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 T790M prior to treatment, but at resistance three molecular subtypes emerged: 6 cases acquired the C797S mutation, 5 cases maintained the T790M mutation but did not acquire the C797S mutation, and 4 cases lost the T790M mutation despite detecting 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 able to overcome resistance mediated by EGFR C797S.

Accurate variant calling in next generation sequencing (NGS) is critical to understand cancer genomes better. Here we present VarDict, a novel and versatile variant caller for both DNA- and RNA-sequencing data. VarDict simultaneously calls SNV, MNV, InDels, complex and structural variants, expanding the detected genetic driver landscape of tumors. It performs local realignments on the fly for more accurate allele frequency estimation. VarDict performance scales linearly to sequencing depth, enabling ultra-deep sequencing used to explore tumor evolution or detect tumor DNA circulating in blood. In addition, VarDict performs amplicon aware variant calling for polymerase chain reaction (PCR)-based targeted sequencing often used in diagnostic settings, and is able to detect PCR artifacts. Finally, VarDict also detects differences in somatic and loss of heterozygosity variants between paired samples. VarDict reprocessing of The Cancer Genome Atlas (TCGA) Lung Adenocarcinoma dataset called known driver mutations in KRAS, EGFR, BRAF, PIK3CA and MET in 16% more patients than previously published variant calls. We believe VarDict will greatly facilitate application of NGS in clinical cancer research.

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.

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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 TKIs 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 to 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 tumours 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 tumours. Further, these findings suggest that NRAS modifications in tumour 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.

BackgroundThis 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.MethodsTumor 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).ResultsWith 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).ConclusionsPPA 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.

Introduction: Osimertinib is the current recommended treatment for EGFR T790M-positive NSCLC after EGFR tyrosine kinase inhibitor therapy. However, resistance to osimertinib therapy is inevitably acquired after a period of effective treatment. We had a patient with EGFR L858R/ T790M-positive NSCLC who initially responded to osimertinib therapy but eventually experienced development of resistance. Plasma cell-free DNA analysis revealed the occurrence of exon 16-skipping HER2, which may have resulted in the erb-b2 receptor tyrosine kinase 2 gene (HER2) splice variant HER2D16. HER2D16 has never been reported in lung cancer, and HER2D16-driven signaling is known to be regulated by Src kinase in breast cancer. We investigated the role of HER2D16 as an osimertinibresistant mechanism.Methods: We constructed and established H1975 cells stably expressing HER2D16. The dimeric formation of HER2D16 was tested by using nonreducing polyacrylamide gel electrophoresis. The effects of the study drugs on signaling transduction were examined by using Western blot. Synergistic effect was assessed by using the Chou-Talalay method.Results: We found that HER2D16 can form a homodimer in NSCLC cells. HER2D16-expressing H1975 cells were resistant to osimertinib treatment. We also found that mutant EGFR and HER2D16 cooperated to activate downstream signaling for osimertinib resistance. In addition, cotreatment with osimertinib and an Src kinase inhibitor failed to reverse resistance, indicating that HER2D16-driven signaling in NSCLC did not occur through a canonical pathway. Finally, we revealed that the combination of osimertinib with the pan-HER small-molecule inhibitor afatinib could synergistically repress cell growth and signaling in H1975-HER2D16 cells. Conclusion:HER2D16 can contribute to osimertinib resistance through an Src-independent pathway. HER2D16 should be included in the molecular diagnosis panel for lung cancer.

BRAF and MEK1/2 inhibitors are effective in melanoma but resistance inevitably develops. Despite increasing the abundance of pro-apoptotic BIM and BMF, ERK1/2 pathway inhibition is predominantly cytostatic, reflecting residual pro-survival BCL2 family activity. Here, we show that uniquely low BCL-XL expression in melanoma biases the pro-survival pool towards MCL1. Consequently, BRAF or MEK1/2 inhibitors are synthetic lethal with the MCL1 inhibitor AZD5991, driving profound tumour cell death that requires BAK/BAX, BIM and BMF, and inhibiting tumour growth in vivo. Combination of ERK1/2 pathway inhibitors with BCL2/BCL-w/BCL-XL inhibitors is stronger in CRC, correlating with a low MCL1:BCL-XL ratio; indeed the MCL1:BCL-XL ratio is predictive of ERK1/2 pathway inhibitor synergy with MCL1 or BCL2/BCL-w/BCL-XL inhibitors. Finally, AZD5991 delays acquired BRAFi/MEKi resistance and enhances the efficacy of an ERK1/2 inhibitor in a model of acquired BRAFi + MEKi resistance. Thus combining ERK1/2 pathway inhibitors with MCL1 antagonists in melanoma could improve therapeutic index and patient outcomes.