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Advanced non-small-cell lung cancer (NSCLC) patients with EGFR T790M-positive tumours benefit from osimertinib, an epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI). Here we show that the size of the EGFR T790M-positive clone impacts response to osimertinib. T790M subclonality, as assessed by a retrospective NGS analysis of 289 baseline plasma ctDNA samples from T790M-positive advanced NSCLC patients from the AURA3 phase III trial, is associated with shorter progression-free survival (PFS), both in the osimertinib and the chemotherapy-treated patients. Both baseline and longitudinal ctDNA profiling indicate that the T790M subclonal tumours are enriched for PIK3CA alterations, which we demonstrate to confer resistance to osimertinib in vitro that can be partially reversed by PI3K pathway inhibitors. Overall, our results elucidate the impact of tumour heterogeneity on response to osimertinib in advanced stage NSCLC patients and could help define appropriate combination therapies in these patients.

Background: Osimertinib is a potent, third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI). The multi-arm phase Ib TATTON study (NCT02143466) was designed to assess the safety and tolerability of osimertinib in combination with other targeted therapies: selumetinib (MEK1/2 inhibitor), savolitinib (MET-TKI), or durvalumab [anti-programmed cell death ligand 1 (anti-PD-L1) monoclonal antibody]., Patients and Methods: Patients with advanced EGFR-mutant non-small-cell lung cancer and disease progression on a prior EGFR-TKI were enrolled and allocated to dose-escalating cohorts combining osimertinib 80 mg orally (p.o.) once a day with selumetinib (25-75 mg p.o. twice a day; continuous or intermittent), savolitinib (600-800 mg p.o. once a day), or durvalumab (3-10 mg/kg intravenous every 2 weeks)., Results: At data cut-off (28 February 2018), 77 patients were enrolled and received osimertinib plus selumetinib (n = 36), savolitinib (n = 18), or durvalumab (n = 23). Most common adverse events (any grade), occurring in ≥20% of patients across dose groups, were: selumetinib arm-diarrhea (75%), rash (58%), nausea (47%); savolitinib arm-nausea (67%), rash (56%), vomiting (50%); durvalumab arm-rash (48%), vomiting (43%), diarrhea (39%). Dose-limiting toxicities were reported in the selumetinib 25 mg (n = 1), 50 mg (n = 1), and 75 mg (n = 4) continuous-dose groups, savolitinib 600 mg (n = 1) and 800 mg dose groups (n = 2), and durvalumab 10 mg/kg (n = 1) dose group. The objective response rate was 42% (95% confidence interval 26% to 59%), 44% (22% to 69%), and 43% (23% to 66%) in the selumetinib, savolitinib, and durvalumab arms, respectively., Conclusion: Our results demonstrate the feasibility of combining osimertinib 80 mg with selumetinib or savolitinib at identified tolerable, active doses. A combination of osimertinib with durvalumab was not feasible due to increased reporting of interstitial lung disease. Osimertinib-based combination therapies represent a compelling approach now being further investigated., Clinical Trials Number: NCT02143466., Competing Interests: Disclosure GO has declared honoraria from Chugai Pharma, Bio-Rad, Sysmex, and Guardant Health, and consultancy fees from AstraZeneca, Inivata, Takeda, LOXO, Ignyta, DropWorks, and GRAIL. JY has declared honoraria from AstraZeneca, Boehringer Ingelheim, Chugai Pharma, MSD, Novartis, and Roche; and consulting or advisory fees from Astellas Pharma, AstraZeneca, AstraZeneca (Inst), Bayer, Boehringer Ingelheim, Boehringer Ingelheim (Inst), Bristol-Myers Squibb, Celgene, Clovis Oncology, Hansoh, Lilly, Merck Serono, MSD Oncology, Novartis, Ono Pharmaceutical, Pfizer, Roche/Genentech, and Yuhan. HY has declared consultancy fees from AstraZeneca and Lilly Oncology; and research funding from AstraZeneca, Daiichi, Lilly Oncology, and Novartis. HS has declared honoraria and research funding from AstraZeneca. LH has declared consultancy fees from AstraZeneca, Lilly, Merck, Roche-Genentech, EMD Serono, Incyte, and Xcovery; and research funding from Boehringer Ingelheim and Xcovery. KG has declared honoraria from Bristol-Myers Squibb, AstraZeneca, Pfizer, Chugai Pharma, Taiho Pharmaceutical, Ono Pharmaceutical, Novartis, Lilly, Boehringher Ingelheim, Quintiles, Merck Serono, Life Technologies, MSD, AbbVie, Riken Genesis, Nippon Kayaku, Takeda, and Otsuka Pharmaceutical; consulting or advisory fees from Otskua; and research funding from MSD, AstraZeneca, Taiho Pharmaceutical, Chugai Pharma, Boehringer Ingelheim, Ono Pharmaceutical, Sumitomo Dainippon, Takeda, Novartis, Daiichi Sankyo, Kyowa Hakko Kirin, Astellas Pharma, Eisai, Lilly, Pfizer, Riken Genesis, Bristol-Myers Squibb, Merck Serono, Ignyta, Life Technologies, Research Triangle Institue d/b/a RTI Health Solutions, Janssen, Xcoo, and Loxo. YO has declared grants and honoraria from AstraZeneca, Bristol-Myers Squibb, Chugai, Pfizer, Lilly, MSD, Novartis, Ono Pharmaceutical; grants from Dainippon-Sumitomo and Kyorin; and honoraria from Bayer, Boehringer Ingelheim, and Nippon Kayaku. SR has declared grants and honoraria from Merck, Tesaro, Amgen, AstraZeneca, Abbvie, Bristol-Myers Squibb, Lilly, Genentech, Takeda, Merck, Luxo, and Tesaro. M-JA has declared honoraria from AstraZeneca, Bristol-Myers Squibb, MSD, Ono Pharmaceutical, and Roche; and consulting or advisory fees from AstraZeneca, Bristol-Myers Squibb, Takeda, MSD, Ono Pharmaceutical, Roche, and Alpha Pharmaceutical. KT is an employee and stockholder at AstraZeneca and Tesaro. HM, MF, KV, and DG are employees and stockholders at AstraZeneca. S-WK has declared no conflicts of interest., (Copyright © 2020 European Society for Medical Oncology. Published by Elsevier Ltd. All rights reserved.)

