Daniel Stetson

Acquired EGFR C797S mutation mediates resistance to AZD9291 in non–small cell lung cancer harboring EGFR T790M

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.

Acquired Resistance to the Mutant-Selective EGFR Inhibitor AZD9291 Is Associated with Increased Dependence on RAS Signaling in Preclinical Models

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.

Osimertinib As First-Line Treatment of EGFR Mutation–Positive Advanced Non–Small-Cell Lung Cancer

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.

Generation of stable PDX derived cell lines using conditional reprogramming

Efforts to develop effective cancer therapeutics have been hindered by a lack of clinically predictive preclinical models which recapitulate this complex disease. Patient derived xenograft (PDX) models have emerged as valuable tools for translational research but have several practical limitations including lack of sustained growth in vitro. In this study, we utilized Conditional Reprogramming (CR) cell technology- a novel cell culture system facilitating the generation of stable cultures from patient biopsies- to establish PDX-derived cell lines which maintain the characteristics of the parental PDX tumor. Human lung and ovarian PDX tumors were successfully propagated using CR technology to create stable explant cell lines (CR-PDX). These CR-PDX cell lines maintained parental driver mutations and allele frequency without clonal drift. Purified CR-PDX cell lines were amenable to high throughput chemosensitivity screening and in vitro genetic knockdown studies. Additionally, re-implanted CR-PDX cells proliferated to form tumors that retained the growth kinetics, histology, and drug responses of the parental PDX tumor. CR technology can be used to generate and expand stable cell lines from PDX tumors without compromising fundamental biological properties of the model. It offers the ability to expand PDX cells in vitro for subsequent 2D screening assays as well as for use in vivo to reduce variability, animal usage and study costs. The methods and data detailed here provide a platform to generate physiologically relevant and predictive preclinical models to enhance drug discovery efforts.

Prioritisation of structural variant calls in cancer genomes

Sensitivity of short read DNA-sequencing for gene fusion detection is improving, but is hampered by the significant amount of noise composed of uninteresting or false positive hits in the data. In this paper we describe a tiered prioritisation approach to extract high impact gene fusion events from existing structural variant calls. Using cell line and patient DNA sequence data we improve the annotation and interpretation of structural variant calls to best highlight likely cancer driving fusions. We also considerably improve on the automated visualisation of the high impact structural variants to highlight the effects of the variants on the resulting transcripts. The resulting framework greatly improves on readily detecting clinically actionable structural variants.

Abstract LB-249: Examination of analytical factors impacting concordance of plasma-tumor testing by next-generation sequencing (NGS)

The increased usage of circulating tumor DNA (ctDNA) sequencing for oncology clinical research demonstrates a critical need for sensitive and specific testing. While we have observed a high degree of concordance between single tumor mutations in tumor and plasma, several recent studies have highlighted a lack of concordance between plasma and tumor panel NGS gene panel testing due to biological and technical factors. To explore further these factors and benchmark ctDNA NGS testing services, a set of matched plasma, tumor, and normal samples from 24 subjects were acquired from three biobanking companies. Replicate 2 ml-plasma samples were tested by four ctDNA sequencing companies, and matching tumor/normal samples were tested by two tumor sequencing companies. Concordance was measured by comparing plasma mutations to tumor mutations as well as comparing mutations among the same plasma tested by the ctDNA companies. While our experience with NGS of matched samples from clinical trials typically identifies ~30% of patients with no detectable mutation and therefore likely not shedding tumor DNA, with the retrospectively collected commercial samples ~60% lacked detectable high confidence mutations, likely due to quality control issues with sample collection. We also found variation in the concordance of ctDNA mutation detection rates among the four vendors, due to significant differences in DNA yield and assay sensitivity. While factors such as tumor heterogeneity and timing of plasma-tumor collection can lower concordance rates, the majority of discordance in our study was due to technical rather than biological variation. Assay analytical variance and the impact of reporting false positive variants are key factors that need to be addressed as plasma-based NGS testing is more widely incorporated into translational and clinical research. Examples illustrating the complexity of the analyses and giving support for confidence in ctDNA testing results will be given. Citation Format: Daniel Stetson, Brian Dougherty, Ambar Ahmed, Tristan Lubinski, Aleksandra Markovets, Kenneth Thress, Robert McEwen, Gaia Schiavon, David Whitston, Barrett Nuttall, J. Carl Barrett. Examination of analytical factors impacting concordance of plasma-tumor testing by next-generation sequencing (NGS) [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 LB-249. doi:10.1158/1538-7445.AM2017-LB-249

