorter than that of patients positive for T790M only. BRAF V600E was detected in one patient at PD under afatinib. MET amplification was not detected in this study. T790M is associated with acquired resistance to afatinib, as with 1st generation EGFR-TKI, but with somewhat lower frequency. The influence of C797S on resistance to afatinib is less than that of T790M, but C797S might cause shorter PFS under osimertinib.

P3.01-45 Multifactorial Gene Alterations in EGFR Bypass Pathway are Induced by Afatinib in T790M-Mutant NSCLC Resistant to Osmertinib

The 3rd generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) was developed to target the EGFR T790M resistance mutation in non-small cell lung cancer patients resistant to 1st or 2nd generation EGFR-TKIs. Although some mechanisms of acquired resistance to 3rd generation EGFR-TKI such as EGFR C797S mutation have been reported, the effect and resistant mechanisms to afatinib followed by osmertinib has not been well-known. Nine patients with EGFR T790M-mutant NSCLC resistance to 3rd generation EGFR-TKI were enrolled in this study and treated with afatinib. Plasma samples were collected before treatment, 4 weeks after treatment, and at disease progression. Mutation profile and tumor mutation burden (TMB) in plasma cell free DNA (cfDNA) were analyzed by CAPP-seq. The objective response rate and median progression-free survival of afatinib were 0% and 2.0 months, respectively. At the time of 4 weeks after treatment, four patients developed disease progression and five patients showed stable disease. A total of 36 somatic mutations or amplification were detected in plasma cfDNA before afatinib treatment; EGFR activating mutations in 8 patients, T790M mutation in 4, TP53 mutations in 6, PIK3CA mutations in 3, BRAF mutations in 3, MET amplification in 3, CTNNB1 mutations in 2, ERBB2 mutations in 2, C797S mutation in 1, SMAD4 mutation in 1, EGFR minor mutation in 1, KRAS mutation in 1, and APC mutation in one patient. EGFR C797S mutation in cfDNA was detected during afatinib treatment in two cases. In the patients having stable disease at 4 weeks after treatment, mutant allele frequency and TMB tended to decline once, and then increased in association with disease progression. Detection of mutant allele frequency and TMB of ctDNA by CAPP-seq could monitor the effectiveness and resistance to afatinib. Resistant mechanisms to afatinib might be characterized by multifactorial bypass pathway activation.

P2.13-12 The deNovo T790M Mutation Recovered by ddPCR is Associated with a Poor Response to 1st and 2nd Generation EGFR-TKI in Lung Adenocarcinoma.

P2.13-12 The deNovo T790M Mutation Recovered by ddPCR is Associated with a Poor Response to 1st and 2nd Generation EGFR-TKI in Lung Adenocarcinoma.

P2.13-22 Resistant Patterns to Osimertinib in Non-Small Cell Lung Cancer Patients with Both T790M and Sensitizing EGFR Mutation

Osimertinib is a 3rd generation, EGFR-TKI which is active for both sensitizing mutations and T790M of EGFR. Although it has proven its efficacy, the acquired resistance eventually occurs requiring the investigation for resistant mechanisms to provide overcoming strategies. We retrospectively reviewed the medical records of T790M+ lung cancer patients treated with osimertinib after acquiring resistance to 1st- or 2nd generation EGFR-TKI between February 2016 and June 2017 to evaluate the efficacy and safety of the drug. In addition, the moleculo-pathologic data of re-biopsy samples after the development of resistance to osimertinib were analyzed. In 23 patients included, the median age was 59.0 years and thirteen (56.5%) were female. The median duration of follow-up was 11.9 months. 17 patients achieved an objective response (73.9%) and the disease was controlled in 22 patients (95.7%). Median progression-free survival was 7.4 months (95% CI 3.6-11.0) and median overall survival was not reached. The adverse effects were minimal except one case of severe pneumonitis. Among 14 patients who finally experienced the disease progression during osimertinib, the re-biopsy was performed in 10 patients. T790M disappeared in 7 patients (70%) and one of them showed the wild-type conversion. Transformation to small cell carcinoma was observed in 2 patients who responded to chemotherapy with etoposide and cisplatin. A newly developed C797S mutation was detected in one patient. Interestingly, C797S disappeared after 6 cycles of pembrolizumab following osimertinib-resistance and the patient responded well to osimertinib again. Osimertinib showed the clinical activity and the favorable toxicity profile comparable to those of clinical trials in real world practice in Korea. The evaluation with repeated biopsy sample after osimertinib-resistance is needed to guide the following treatment strategy.