Background: This study assesses different technologies for detecting epidermal growth factor receptor (EGFR) mutations from circulating tumor DNA in patients with EGFR T790M-positive advanced non-small cell lung cancer (NSCLC) from the AURA3 study (NCT02151981), and it evaluates clinical responses to osimertinib and platinum-pemetrexed according to the plasma T790M status., Methods: Tumor tissue biopsy samples were tested for T790M during screening with the cobas EGFR Mutation Test (cobas tissue). Plasma samples were collected at screening and at the baseline and were retrospectively analyzed for EGFR mutations with the cobas EGFR Mutation Test v2 (cobas plasma), droplet digital polymerase chain reaction (ddPCR; Biodesix), and next-generation sequencing (NGS; Guardant360, Guardant Health)., Results: With cobas tissue test results as a reference, the plasma T790M positive percent agreement (PPA) was 51% (110 of 215 samples) by cobas plasma, 58% (110 of 189) by ddPCR, and 66% (136 of 207) by NGS. Plasma T790M detection was associated with a larger median baseline tumor size (56 mm for T790M-positive vs 39 mm for T790M-negative; P < .0001) and the presence of extrathoracic disease (58% for M1b-positive vs 39% for M0-1a-positive; P = .002). Progression-free survival (PFS) was prolonged in randomized patients (tissue T790M-positive) with a T790M-negative cobas plasma result in comparison with those with a T790M-positive plasma result in both osimertinib (median, 12.5 vs 8.3 months) and platinum-pemetrexed groups (median, 5.6 vs 4.2 months)., Conclusions: PPA was similar between ddPCR and NGS assays; both were more sensitive than cobas plasma. All 3 test platforms are suitable for routine clinical practice. In patients with tissue T790M-positive NSCLC, an absence of detectable plasma T790M at the baseline is associated with longer PFS, which may be attributed to a lower disease burden., (© 2019 American Cancer Society.)

Importance: Osimertinib mesylate is used globally to treat EGFR-mutant non-small cell lung cancer (NSCLC) with tyrosine kinase inhibitor resistance mediated by the EGFR T790M mutation. Acquired resistance to osimertinib is a growing clinical challenge that is poorly understood., Objective: To understand the molecular mechanisms of acquired resistance to osimertinib and their clinical behavior., Design, Setting, and Participants: Patients with advanced NSCLC who received osimertinib for T790M-positive acquired resistance to prior EGFR tyrosine kinase inhibitor were identified from a multi-institutional cohort (n = 143) and a confirmatory trial cohort (NCT01802632) (n = 110). Next-generation sequencing of tumor biopsies after osimertinib resistance was performed. Genotyping of plasma cell-free DNA was studied as an orthogonal approach, including serial plasma samples when available. The study and analysis were finalized on November 9, 2017., Main Outcomes and Measures: Mechanisms of resistance and their association with time to treatment discontinuation on osimertinib., Results: Of the 143 patients evaluated, 41 (28 [68%] women) had tumor next-generation sequencing after acquired resistance to osimertinib. Among 13 patients (32%) with maintained T790M at the time of resistance, EGFR C797S was seen in 9 patients (22%). Among 28 individuals (68%) with loss of T790M, a range of competing resistance mechanisms was detected, including novel mechanisms such as acquired KRAS mutations and targetable gene fusions. Time to treatment discontinuation was shorter in patients with T790M loss (6.1 vs 15.2 months), suggesting emergence of pre-existing resistant clones; this finding was confirmed in a validation cohort of 110 patients with plasma cell-free DNA genotyping performed after osimertinib resistance. In studies of serial plasma levels of mutant EGFR, loss of T790M at resistance was associated with a smaller decrease in levels of the EGFR driver mutation after 1 to 3 weeks of therapy (100% vs 83% decrease; P = .01)., Conclusions and Relevance: Acquired resistance to osimertinib mediated by loss of the T790M mutation is associated with early resistance and a range of competing resistance mechanisms. These data provide clinical evidence of the heterogeneity of resistance in advanced NSCLC and a need for clinical trial strategies that can overcome multiple concomitant resistance mechanisms or strategies for preventing such resistance.