Abstract 397: Whole genome copy number variation analysis using a SNP-focused targeted sequencing panel for tumor analysis

Accurate genome-wide copy number variation (CNV) analysis is critical for disease and cancer research. Current approaches for CNV analysis include fluorescence in situ hybridization (FISH), array comparative genomic hybridization (array CGH), and SNP arrays. Unfortunately, these methods are not sensitive enough for real world cancer samples because of tumor ploidy, purity and heterogeneity. NGS-based targeted sequencing is increasingly being used for CNV analysis due to throughput, coverage, cost, and sample input requirements. For CNV analysis, detection power is improved by combining both read depth and SNP allele frequency analysis, particularly for copy-neutral events such as loss of heterozygosity. A custom xGen Lockdown CNV backbone panel was developed for broad, uniform genome coverage and to enrich for population-based SNPs. We demonstrate use of the panel as an addition to the xGen Exome Research Panel and a custom cancer focused panel. Downstream analysis incorporates both read depth and observed minor allele frequencies to determine CNVs with enhanced sensitivity. To increase the resolution for large-scale alterations of chromosome 7, a hot-spot for disease-associated CNVs, probe density was increased 6 fold. A known standard, NA12878, was used to validate the panel’s ability to detect heterozygous SNPs with high confidence. In addition, mixtures of cancer cell lines from the Cancer Cell Line Encyclopedia (CCLE) were tested with varying levels of background copy-neutral genomic DNA. The sensitivity and specificity of the panel to detect CNV and LOH events with was assessed using deep exome and Affymetrix SNP array data. The ability to detect copy number alterations with high resolution and accuracy would be a valuable resource for disease and cancer research. Citation Format: Jiashi Wang, Kristina Giorda, Zhongwu Lai, Daniel Stetson, Mirna Jarosz. Whole genome copy number variation analysis using a SNP-focused targeted sequencing panel for tumor analysis [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 397. doi:10.1158/1538-7445.AM2017-397

Abstract 641: Use of conditional reprogramming to develop and characterize cell cultures from patient-derived xenograft (PDX) models of human lung and ovarian cancer

Patient-derived xenografts (PDX) are widely recognized as a more physiologically relevant preclinical model to standard cell line xenografts. PDX models faithfully recapitulate the original patient genetic profile, gene expression patterns and tissue histology. Despite their benefits, PDX models are limited by their inherent variability, lower throughput and lack of growth in vitro. The ability to generate cell lines from PDX models would enable high throughput chemosensitivity screens, ex vivo genetic manipulation and the development of novel orthotopic models. Development of stable PDX cell lines remains a challenge due to murine stromal outgrowth, lineage commitment and limited differentiation potential. Conditional reprogramming (CR) cell technology is a novel cell culture system facilitating the generation of stable cultures without genetic manipulation (Liu, Am J Pathol, 2012). The success of CR cell technology is dependent upon the combination of feeder cell-derived factors and Rho Kinase (ROCK) inhibitor. CR cells, therefore, represent a new class of progenitor-like cells, distinct from the phenotype of embryonic stem (ES) cells and induced pluripotent stem (iPS) cells. The purpose of this study was to identify the advantages, limitations and potential applications of CR technology for derivation of PDX explant cell lines. Early passage human lung and ovarian PDX tumors were cultured in CR conditions to create stable explant cell lines. Cell lines were established from 5/8 (63%) PDX tumors and were expanded over 6 months in culture with varying morphologies and growth kinetics. Due to normal outgrowth of murine stromal cells, early CR cultures contained mixed populations and required murine depletion to enrich for human cells. Key oncogenic mutations in a model of ovarian papillary serous adenocarcinoma were preserved in the enriched tumor cell population. While purified CR PDX cell lines were amenable to high throughput chemosensitivity screens, in vitro chemosensitivity did not consistently predict response in in vivo murine models. The CR PDX cell lines were additionally assessed for genetic manipulation and ability to form tumors in vivo. Collectively, these results demonstrate the applications of CR technology for the generation of stable explant cell lines from PDX models for preclinical studies. Citation Format: Alexandra Borodovsky, Travis J. McQuiston, Brian Dougherty, Ambar Ahmed, David Whitston, Daniel Stetson, Gretchen K. Hubbard, Sharon S. Challberg, Brian A. Pollok, Celina M. D’Cruz. Use of conditional reprogramming to develop and characterize cell cultures from patient-derived xenograft (PDX) models of human lung and ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 641.