Abstract 4576: Combination of a label independent CTC enrichment platform with fluorescence in situ hybridization for the molecular characterization of CTCs in non-small cell lung cancer patients

Abstract Background: Patients with EGFRmt non-small cell lung cancer (NSCLC) develop resistance to first (erlotinib/ gefitinib) and second (afatinib) generation EGFR tyrosine kinase inhibitors (EGFR TKIs). Circulating tumor cells (CTCs) may be used as a potential biological material to monitor genetic alterations during the clinical course of the disease. We used the ParsortixTM (ANGLE) system, a label-independent microfluidic-based technology platform, to enrich CTCs from whole blood of NSCLC patients to evaluate the presence of HER-2 and c-MET amplification using fluorescence in situ hybridization (FISH). Materials and methods: Peripheral blood (PB) (15mL) from 42 patients with EGFRmt and resistant to 1st and 2nd generation EGFR TKIs NSCLC was obtained before, during and after treatment with osimertinib. CTCs were isolated using the ParsortixTM system and were further analyzed for HER-2 and c-MET amplification by FISH. Results: PB samples were obtained before (n=42), after one cycle (n=12) and at PD (n=7) during treatment with Osimertinib. One patient had HER-2 amplified CTCs whereas in total, 5 patients had marginally HER-2-positive CTCs; HER-2-positive CTCs were observed in 1/42 baseline sample but in none post-1st cycle and PD samples. Similarly, c-MET amplification was detected in 3/42 patients at baseline whereas in total 4 patients had marginally c-MET-positive CTCs. Treatment with Osimertinib resulted in a decrease of the number of c-MET amplified CTCs in these patients. In all c-MET and HER-2 amplified CTCs there was no HER-2 or c-MET amplification in the primary tumor. c-MET amplification in the primary tumor was detected only in one patient, however this patient had no detectable CTCs. Conclusions: Our preliminary results indicate that in selected patients with EGFRmt NSCLC, detection of HER-2 or c-MET amplified CTCs may be correlated with tumor resistance to EGFR TKIs. The ParsortixTM system can be successfully combined with downstream FISH analysis for the molecular characterization of CTCs in NSCLC patients. More samples are currently being analyzed in order to reveal the implication of HER-2 and c-MET amplification in resistance to therapy with first, second and third generation EGFR TKIs. Citation Format: Aliki Ntzifa, Martha Zavridou, Pavlos Pollakis, Georgia Bardi, Vasilis Georgoulias, Athanasios Kotsakis, Evi Lianidou. Combination of a label independent CTC enrichment platform with fluorescence in situ hybridization for the molecular characterization of CTCs in non-small cell lung cancer patients [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 4576.

MA 02.11 A Phase I Trial of Erlotinib and Onalespib in EGFR-mutant NSCLC: Focus on EGFR Exon 20 Insertions

Onalespib (AT13387) is a non-ansamycin small molecule that inhibits heat shock protein-90 (Hsp90). Hsp90 inhibitors (Hsp90i) preferentially degrade overexpressed and mutated oncoproteins including those that mediate resistance to EGFR-TKIs. Previous Hsp90i studies demonstrated activity in EGFR-mutant NSCLC including EGFR Exon 20 insertions (EGFRex20ins) - uncommon EGFR mutations typically refractory to 1st and 2nd generation EGFR-TKIs. A phase I study of onalespib plus erlotinib was conducted to determine the MTD, DLT, RP2D, pharmacokinetics (PK) and preliminary antitumor activity for a planned phase 2 trial in EGFR-mutant NSCLC including EGFRex20ins. Using a 3 + 3, dose escalation design, onalespib was examined at 2 dose levels (DL) from 150 (DL0) to 120 (DL-1) mg/m2 IV weekly (D1, D8, D15 on a q28 day cycle). Daily erlotinib was given at 150 mg at both DL. Key eligibility: NSCLC with EGFR activating mutation including EGFRex20ins, age ≥ 18, ECOG PS≤2, acceptable organ function, and ≥1 systemic therapy for advanced disease (platinum-based chemotherapy for EGFRex20ins and EGFR-TKI for other EGFR-mutations). Plasma for PK and ctDNA for next-generation sequencing of ∼70 cancer related genes was collected at relevant timepoints. 9 pts have been treated on 2 DL (3 DL0, 6 DL-1). Pt characteristics: median age 65, M/F (2/7), ECOG PS 0-1 (4/5), EGFRex20ins (8), EGFR E19del (1). 7 pts completed ≥1 cycle. Two DLTs (grade (Gr) 3 maculopapular rash and Gr 3 hypophosphatemia) occurred in DL0. Common drug-related adverse events (AE) of any Gr were diarrhea (100%) and rash (44%), fatigue (55%), increased bilirubin (22%), nausea (44%) and vomiting (33%). Drug-related Gr 3 AEs were diarrhea (55%), maculopapular rash (11%) and hypophosphatemia (11%). At the planned 2-month evaluation, 5 pts had SD, 3 PD, and 1 had withdrawn for toxicity. Of the 5 pts continuing, 2 had SD and 1 PD at the 4-month evaluation. Kaplan-Meier estimate on therapy without progression at the second evaluation is 30% (95% CI: 10 to 87%). In patients with EGFR-mutant NSCLC, onalespib plus erlotinib is feasible, tolerable and demonstrates disease control in EGFRex20ins, thereby addressing a key unmet need in NSCLC. The RP2D is erlotinib 150 mg PO daily and onalespib 120 mg/m2 weekly (D1, D8, D15 q28days). Diarrhea was the most common AE, and generally manageable with supportive care and dose reduction to DL-1. Updated results including PK as well as ctDNA for EGFR-mutation and relevant bypass tracts mediating EGFR-TKI resistance will be presented.