Purpose The AURA study ( ClinicalTrials.gov identifier: NCT01802632) included two cohorts of treatment-naïve patients to examine clinical activity and safety of osimertinib (an epidermal growth factor receptor [EGFR] -tyrosine kinase inhibitor selective for EGFR-tyrosine kinase inhibitor sensitizing [ EGFRm] and EGFR T790M resistance mutations) as first-line treatment of EGFR-mutated advanced non-small-cell lung cancer (NSCLC). Patients and Methods Sixty treatment-naïve patients with locally advanced or metastatic EGFRm NSCLC received osimertinib 80 or 160 mg once daily (30 patients per cohort). End points included investigator-assessed objective response rate (ORR), progression-free survival (PFS), and safety evaluation. Plasma samples were collected at or after patients experienced disease progression, as defined by Response Evaluation Criteria in Solid Tumors (RECIST), to investigate osimertinib resistance mechanisms. Results At data cutoff (November 1, 2016), median follow-up was 19.1 months. Overall ORR was 67% (95% CI, 47% to 83%) in the 80-mg group, 87% (95% CI, 69% to 96%) in the 160-mg group, and 77% (95% CI, 64% to 87%) across doses. Median PFS time was 22.1 months (95% CI, 13.7 to 30.2 months) in the 80-mg group, 19.3 months (95% CI, 13.7 to 26.0 months) in the 160-mg group, and 20.5 months (95% CI, 15.0 to 26.1 months) across doses. Of 38 patients with postprogression plasma samples, 50% had no detectable circulating tumor DNA. Nine of 19 patients had putative resistance mechanisms, including amplification of MET (n = 1); amplification of EGFR and KRAS (n = 1); MEK1, KRAS, or PIK3CA mutation (n = 1 each); EGFR C797S mutation (n = 2); JAK2 mutation (n = 1); and HER2 exon 20 insertion (n = 1). Acquired EGFR T790M was not detected. Conclusion Osimertinib demonstrated a robust ORR and prolonged PFS in treatment-naïve patients with EGFRm advanced NSCLC. There was no evidence of acquired EGFR T790M mutation in postprogression plasma samples.

Background: The identification and characterization of molecular biomarkers has helped to revolutionize non-small-cell lung cancer (NSCLC) management, as it transitions from target-focused to patient-based treatment, centered on the evolving genomic profile of the individual. Determination of epidermal growth factor receptor ( EGFR ) mutation status represents a critical step in the diagnostic process. The recent emergence of acquired resistance to "third-generation" EGFR tyrosine kinase inhibitors (TKIs) via multiple mechanisms serves to illustrate the important influence of tumor heterogeneity on prognostic outcomes in patients with NSCLC., Design: This literature review examines the emergence of TKI resistance and the course of disease progression and, consequently, the clinical decision-making process in NSCLC., Results: Molecular markers of acquired resistance, of which T790M and HER2 or MET amplifications are the most common, help to guide ongoing treatment past the point of progression. Although tissue biopsy techniques remain the gold standard, the emergence of liquid biopsies and advances in analytical techniques may eventually allow "real-time" monitoring of tumor evolution and, in this way, help to optimize targeted treatment approaches., Conclusions: The influence of inter- and intra-tumor heterogeneity on resistance mechanisms should be considered when treating patients using resistance-specific therapies. New tools are necessary to analyze changes in heterogeneity and clonal composition during drug treatment. The refinement and standardization of diagnostic procedures and increased accessibility to technology will ultimately help in personalizing the management of NSCLC., Competing Interests: CONFLICTS OF INTEREST C. Heydt: nothing to disclose. S. Michels: consultancy for, and honoraria from, AstraZeneca, Novartis, Boehringer Ingelheim, and Pfizer; research support from Pfizer and Novartis; and travel expenses from Novartis. K.S. Thress: employee of, and holds shares in, AstraZeneca. S. Bergner: employee of, and holds shares in, AstraZeneca. J. Wolf: advisory boards (compensated) for, and lecture fees from, AstraZeneca, BMS, Boehringer Ingelheim, MSD, Clovis, Novartis, Pfizer, and Roche; and research support from Novartis, Pfizer, BMS, and MSD. R. Buettner advisory boards (compensated) for, and lecture fees from AstraZeneca, BMS, Boehringer Ingelheim, MSD, Novartis, Pfizer, Qiagen, and Roche; and co-founder and chief scientific advisor for Targos Molecular Pathology, Inc (Kassel, Germany).