Abstract PR05: ERK pathway activation is associated with acquired resistance to AZD9291, a third-generation irreversible inhibitor targeting EGFR sensitizing (EGFRm+) and resistance (T790M) mutations in NSCLC

First- and second-generation EGFR tyrosine kinase inhibitors (TKIs) are established first line therapies for patients with advanced NSCLC with activating/sensitising mutations in EGFR. Unfortunately, patients ultimately develop disease progression with acquisition of a second-site EGFR T790M mutation in more than half of cases. This has led to the development of third generation EGFR TKIs such as AZD9291 which inhibit both the EGFRm+ and T790M mutations in preclinical models, and are showing activity in patients with TKI-resistant tumors harbouring T790M in Phase I studies. Despite the potential improvements brought by third generation EGFR-TKIs, advanced EGFRm+ tumor cells will still remain highly adaptable, and the inevitability of further resistance will potentially limit the effectiveness of these drugs. As such, the identification of resistance mechanisms to these agents is essential to guide future therapeutic strategies and identify novel:novel combinations. To interrogate resistance to AZD9291, we have generated panels of EGFRm+ cell populations resistant to gefitinib (first generation TKI), and EGFRm+ and EGFRm+/T790M cell populations resistant to afatinib (second generation TKI) and WZ4002 or AZD9291 (third generation TKIs). Subsequently, we have characterized the cell lines using a phenotypic screen to compare the sensitivity of small molecule inhibitors of canonical signaling pathways between the resistant and parental cell populations. In addition we have used a variety of molecular profiling techniques to determine the DNA mutation and copy number status and mRNA expression profile of a panel of cancer associated genes within the resistant cell populations. The effects on cell survival across the range of resistant models by a panel of pathway inhibitors, in combination with the originating TKI, indicated that resistance to the EGFR inhibitors was frequently associated with increased sensitivity to selumetinib (AZD6244; ARRY-142886) (MEK1/2 inhibitor), suggesting that ERK signaling is commonly reactivated to circumvent inhibition of the EGFR pathway. Further, molecular analysis indicated the presence of mutations in NRAS, including a novel E63K mutation, or increased copy number of NRAS or KRAS within 12/25 and 3/9 resistant cell populations representing resistance in EGFRm+ and EGFRm+/T790M settings respectively. Analysis of the functional consequence of the observed RAS modifications confirmed their role in driving the survival of EGFR pathway addicted cells when EGFR signaling is inhibited. Collectively, these data suggest that ERK pathway activation, in particular as a result of increased RAS activation, is frequently associated with acquired resistance to AZD9291 and other EGFR TKI inhibitors. In addition the data suggests that combining AZD9291 with selumetinib could prevent or delay resistance, and therefore potentially drive superior duration of benefit compared to TKI alone. Citation Format: Cath Eberlein, Katie Al-Kadhimi, Sarah Ross, Henry Brown, Paul Fisher, Daniel Stetson, Zhongwu Lai, Kenneth Thress, Brian Dougherty, William Pao, Darren Cross. ERK pathway activation is associated with acquired resistance to AZD9291, a third-generation irreversible inhibitor targeting EGFR sensitizing (EGFRm+) and resistance (T790M) mutations in NSCLC. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr PR05.

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

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