Abstract 3157: Osimertinib (AZD9291) is effective against NSCLC cells harboring EGFR exon 20 insertion mutations

Abstract Background: Although EGFR exon 20 insertion mutation is the third most common among EGFR-mutant NSCLC, this mutant clones are resistant to the first-generation EGFR TKIs. In addition, the efficacy of the second or third-generation EGFR TKIs is unknown in NSCLC with EGFR exon 20 insertion mutation. Methods: Various EGFR exon 20 insertion mutations were introduced into Ba/F3 cells as follows: A763_Y764insFQEA in C-helix; and V769_D770insASV, D770_N771insNPG, D770_N771insSVD, P772_H773insPR, H773_V774insNPH, H773_V774insH, and H773_V774insAH in loop following C-helix. The patient-derived SNU-3173 cell line was developed in a patient with EGFRH773_V774insAH mutation. Ba/F3 cells transfected with EGFR exon 20 insertion mutations and SNU-3173 were exposed to the EGFR TKIs (the 1st-generation gefitinib and erlotinib; the 2nd-generation dacomitinib and afatinib; and the 3rd-generation rociletinib, olmutinib, nazartinib, and osimertinib) and cell viability assays were performed. In addition, immunoblotting was performed to evaluate the downstream signals of EGFR pathway. Results: Cell viability assay showed that EGFR exon 20 insertion mutants are resistant to the 1st-generation EGFR TKIs, except for EGFRA763_Y764insFQEA-mutant Ba/F3 cells. IC50 values of the 2nd-generation EGFR TKIs are five to hundred-folds lower than the 1st-generation EGFR TKIs. The 2nd-generation EGFR TKIs were effective against EGFR exon 20 inserted Ba/F3 cells and down-regulated EGFR downstream signals. The 3rd-generation EGFR mutation-selective inhibitors showed a moderate efficacy except osimertinib that was effective and down-regulated EGFR downstream signals. In addition, SNU-3173 cells were moderately toxic to afatinib (IC50 46.2±5.2nM) as well as osimertinib (IC50 403.2±3.0nM). IC50 values of EGFR exon 20 insertion mutation Ba/F3 cellsIC50 (nM)FQEAASVSVDNPGPRHAHNPHGefitinib32.7525.7752.71597.01363.02581.02259.01701.0Erlotinib22.3486.9338.3765.7798.21523.01438.0562.1Dacomitinib0.00243.55.715.0205.0608.4556.312.3Afatinib0.0082.344.911.494.7297.7204.425.4Rociletinib219.7367.191.5131.21093.93710.05085.4498.7Olmutinib76.082.195.664.5332.6952.41406.6337.1Osimertinib3.68.18.9128.2247.2128.2880.338.2Nazartinib28.322.336.657.6747.8872.11606.8118.0 Conclusions: Overall, osimertinib showed effectiveness against NSCLC cells with EGFR exon 20 insertion mutations. Our results warrant a clinical trial of osimertinib in NSCLC patients with EGFR exon 20 insertion mutations. Citation Format: Yusoo Lee, Tae Min Kim, Dong-Wan Kim, Yong-Oon Ahn, Soyeon Kim, Bhumsuk Keam, Miso Kim, Dae Seog Heo. Osimertinib (AZD9291) is effective against NSCLC cells harboring EGFR exon 20 insertion mutations [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 3157. doi:10.1158/1538-7445.AM2017-3157

Dacomitinib versus gefitinib for the first-line treatment of advanced EGFR mutation positive non-small cell lung cancer (ARCHER 1050): A randomized, open-label phase III trial.