Background: Osimertinib is approved for the treatment of non-small-cell lung cancer in patients who develop the EGFR Thr790Met mutation after treatment with epidermal growth factor receptor (EGFR) tyrosine-kinase inhibitors (TKIs). We assessed outcomes in patients with non-small-cell lung cancer and the EGFR Thr790Met mutation who were treated with osimertinib, a third-generation EGFR TKI, after previous treatment failure with one or more other EGFR TKIs., Methods: Eligible patients had been enrolled at one centre in the AURA study, had shown resistance to a previous EGFR TKI, and had EGFR-activating mutations and acquired Thr790Met mutation detectable in tumour tissue or plasma. Patients took 20-240 mg osimertinib per day until disease progression or development of intolerable side-effects. Plasma samples were collected every 6 weeks and tumour tissue biopsy was done at study entry and was optional after disease progression. We tested samples for resistance mechanisms, including EGFR-activating, Thr790Met, and Cys797Ser mutations, and assessed associations with overall survival, progression-free survival, and survival after disease progression., Findings: Of 71 patients enrolled in AURA, 53 were eligible for this analysis. Median progression-free survival was 11·1 months (95% CI 8·4-13·9) and overall survival was 16·9 months (11·7-29·1). 47 patients had disease progression. Median overall survival after osimertinib progression was 5·4 months (95% CI 4·1-10·0). Plasma samples were available for 40 patients after disease progression. 12 (30%) of these had the Thr790Met mutation (four of whom also had Cys797Ser mutations). Patients without detectable EGFR-activating mutations in plasma before treatment had the best overall and post-progression survival (22·4 months, 95% CI 15·6-not reached, and 10·8 months, 7·2-not reached, respectively). Loss of the Thr790Met mutation but presence of EGFR-activating mutations in plasma were associated with the shortest progression-free survival (median 2·6 months, 95% CI 1·3-not reached). In 22 post-progression tumour samples, we found one squamous cell and two small-cell transformations. We detected Thr790Met in nine (50%) of 18 samples, Cys797Ser in two (17%) of 12, cMET amplification in five (50%) of ten, BRAF mutation in one (8%) of 13, and KRAS mutation in one (8%) of 13., Interpretation: Heterogeneous resistance mechanisms developed in patients receiving osimertinib. Differences in resistance mechanisms might dictate future development strategies for osimertinib in clinical trials., Funding: AstraZeneca, Taiwan Ministry of Science and Technology., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Previously, the chromosomal translocation t(12;15)(p13;q25) has been found to recurrently occur in both solid tumors and leukemias. This translocation leads to ETV6-NTRK3 (EN) gene fusions resulting in ectopic expression of the NTRK3 neurotropic tyrosine receptor kinase moiety as well as oligomerization through the donated ETV6-sterile alpha motif domain. As yet, no in vitro cell line model carrying this anomaly is available. Here we genetically characterized the acute promyelocytic leukemia (APL) cell line AP-1060 and, by doing so, revealed the presence of a t(12;15)(p13;q25). Subsequently, we evaluated its suitability as a model for this important clinical entity., Methods: Spectral karyotyping, fluorescence in situ hybridization (FISH), and genomic and transcriptomic microarray-based profiling were used to screen for the presence of EN fusions. qRT-PCR was used for quantitative expression analyses. Responses to AZ-23 (NTRK) and wortmannin (PI3K) inhibitors, as well as to arsenic trioxide (ATO), were assessed using colorimetric assays. An AZ-23 microarray screen was used to define the EN targetome, which was parsed bioinformatically. MAPK1 and MALAT1 activation were assayed using Western blotting and RNA-FISH, respectively, whereas an AML patient cohort was used to assess the clinical occurrence of MALAT1 activation., Results: An EN fusion was detected in AP1060 cells which, accordingly, turned out to be hypersensitive to AZ-23. We also found that AZ-23 can potentiate the effect of ATO and inhibit the phosphorylation of its canonical target MAPK1. The AZ-23 microarray screen highlighted a novel EN target, MALAT1, which also proved sensitive to wortmannin. Finally, we found that MALAT1 was massively up-regulated in a subset of AML patients., Conclusions: From our data we conclude that AP-1060 may serve as a first publicly available preclinical model for EN. In addition, we conclude that these EN-positive cells are sensitive to the NTRK inhibitor AZ-23 and that this inhibitor may potentiate the therapeutic efficacy of ATO. Our data also highlight a novel AML EN target, MALAT1, which was so far only conspicuous in solid tumors.

