Suzanne Jenkins

EGFR mutation detection in ctDNA from NSCLC patient plasma: A cross-platform comparison of leading technologies to support the clinical development of AZD9291.

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.

High-Throughput SNP Genotyping

Whole genome approaches using single nucleotide polymorphism (SNP) markers have the potential to transform complex disease genetics and expedite pharmacogenetics research. This has led to a requirement for high-throughput SNP genotyping platforms. Development of a successful high-throughput genotyping platform depends on coupling reliable assay chemistry with an appropriate detection system to maximise efficiency with respect to accuracy, speed and cost. Current technology platforms are able to deliver throughputs in excess of 100 000 genotypes per day, with an accuracy of >99%, at a cost of 20–30 cents per genotype. In order to meet the demands of the coming years, however, genotyping platforms need to deliver throughputs in the order of one million genotypes per day at a cost of only a few cents per genotype. In addition, DNA template requirements must be minimised such that hundreds of thousands of SNPs can be interrogated using a relatively small amount of genomic DNA. As such, it is predicted that the next generation of high-throughput genotyping platforms will exploit large-scale multiplex reactions and solid phase assay detection systems.

Plasma ctDNA Analysis for Detection of the EGFR T790M Mutation in Patients with Advanced non-small Cell Lung Cancer.

Introduction: Tumor biopsies for detecting EGFR mutations in advanced NSCLC are invasive, costly, and not always feasible for patients with late‐stage disease. The clinical utility of the cobas EGFR Mutation Test v2 (Roche Molecular Systems, Inc., Pleasanton, CA) with plasma samples from patients with NSCLC at disease progression after previous EGFR tyrosine kinase inhibitor therapy was investigated to determine eligibility for osimertinib treatment. Methods: Matched tumor tissue and plasma samples from patients screened for the AURA extension and AURA2 phase II studies were tested for EGFR mutations by using tissue‐ and plasma‐based cobas EGFR mutation tests. Plasma test performance was assessed by using the cobas tissue test and a next‐generation sequencing method (MiSeq [Illumina Inc., San Diego, CA]) as references. The objective response rate, measured by blinded independent central review, was assessed in patients receiving osimertinib with a plasma T790M mutation–positive status. Results: During screening, 551 patients provided matched tumor tissue and plasma samples. Pooled analysis of the positive and negative percent agreements between the cobas plasma and tissue tests for detection of T790M mutation were 61% and 79%, respectively. Comparing cobas plasma test with next‐generation sequencing demonstrated positive and negative percent agreements of 90% or higher. The objective response rate was 64% (95% confidence interval: 57–70) in T790M mutation–positive patients by both cobas tissue and plasma tests (evaluable for response). Conclusions: The cobas plasma test detected the T790M mutation in 61% of tumor tissue T790M mutation–positive patients. To mitigate the risk of false‐negative plasma results, patients with a negative plasma result should undergo a tissue test where feasible.

134O_PR: Plasma ctDNA analysis for detection of EGFR T790M mutation in patients (pts) with EGFR mutation-positive advanced non-small cell lung cancer (aNSCLC)

investigator assessed ORR was 71% (43/61; 95%CI 57, 82), median DoR was 9.6 months (95%CI 7.7, 15.6) and median PFS was 9.7 months (95%CI 8.3, 13.6). In the AURA pooled P2, by blinded independent central review (BICR), ORR was 66% (262/397; 95%CI 61, 71), median DoR was 12.5 months (95%CI 11.1, not calculable), median PFS was 11.0 months (95%CI 9.6, 12.4) and the proportion of pts progression free at 12 months was 47.5% (95%CI 42.4, 52.5). In AURA P1 the most common causally-related adverse events (AEs) were rash grouped terms (37% [no Grade 3 (G3)]) and diarrhoea (35% [2%]). Rash grouped terms and diarrhoea were also the most common causally-related AEs in AURA pooled P2 (41% [G3 1%] and 38% [G3 1%], respectively). Conclusions: In these updated analyses, the AURA pooled P2 results by BICR were found to confirm the findings of AURA P1. Osimertinib provides a high ORR, with encouraging DoR and PFS, and a manageable tolerability profile. Clinical trial identification: NCT01802632 (23 February 2013) and NCT02094261 (17 March 2014) Legal entity responsible for the study: AstraZeneca Funding: AstraZeneca Disclosure: J. Yang: Advisory boards: Boehringer Ingelheim, Eli Lilly, Bayer, Roche/Genentech, AstraZeneca, Astellas, Bayer, MSD, Merck Serono, Pfizer, Novartis, Clovis Oncology, Celgene. S.S. Ramalingam: Consultancy fees: AstraZeneca, Boehringer Ingelheim, Celgene, Genentech, Novartis, Lilly, Merck, Bristol Myer Squibb. P.A. Jänne: Consultancy fees: AstraZeneca, Pfizer, Roche. Research support: AstraZeneca, Astellas Pharma. Stock ownership: Gatekeeper Pharma. Other: Post marketing royalties on Dana-Farber Cancer Institute owned patent on EGFR mutations licensed to Lab Corp. M. Cantarini: Employment: AstraZeneca. Shareholder: AstraZeneca. T. Mitsudomi: Advisory board AstraZeneca, Boehringer-Ingelheim, Chugai, Pfizer; Honoraria AstraZeneca, Chugai, Boehringer-Ingelheim, Pfizer; Research fund Boehringer-Ingelheim, Chugai, Pfizer.