LBA9007 Background: Dacomitinib is a 2nd generation EGFR TKI with encouraging clinical activity as 1st-line therapy in patients with EGFR-activating mutation positive advanced NSCLC (Jänne, Lancet Oncol 2014). We performed the first randomized phase III trial, currently ongoing, comparing dacomitinib (D) with gefitinib (G) as 1st-line therapy (1L) (NCT01774721). Methods: Patients (pts) with newly diagnosed stage IIIB/IV/ recurrent NSCLC harboring an EGFR- activating mutation (exon 19 del or exon 21 L858R mu +/- exon 20 T790M mu) were randomized 1:1 to D 45 mg PO QD or G 250mg PO QD; stratification was by race and EGFR mutation subtype. The primary endpoint was PFS per blinded independent review (IRC) analyzed by Kaplan-Meier method with log-rank test and Cox model. Secondary endpoints included: overall survival, objective response rate (ORR), duration of response (DR), PFS per Investigator (INV), time to treatment failure (TTF), restricted mean survival time (RMST) for PFS, safety and patient-reported outcomes (PROs). Results: The ITT population included 452 pts with baseline characteristics fairly well balanced between the arms. ORR per IRC was similar between arms (75% [95% CI: 69, 80] for D and 72% [95% CI: 65, 77]; 2-sided p-value = 0.39; OS is not mature. The most commonly reported grade 3 adverse events with dacomitinib were dermatitis acneiform (13.7%) and diarrhea (8.4%) and with gefitinib ALT (8.5%); no new safety signals were identified. Conclusions: ARCHER 1050 was the first phase III head-to-head study of EGFR TKIs and demonstrated statistically significant and clinically meaningful improvement in efficacy of D vs. G in 1L NSCLC pts with EGFR-activating mutations with a manageable safety profile, to offer a new 1L treatment option. Clinical trial information: NCT01774721. [Table: see text]

Antitumor activity of osimertinib in NSCLC harboring EGFR exon 20 insertions.

9030 Background: EGFR exon 20 insertions (Ex20Ins), the 3rd most common EGFR activating mutation, are generally unresponsive to 1st and 2nd generation EGFR-TKIs. Development of EGFR-TKIs that effectively target NSCLC with Ex20Ins mutations represents a major unmet need. Osimertinib is an EGFR TKI approved for the treatment of advanced NSCLC harboring EGFR T790M, but the potential of osimertinib remains to be fully assessed in patients (pts) with Ex20Ins NSCLC. Methods: CRISPR engineered Ex20Ins cell line xenografts representing the two most common Ex20Ins (D770_N771InsSVD and V769_D770InsASV) and pt derived xenograft (PDX) of 3 EGFR Ex20Ins (V769_D770InsASV, M766_A767insASV, H773_V774insNPH) were used for in vivo experiments. Xenografts were treated by oral gavage with vehicle, erlotinib (50 mg/kg/day) or afatinib (20 mg/kg/day), osimertinib metabolite AZ5104 (50 mg/kg/day) and osimertinib (25 mg/kg/day) and assessed for tumor growth inhibition (TGI). Immunoblotting was performed for EGFR and relevant signaling pathways. A pt from whom the V769_D770InsASV Ex20ins PDX was derived was treated on a UC Davis IRB approved protocol with osimertinib at 160 mg PO once-daily (QD). Results: At completion of treatment, QD administration of osimertinib or AZ5104 induced significant TGI in xenografts across the 4 EGFR Ex20ins tested (range 60-95% TGI, p &lt; 0.001 compared to control for all models) that was superior to either afatinib or erlotinib. Robust decrease in p-EGFR, p-ERK, p-Akt, p-Stat3 was observed with osimertinib treatment. The patient corresponding to the V769_D770InsASV Ex20ins PDX treated with osimertinib exhibited clinical improvement and tumor shrinkage; unfortunately he was found to have interstitial pneumonitis that necessitated drug discontinuation. Conclusions: Osimertinib at clinically representative doses has in vivo activity across multiple EGFR Ex20ins that comprising the most common Ex20ins detected in patients (~50% prevalence); metabolite AZ5104 may contribute to efficacy. Tumor shrinkage was observed in a patient with lung cancer harboring an Ex20ins treated for a limited time with osimertinib. Based on this in vivo xenograft and pt data, osimertinib warrants further study in pts with EGFR Ex20ins NSCLC.