Purpose The Iressa Mutation-Positive Multicentre Treatment Beyond ProgRESsion Study (IMPRESS) compared the continuation of gefitinib plus chemotherapy with placebo plus chemotherapy in patients with epidermal growth factor receptor ( EGFR) mutation-positive advanced non-small-cell lung cancer with progression (Response Evaluation Criteria in Solid Tumors 1.1) after first-line gefitinib. Primary results indicated no difference between treatments in terms of progression-free survival (PFS). The current analysis presents final, mature, overall survival (OS) data, together with exploratory analyses that examined whether specific biomarkers, including T790M mutation status, were able to differentiate a relative treatment effect. Patients and Methods Patients were randomly assigned to gefitinib 250 mg or placebo, in addition to cisplatin 75 mg/m 2 plus pemetrexed 500 mg/m 2 (maximum of six cycles of chemotherapy). EGFR mutation status was determined from plasma-derived circulating free tumor-derived DNA samples (beads, emulsification, amplification, and magnetics digital polymerase chain reaction assay, allelic fraction analysis). Results A total of 265 patients with non-small-cell lung cancer were randomly assigned, and overall data maturity was 66%. Continuation of gefitinib plus cisplatin and pemetrexed was detrimental to OS when compared with placebo plus cisplatin and pemetrexed (hazard ratio [HR], 1.44; 95% CI, 1.07 to 1.94; P = .016; median OS, 13.4 v 19.5 months). The detriment was statistically significant in patients with T790M mutation-positive plasma samples (HR, 1.49; 95% CI, 1.02 to 2.21), whereas statistical significance was not reached in T790M mutation-negative patients (HR, 1.15; 95% CI, 0.68 to 1.94). PFS in T790M mutation-positive patients was similar between treatments, and the difference observed in T790M mutation-negative patients did not reach statistical significance (HR, 0.67; 95% CI, 0.43 to 1.03; P = .0745). Conclusion Final OS data from IMPRESS are supportive of earlier PFS results and are sufficient to warn physicians against the continuation of treatment with first-generation EGFR tyrosine kinase inhibitors beyond radiologic disease progression when chemotherapy is initiated. Plasma biomarker analyses suggest that this effect may be driven by T790M-positive status.

Introduction: Osimertinib is an oral, potent, irreversible EGFR tyrosine kinase inhibitor (TKI) selective for EGFR TKI and T790M resistance mutations. To enhance understanding of osimertinib's mechanism of action, we aimed to evaluate the modulation of key molecular biomarkers after osimertinib treatment in paired clinical samples from the phase I AURA trial., Methods: Paired tumor biopsy samples were collected before the study and after 15 plus or minus 7 days of osimertinib treatment (80 or 160 mg daily). Clinical efficacy outcomes were assessed according to whether viable paired biopsy samples could be collected; safety was also assessed. Immunohistochemical analyses assessed key pathway and tumor/immune-relevant markers (phospho-EGFR, phospho-S6, phospho-AKT, programmed death ligand 1, and CD8), with samples scored by image analysis or a pathologist blinded to treatment allocation., Results: Predose tumor biopsy samples were collected from 61 patients with EGFR T790M tumors; 29 patients had no viable postdose biopsy sample because of tumor regression or insufficient tumor sample. Evaluable predose and postdose tumor biopsy samples were collected from 24 patients. Objective response rate (ORR) and median progression-free survival (mPFS) were improved in patients from whom a postdose biopsy sample could not be collected (ORR 62% and mPFS 9.7 months [p = 0.027]) compared with those from whom paired samples were collected (ORR 29% and mPFS 6.6 months). Osimertinib modulated key EGFR signaling pathways and led to increased immune cell infiltration., Conclusions: Collection of paired biopsy samples was challenging because of rapid tumor regression after osimertinib treatment, highlighting the difficulties of performing on-study biopsies in patients treated with highly active drugs., (Copyright © 2017 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.)

Introduction: Osimertinib is an oral, central nervous system-active, EGFR tyrosine kinase inhibitor (TKI) for the treatment of EGFR T790M-positive advanced NSCLC. Here we have evaluated EGFR mutation frequencies in two phase II studies of osimertinib (AURA extension and AURA2)., Methods: After progression while receiving their latest line of therapy, patients with EGFR mutation-positive advanced NSCLC provided tumor samples for mandatory central T790M testing for the study selection criteria. Tumor tissue mutation analysis for patient selection was performed with the Roche cobas EGFR Mutation Test (European Conformity-in vitro diagnostic, labeled investigational use only) (Roche Molecular Systems, Pleasanton, CA). Patients should not have been prescreened for T790M mutation status. The cobas test results were compared with those of the MiSeq next-generation sequencing system (Illumina, San Diego, CA), which was used as a reference method., Results: Samples from 324 and 373 patients screened for AURA extension and AURA2, respectively, produced valid cobas test results. The T790M detection rates were similar between AURA extension and AURA2 (64% and 63%, respectively). The pooled T790M rate was 63%, with no difference by ethnicity (63% for Asian and non-Asian patients alike) or immediately prior treatment with an EGFR TKI (afatinib, 69%; erlotinib, 69%; and gefitinib, 63%). A higher proportion of patients had T790M detected against a background of exon 19 deletions versus L858R mutation (73% versus 58% [p = 0.0002]). In both trials the cobas test demonstrated high sensitivity (positive percent agreement) and specificity (negative percent agreement) for T790M detection when compared with the next-generation sequencing reference method: positive percent agreement of 91% versus 89% and negative percent agreement of 97% versus 98%., Conclusions: In both trials, the rate of detection of T790M mutation in patients with advanced NSCLC was approximately 63% and was unaffected by immediately prior treatment with an EGFR TKI or ethnicity., (Copyright © 2017 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.)

