Emma Green

Osimertinib benefit in EGFR-mutant NSCLC patients with T790M-mutation detected by circulating tumour DNA.

Background Approximately 50% of epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer (NSCLC) patients treated with EGFR tyrosine kinase inhibitors (TKIs) will acquire resistance by the T790M mutation. Osimertinib is the standard of care in this situation. The present study assesses the efficacy of osimertinib when T790M status is determined in circulating cell-free tumour DNA (ctDNA) from blood samples in progressing advanced EGFR-mutant NSCLC patients. Material and methods ctDNA T790M mutational status was assessed by Inivata InVision™ (eTAm-Seq™) assay in 48 EGFR-mutant advanced NSCLC patients with acquired resistance to EGFR TKIs without a tissue biopsy between April 2015 and April 2016. Progressing T790M-positive NSCLC patients received osimertinib (80 mg daily). The objectives were to assess the response rate to osimertinib according to Response Evaluation Criteria in Solid Tumours (RECIST) 1.1, the progression-free survival (PFS) on osimertinib, and the percentage of T790M positive in ctDNA. Results The ctDNA T790M mutation was detected in 50% of NSCLC patients. Among assessable patients, osimertinib gave a partial response rate of 62.5% and a stable disease rate of 37.5%. All responses were confirmed responses. After median follow up of 8 months, median PFS by RECIST criteria was not achieved (95% CI: 4–NA), with 6- and 12-months PFS of 66.7% and 52%, respectively. Conclusion(s) ctDNA from liquid biopsy can be used as a surrogate marker for T790M in tumour tissue.

Abstract 3639: Analytical performance and validation of an enhanced TAm-Seq circulating tumor DNA sequencing assay

Circulating tumor DNA (ctDNA) is becoming established as a tool to supplement conventional biopsies for molecular characterization and monitoring of solid cancers, especially for cancers where tumor tissue is difficult to obtain or is only available at limiting quantity. This requires reliable identification, in patient plasma, of tumor-specific DNA alterations that in some cases may be present as a small fraction of the total cell-free DNA molecules. To overcome these technical challenges, we have developed an enhanced platform for tagged-amplicon deep sequencing (TAm-Seq™). Using a combination of efficient library preparation and statistically-based analysis algorithms, this platform can be used to sequence, identify and quantify cancer mutations across a gene panel including both cancer hotspots, as well as entire coding regions of selected genes. This poster will present validated performance specifications of this multi-gene ctDNA sequencing assay. To perform analytical validation, we used reference standards and plasma DNA controls to demonstrate the sensitivity, specificity and quantitative accuracy of this ctDNA analysis platform. We found that our workflow, using 4 mL input plasma, yields very high sensitivity for variants that are present at allele fraction 0.25% or higher in plasma, and retains substantial sensitivity at allele fractions as low as 0.1%. Using dilution mixtures of well-characterised reference samples, we show that the assay accurately quantifies allele fractions with precision predominantly limited by stochastic sampling. Analysis of plasma samples from control individuals demonstrates a low false positive rate. The assay also detects DNA amplifications (including in ERBB2, MYC, KRAS, EGFR, MET, FGFR1, FGFR2) when the ctDNA are sufficiently high. Together, these data demonstrate the analytical validity and robustness of the TAm-Seq assay and support its use as a basis for clinical applications. Citation Format: Davina Gale, Vincent Plagnol, Andrew Lawson, Michelle Pugh, Sarah Smalley, Karen Howarth, Mikidache Madi, Bradley Durham, Vasudev Kumanduri, Kitty Lo, James Clark, Emma Green, Nitzan Rosenfeld, Tim Forshew. Analytical performance and validation of an enhanced TAm-Seq circulating tumor DNA sequencing assay. [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 3639.