EGFR T790M mutation testing within the osimertinib AURA phase I study.

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.

1270PLEVELS OF EGFR T790M IN PLASMA DNA AS A PREDICTIVE BIOMARKER FOR RESPONSE TO AZD9291, A MUTANT-SELECTIVE EGFR KINASE INHIBITOR

ABSTRACT Aim: AZD9291 is in clinical development to address T790M-mediated drug resistance associated with treatment of EGFR-mutant NSCLC. Genotyping of circulating plasma DNA may provide an option to identify patients (pts) unable to provide tissue biopsies. We aimed to study plasma levels of EGFR T790M in relation to AZD9291 drug activity. Methods: 101 pts with advanced NSCLC consented to plasma analysis as part of the Phase I study of AZD9291(NCT01802632). Central tumour genotyping was performed when feasible using the cobas™ assay. After pre-amplification of plasma DNA, levels of pre-dose T790M, L858R and exon 19 del variants were quantified using an emulsion PCR assay (Sysmex). Cases with no detectable plasma-sensitising mutation were omitted from T790M analysis. Response rate (RR) as of 16 Jan 2014 was calculated as the proportion of pts with ≥1 follow-up tumour measurement having a diameter decrease ≥30%. Results: Plasma assays for L858R and 19 del were studied in 44 pts with 19 del and 28 pts with L858R centrally confirmed in their tumour. A threshold of 0.05% mutant had 0% false positive rate (FPR) and 83% sensitivity for these mutations. In 64 pts with central T790M results, the same threshold applied to plasma T790M had 50% FPR, 89% sensitivity. Notably, of 6 cases falsely T790M+ in plasma, 5 were also T790M+ using a second plasma assay (cobas™). RR was 61% (25/41) and 24% (5/21) in 62 pts with central T790M+ and T790M- tumour genotyping (p = 0.008). Similarly, RR was 65% (33/51) and 38% (8/21) in 72 pts with T790M+ and T790M- plasma genotyping (p Conclusions: Plasma levels of EGFR-TKI-sensitising mutations correlate more closely with tumour genotype than T790M, likely due to the greater genomic heterogeneity of resistance. RR is higher in T790M+ pts than in T790M- pts, either using tumour or plasma genotyping. Plasma T790M genotyping using emulsion PCR may be an attractive alternative to rebiopsy for tumour genotyping of NSCLC pts with acquired EGFR resistance. Disclosure: K. Thress: Employment and stock ownership: AstraZeneca; J.C. Yang: Advisory boards: Boehringer Ingelheim, Eli Lilly, Pfizer, Novartis, Roche/Genentech, AstraZeneca, Merck, Bayer, Clovis Oncology. Corporate-sponsored research: Boehringer Ingelheim; D. Planchard: Advisory boards: AstraZeneca, Boehringer Ingelheim, Lilly, Novartis, Pfizer, Roche, BMS; R. Brant: Employment and stock ownership: AstraZeneca; H. Carr: Employment and stock ownership: AstraZeneca; S. Dearden: Employment and stock ownership: AstraZeneca; S. Jenkins: Employment and stock ownership: AstraZeneca; M. Cantarini: Employment and stock ownership: AstraZeneca; S. Ghiorghiu: Employment and stock ownership: AstraZeneca; J.C. Barrett: Employment and stock ownership: AstraZeneca; P.A. Janne: Consultant or advisory role: AstraZeneca, Boehringer Ingelheim, Clovis Oncology, Pfizer, Merrimack Pharmaceuticals, Chugai, Immunogen. Stock ownership: Gatekeeper. Other: Lab Corp; G.R. Oxnard: Compensated consultancy: Astra-Zeneca, Boehringer Ingelheim, Clovis, Genentech, Novartis, Sanofi. All other authors have declared no conflicts of interest.

EGFR Mutation Analysis for Prospective Patient Selection in Two Phase II Registration Studies of Osimertinib

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.