Objectives: Reliable epidermal growth factor receptor (EGFR) mutation testing techniques are required to identify eligible patients with EGFR mutation/T790M positive advanced non-small cell lung cancer (NSCLC), for treatment with osimertinib (AZD9291), an oral, potent, irreversible EGFR tyrosine kinase inhibitor (TKI) selective for EGFR-TKI-sensitizing and T790M resistance mutations over wild-type EGFR. There is no current consensus regarding the best method to detect EGFR T790M mutations. The aim of this study was to describe the concordance between local testing, which used a variety of methods, and central testing, using the cobas ® EGFR Mutation Test, for EGFR-sensitizing mutations and the T790M resistance mutation., Materials and Methods: Tumor samples were obtained from all patients screened for inclusion onto the osimertinib Phase I expansion component of the AURA Phase I/II study (NCT01802632). Samples underwent central laboratory testing for EGFR-sensitizing mutations and T790M resistance mutation using the cobas ® EGFR Mutation Test. Results were compared with local laboratory test results, based on other testing methodologies including Sanger sequencing, therascreen ® , PNAClamp™, and Sequenom MassARRAY ® ., Results: Central laboratory testing was successful in 99% of samples passing histopathology review and testing success rates were comparable across the three central laboratories. Concordance between central and local testing for common sensitizing mutations was high (>98%) and concordance for the T790M mutation was also high (>90%). Tumor heterogeneity, along with other technical factors may have influenced this result., Conclusions: Within the osimertinib AURA Phase I study, EGFR mutation testing across three centralized laboratories using the cobas ® EGFR Mutation Test was feasible and successful, with strong concordance between local and central laboratory results, including for T790M. The cobas ® EGFR Mutation Test has subsequently been approved as the companion diagnostic test for osimertinib in the USA and Japan., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Cancer treatment is evolving towards therapies targeted at specific molecular abnormalities that drive tumor growth. Consequently, to determine which patients are eligible, accurate assessment of molecular aberrations within tumors is required. Obtaining sufficient tumor tissue for molecular testing can present challenges; therefore, circulating free tumor-derived DNA (ctDNA) found in blood plasma has been proposed as an alternative source of tumor DNA. The diagnostic utility of ctDNA for the detection of epidermal growth factor receptor (EGFR) mutations harbored in tumors of patients with advanced non-small-cell lung cancer (NSCLC) is supported by the results of several large studies/meta-analyses. However, recent real-world studies suggest that the performance of ctDNA testing varies between geographic regions/laboratories, demonstrating the need for standardized guidance. In this review, we outline recommendations for obtaining an accurate result using ctDNA, relating to pre-analytical plasma processing, ctDNA extraction, and appropriate EGFR mutation detection methods, based on clinical trial results. We conclude that there are several advantages associated with ctDNA, including the potential for repeated sampling - particularly following progression after first-line tyrosine kinase inhibitor (TKI) therapy, as TKIs targeting resistance mutations (eg T790M) are now approved for use in the USA/EU/Japan (at time of writing). However, evidence suggests that ctDNA does not allow detection of EGFR mutations in all patients with known mutation-positive NSCLC. Therefore, although tumor tissue should be the first sample choice for EGFR testing at diagnosis, ctDNA is a promising alternative diagnostic approach.

Purpose: Approximately one-third of patients with non-small cell lung cancer (NSCLC) harboring tumors with EGFR-tyrosine kinase inhibitor (TKI)-sensitizing mutations (EGFRm) experience disease progression during treatment due to brain metastases. Despite anecdotal reports of EGFR-TKIs providing benefit in some patients with EGFRm NSCLC brain metastases, there is a clinical need for novel EGFR-TKIs with improved efficacy against brain lesions., Experimental Design: We performed preclinical assessments of brain penetration and activity of osimertinib (AZD9291), an oral, potent, irreversible EGFR-TKI selective for EGFRm and T790M resistance mutations, and other EGFR-TKIs in various animal models of EGFR-mutant NSCLC brain metastases. We also present case reports of previously treated patients with EGFRm-advanced NSCLC and brain metastases who received osimertinib in the phase I/II AURA study (NCT01802632)., Results: Osimertinib demonstrated greater penetration of the mouse blood-brain barrier than gefitinib, rociletinib (CO-1686), or afatinib, and at clinically relevant doses induced sustained tumor regression in an EGFRm PC9 mouse brain metastases model; rociletinib did not achieve tumor regression. Under positron emission tomography micro-dosing conditions, [ 11 C]osimertinib showed markedly greater exposure in the cynomolgus monkey brain than [ 11 C]rociletinib and [ 11 C]gefitinib. Early clinical evidence of osimertinib activity in previously treated patients with EGFRm-advanced NSCLC and brain metastases is also reported., Conclusions: Osimertinib may represent a clinically significant treatment option for patients with EGFRm NSCLC and brain metastases. Further investigation of osimertinib in this patient population is ongoing. Clin Cancer Res; 22(20); 5130-40. ©2016 AACR., (©2016 American Association for Cancer Research.)