Development of a highly sensitive liquid biopsy platform to detect clinically-relevant cancer mutations at low allele fractions in cell-free DNA

Introduction Detection and monitoring of circulating tumor DNA (ctDNA) is rapidly becoming a diagnostic, prognostic and predictive tool in cancer patient care. A growing number of gene targets have been identified as diagnostic or actionable, requiring the development of reliable technology that provides analysis of multiple genes in parallel. We have developed the InVision™ liquid biopsy platform which utilizes enhanced TAm-Seq™ (eTAm-Seq™) technology, an amplicon-based next generation sequencing method for the identification of clinically-relevant somatic alterations at low frequency in ctDNA across a panel of 35 cancer-related genes. Materials and methods We present analytical validation of the eTAm-Seq technology across two laboratories to determine the reproducibility of mutation identification. We assess the quantitative performance of eTAm-Seq technology for analysis of single nucleotide variants in clinically-relevant genes as compared to digital PCR (dPCR), using both established DNA standards and novel full-process control material. Results The assay detected mutant alleles down to 0.02% AF, with high per-base specificity of 99.9997%. Across two laboratories, analysis of samples with optimal amount of DNA detected 94% mutations at 0.25%-0.33% allele fraction (AF), with 90% of mutations detected for samples with lower amounts of input DNA. Conclusions These studies demonstrate that eTAm-Seq technology is a robust and reproducible technology for the identification and quantification of somatic mutations in circulating tumor DNA, and support its use in clinical applications for precision medicine.

Abstract 3192: Liquid biopsies for molecular profiling of mutations in non-small cell lung cancer patients lacking tissue samples

Introduction: Approximately 30% of patients with an adenocarcinoma of the lung have an actionable driver mutation. Further understanding the molecular mechanisms of acquired resistance to targeted therapies provides key information for determining subsequent treatment options. Access to tumor tissue to perform either the initial molecular profile or at the point of acquired resistance, however, is often limited. Circulating tumor DNA (ctDNA) can be used as a minimally invasive method for the detection and quantification of molecular abnormalities. We performed a prospective study to assess molecular alterations in the ctDNA of NSCLC patients in whom the initial molecular profile or profile at acquired resistance was unknown due to lack of tumor tissue biopsy or insufficient cellularity in the biopsy. Methods: Plasma samples were collected from 52 pre-treated advanced NSCLC patients at the Gustave Roussy. DNA was extracted from TM assay covering regions from 35 cancer-related genes. Sequences were generated using Illumina sequencing. We also analysed plasma taken following treatments prescribed after the original molecular profile detected using plasma ctDNA. Results: From July 2015 to October 2015, 52 patients were included (63% female, 37% never-smoker, 95% diagnosed with an adenocarcinoma subtype, 95% with stage IV disease, and 54% had EGFR mutant tumors of which 68% had mutations in exon 19 and 32% had mutations in exon 21). ctDNA profiling was successfully performed for all patients, and mutations were detected in 38 of 52 patients. The median number of mutations detected in plasma samples was 1. Within the EGFR mutant subpopulation, T790M mutations were identified including 8 acquired cases (with a concomitant C797S mutation in 1 case) and 1 primary T790M mutation. Of these patients, 5 started personalised treatment with AZD9291 based on the results of ctDNA analysis. In the other 18 patients with EGFR mutant tumors, no acquired mutations associated with resistance were detected. Other results encompassed: 2 plasma samples with EGFR mutation exon 18 (G719A, G719C) leading to initiation of afatinib in one case, 1 case with EGFR mutation exon 21 (L861Q), 1 patients with ERBB2 exon 20 insertion, 3 KRAS mutant detected in plasma (G12C, G12S, G12F), 2 STK11 mutant samples, and 1 patient with a MET mutation (exon 14) who subsequently started crizotinib. Conclusions: ctDNA analysis with Inivata9s enhanced TAm-Seq™ provides an alternative method of ‘liquid biopsy’ for obtaining molecular profile of mutations present in NSCLC patients in the absence of an invasive tissue biopsy. Liquid biopsy identified cancer mutations in 73% of the study population, and 18% of those patients subsequently received treatment tailored to the plasma ctDNA detected mutations. An update on the analysis of 75 patients will be presented during the conference. Citation Format: Jordi Remon, Jean Charles Soria, David Planchard, Cecile Jovelet, Chloe Pannet, Ludovic Lacroix, Annas Gazzah, Andrew Lawson, Sarah Smalley, Kenth Howarth, David Gale, Emma Green, Vincent Plagnol, Nitzan Rosenfeld, Ken Oulassen, Nathalie Chaput, Benjamin Besse. Liquid biopsies for molecular profiling of mutations in non-small cell lung cancer patients lacking tissue samples. [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 3192.