Abstract B105: Characterization of the activity of AZD9291 in patients (pts) with T790M ‘negative’ advanced non-small cell lung cancer (aNSCLC)

Objective AZD9291 is an oral, selective, irreversible EGFR-TKI, effective against EGFR-TKI-sensitizing (EGFRm) and resistance T790M mutations. Pre-study tumor samples from pts enrolled into expansion cohorts of a PhI study (AURA, NCT01802632) were tested centrally for T790M status. Pts with T790M positive tumor samples had higher objective response rate (ORR) than pts with no detectable T790M (T790M ‘negative’): 78/127 (61%) and 13/61 (21%), respectively (Janne et al 2015). Exploratory analyses to characterize AZD9291 activity in pts whose tumors were T790M negative by central test were performed. Methods Pts with EGFRm aNSCLC and acquired resistance to EGFR-TKIs were enrolled into expansion cohorts of the AURA study. The T790M status of pre-study tumor samples was determined locally (any method allowed) and/or centrally using the cobas TM EGFR mutation test (Roche Molecular Systems, Inc.). Presence of T790M in plasma derived ctDNA was determined by digital PCR (BEAMing). Pts with a centrally determined tumor based T790M negative status were then classified into one of nine groups according to local tumor status (T790M positive, negative, or unknown) and ctDNA status (T790M positive, negative, or unknown). Clinical outcome is reported for each group. Results 69 pts with T790M negative tumor status were determined centrally, and received AZD9291; 35 and 21 were also negative by local test and ctDNA, respectively and 7 were negative by all 3 tests; despite a negative central test, T790M positive tumor status was identified in 11 pts by prior local test and 26 by ctDNA. 17 pts with a central T790M negative status responded to AZD9291 treatment. ORR was 55% (6/11) in pts with a central T790M negative/local positive test result. No objective responses (0/7) were observed in pts with T790M negative tumor status by all three tests. Additional analysis of the sub-groups, by immediate prior TKI, EGFRm status, and duration of response will be available for presentation. Discussion In 69 pts with centrally identified T790M negative tumor status, 17 achieved a confirmed response. Of these, 12 were T790M positive by local test, ctDNA, or both. Different sensitivity and specificity of assays, tumor heterogeneity, and ctDNA shedding may explain positive results in this small group of pts classified as central T790M negative. The data support the hypothesis that T790M directed inhibitors are active against T790M driven resistance. Citation Format: Pasi A. Janne, Enriqueta Felip, Dong-Wan Kim, Thomas John, Daniel E. Haggstrom, Myung-Ju Ahn, Kenneth S. Thress, Suzanne C. Jenkins, Helen Mann, Mireille V. Cantarini, Simon P. Dearden, James Chih-Hsin Yang. Characterization of the activity of AZD9291 in patients (pts) with T790M ‘negative’ advanced non-small cell lung cancer (aNSCLC). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B105.

Abstract C196: EGFR mutation detection in ctDNA isolated from NSCLC patient plasma; a cross-platform comparison of leading technologies.

Emerging evidence suggests that circulating tumor DNA (ctDNA) provides an alternative diagnostic sample when surgical biopsies are inaccessible, and could enable disease diagnosis, frequent assessment of disease progression, and almost real-time monitoring of the emergence of resistance mutations. We hypothesized that novel highly sensitive technologies enable EGFR T790M mutation detection in ctDNA from patients that had previously been treated with and become resistant to EGFR-TKIs. We have used 3 technologies to assess T790M mutation detection rate. Samples comprised patients with sensitizing EGFR mutations and wild type EGFR patients who had been previously treated with EGFR TKI therapies. In approximately 50% of EGFR mutation positive cases, acquired resistance to EGFR-TKIs is mediated by an EGFR T790M mutation. Reference data for T790M mutations from tumor material was unavailable.We undertook a comprehensive cross-technology comparison of three technology platforms: ARMS based detection using the Roche cobas EGFR mutation detection kit; digital droplet PCR using the BioRad ddPCR instrument (by MolecularMD) and bead based digital PCR using the Inostics BEAMing technology for the detection of T790M mutations. In total, 135 frozen plasma samples, obtained from 2 clinical studies, were used to estimate T790M detection rates in ctDNA. Within the study group, 72 samples were taken from EGFR mutation positive patients, of which around 36 (expected range 28-44) would be expected to have T790M mutations, the remaining samples would be expected to be T790M negative. Each individual patient plasma sample was split and evaluated across multiple platforms. In addition, the suitability of 3 ctDNA preparation kits (cobas Circulating DNA Isolation kit, QIAamp Circulating Nucleic Acid kit and QIAamp DNA Mini kit) in terms of DNA yield was evaluated. The QIAamp Circulating Nucleic Acid kit and cobas Circulating DNA Isolation kit were equally effective in preparing ctDNA from plasma samples. Overall, concordance of EGFR T790M mutation status was high between all 3 methods. In addition, the false positive rate in the known EGFR mutation negative cases was low for all 3 methods. However, differences were seen in the rate of false negative results (assay sensitivity) between methods. DNA preparation kits specifically designed for use with ctDNA samples are preferable to other methods. Current technologies for the detection of T790M mutations in ctDNA, where patient tumor material is not available, still require further development to increase detection rates in these samples. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C196. Citation Format: Helen Brown, Roz Brant, Simon Dearden, Suzanne Jenkins, Kenneth Thress, Alain Horvais, Ruth March, J. Carl Barrett. EGFR mutation detection in ctDNA isolated from NSCLC patient plasma; a cross-platform comparison of leading technologies. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C196.