Purpose: Third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have demonstrated potent activity against TKI resistance mediated by EGFR T790M. We studied whether noninvasive genotyping of cell-free plasma DNA (cfDNA) is a useful biomarker for prediction of outcome from a third-generation EGFR-TKI, osimertinib., Methods: Plasma was collected from all patients in the first-in-man study of osimertinib. Patients who were included had acquired EGFR-TKI resistance and evidence of a common EGFR-sensitizing mutation. Genotyping of cell-free plasma DNA was performed by using BEAMing. Plasma genotyping accuracy was assessed by using tumor genotyping from a central laboratory as reference. Objective response rate (ORR) and progression-free survival (PFS) were analyzed in all T790M-positive or T790M-negative patients., Results: Sensitivity of plasma genotyping for detection of T790M was 70%. Of 58 patients with T790M-negative tumors, T790M was detected in plasma of 18 (31%). ORR and median PFS were similar in patients with T790M-positive plasma (ORR, 63%; PFS, 9.7 months) or T790M-positive tumor (ORR, 62%; PFS, 9.7 months) results. Although patients with T790M-negative plasma had overall favorable outcomes (ORR, 46%; median PFS, 8.2 months), tumor genotyping distinguished a subset of patients positive for T790M who had better outcomes (ORR, 69%; PFS, 16.5 months) as well as a subset of patients negative for T790M with poor outcomes (ORR, 25%; PFS, 2.8 months)., Conclusion: In this retrospective analysis, patients positive for T790M in plasma have outcomes with osimertinib that are equivalent to patients positive by a tissue-based assay. This study suggests that, upon availability of validated plasma T790M assays, some patients could avoid a tumor biopsy for T790M genotyping. As a result of the 30% false-negative rate of plasma genotyping, those with T790M-negative plasma results still need a tumor biopsy to determine presence or absence of T790M., (© 2016 by American Society of Clinical Oncology.)

Purpose: To assess the safety and efficacy of fostamatinib in patients with relapsed or refractory diffuse large B-cell lymphoma (DLBCL)., Experimental Design: Relapsed or refractory DLBCL patients originally received the oral spleen tyrosine kinase inhibitor, fostamatinib in a two-arm, randomised, double-blinded manner at either 100 mg twice a day (BID) or 200 mg BID until disease progression or unacceptable toxicity. The primary objective was to assess the overall response rate (ORR). Preliminary analysis showed limited efficacy and all subsequent patients were treated at 200 mg BID. Previously randomised patients were unblinded and given the opportunity to receive 200 mg BID., Results: Sixty-eight patients were treated (47 at 200 mg BID, 21 at 100 mg BID). Cell of origin analysis showed 58% germinal B-cell (GCB) origin, 30% activated B-cell (ABC) origin and 12% with an intermediate cell of origin signature. The most common treatment-related adverse events of all patients were diarrhoea (21% total, 6% grade 3/4), nausea (19% total, 3% grade 3/4), and, fatigue (18% total, 9% grade 3/4). The ORR rate was 3% across both arms and clinical benefit (≥ stable disease) was achieved for 13% of all patients. The cell of origin for patients with clinical benefit was GCB (4 patients), intermediate (4 patients) or unknown (1 patient). None of the patients with clinical benefit had ABC genotype., Conclusions: While fostamatinib was generally well tolerated in this patient population, efficacy at these doses and schedule was poor. Unlike data with other B-cell antigen receptor pathway inhibitors, responses were not observed in the ABC genotype., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Objectives: To assess the ability of different technology platforms to detect epidermal growth factor receptor (EGFR) mutations, including T790M, from circulating tumor DNA (ctDNA) in advanced non-small cell lung cancer (NSCLC) patients., Materials and Methods: A comparison of multiple platforms for detecting EGFR mutations in plasma ctDNA was undertaken. Plasma samples were collected from patients entering the ongoing AURA trial (NCT01802632), investigating the safety, tolerability, and efficacy of AZD9291 in patients with EGFR-sensitizing mutation-positive NSCLC. Plasma was collected prior to AZD9291 dosing but following clinical progression on a previous EGFR-tyrosine kinase inhibitor (TKI). Extracted ctDNA was analyzed using two non-digital platforms (cobas(®) EGFR Mutation Test and therascreen™ EGFR amplification refractory mutation system assay) and two digital platforms (Droplet Digital™ PCR and BEAMing digital PCR [dPCR])., Results: Preliminary assessment (38 samples) was conducted using all four platforms. For EGFR-TKI-sensitizing mutations, high sensitivity (78-100%) and specificity (93-100%) were observed using tissue as a non-reference standard. For the T790M mutation, the digital platforms outperformed the non-digital platforms. Subsequent assessment using 72 additional baseline plasma samples was conducted using the cobas(®) EGFR Mutation Test and BEAMing dPCR. The two platforms demonstrated high sensitivity (82-87%) and specificity (97%) for EGFR-sensitizing mutations. For the T790M mutation, the sensitivity and specificity were 73% and 67%, respectively, with the cobas(®) EGFR Mutation Test, and 81% and 58%, respectively, with BEAMing dPCR. Concordance between the platforms was >90%, showing that multiple platforms are capable of sensitive and specific detection of EGFR-TKI-sensitizing mutations from NSCLC patient plasma., Conclusion: The cobas(®) EGFR Mutation Test and BEAMing dPCR demonstrate a high sensitivity for T790M mutation detection. Genomic heterogeneity of T790M-mediated resistance may explain the reduced specificity observed with plasma-based detection of T790M mutations versus tissue. These data support the use of both platforms in the AZD9291 clinical development program., (Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.)

Resistance to targeted EGFR inhibitors is likely to develop in EGFR-mutant lung cancers. Early identification of innate or acquired resistance mechanisms to these agents is essential to direct development of future therapies. We describe the detection of heterogeneous mechanisms of resistance within populations of EGFR-mutant cells (PC9 and/or NCI-H1975) with acquired resistance to current and newly developed EGFR tyrosine kinase inhibitors, including AZD9291. We report the detection of NRAS mutations, including a novel E63K mutation, and a gain of copy number of WT NRAS or WT KRAS in cell populations resistant to gefitinib, afatinib, WZ4002, or AZD9291. Compared with parental cells, a number of resistant cell populations were more sensitive to inhibition by the MEK inhibitor selumetinib (AZD6244; ARRY-142886) when treated in combination with the originating EGFR inhibitor. In vitro, a combination of AZD9291 with selumetinib prevented emergence of resistance in PC9 cells and delayed resistance in NCI-H1975 cells. In vivo, concomitant dosing of AZD9291 with selumetinib caused regression of AZD9291-resistant tumors in an EGFRm/T790M transgenic model. Our data support the use of a combination of AZD9291 with a MEK inhibitor to delay or prevent resistance to AZD9291 in EGFRm and/or EGFRm/T790M tumors. Furthermore, these findings suggest that NRAS modifications in tumor samples from patients who have progressed on current or EGFR inhibitors in development may support subsequent treatment with a combination of EGFR and MEK inhibition., (©2015 American Association for Cancer Research.)

Here we studied cell-free plasma DNA (cfDNA) collected from subjects with advanced lung cancer whose tumors had developed resistance to the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) AZD9291. We first performed next-generation sequencing of cfDNA from seven subjects and detected an acquired EGFR C797S mutation in one; expression of this mutant EGFR construct in a cell line rendered it resistant to AZD9291. We then performed droplet digital PCR on serial cfDNA specimens collected from 15 AZD9291-treated subjects. All were positive for the T790M mutation before treatment, but upon developing AZD9291 resistance three molecular subtypes emerged: six cases acquired the C797S mutation, five cases maintained the T790M mutation but did not acquire the C797S mutation and four cases lost the T790M mutation despite the presence of the underlying EGFR activating mutation. Our findings provide insight into the diversity of mechanisms through which tumors acquire resistance to AZD9291 and highlight the need for therapies that are able to overcome resistance mediated by the EGFR C797S mutation.

Purpose: Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease with distinct molecular subtypes. The most established subtyping approach, the "Cell of Origin" (COO) algorithm, categorizes DLBCL into activated B-cell (ABC) and germinal center B-cell (GCB)-like subgroups through gene expression profiling. Recently developed immunohistochemical (IHC) techniques and other established methodologies can deliver discordant results and have various technical limitations. We evaluated the NanoString nCounter gene expression system to address issues with current platforms., Experimental Design: We devised a scoring system using 145 genes from published datasets to categorize DLBCL samples. After cell line validation, clinical tissue segmentation was tested using commercially available diagnostic DLBCL samples. Finally, we profiled biopsies from patients with relapsed/refractory DLBCL enrolled in the fostamatinib phase IIb clinical trial using three independent RNA expression platforms: NanoString, Affymetrix, and qNPA., Results: Diagnostic samples showed a typical spread of subtypes with consistent gene expression profiles across matched fresh, frozen, and formalin-fixed paraffin-embedded tissues. Results from biopsy samples across platforms were remarkably consistent, in contrast to published IHC data. Interestingly, COO segmentation of longitudinal fostamatinib biopsies taken at initial diagnosis and then again at primary relapse showed 88% concordance (15/17), suggesting that COO designation remains stable over the course of disease progression., Conclusions: DLBCL segmentation of patient tumor samples is possible using a number of expression platforms. However, we found that NanoString offers the most flexibility and fewest limitations in regards to robust clinical tissue subtype characterization. These subtype distinctions should help guide disease prognosis and treatment options within DLBCL clinical practice., (©2014 American Association for Cancer Research.)