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A.K. Krug
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P2.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 234)
- Event: WCLC 2015
- Type: Poster
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:
- Coordinates: 9/08/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P2.04-001 - EGFR Activating and T790M Resistance Mutation in Plasma exoRNA and cfDNA, Detected with Single-Step Isolation Columns and Targeted Resequencing (ID 2618)
09:30 - 09:30 | Author(s): A.K. Krug
- Abstract
Background:
After initial responses to tyrosine kinase inhibitors (TKIs), NSCLC patients harboring EGFR activating mutations inevitably show progression, a consequence of acquired resistance (AR). Secondary mutations in the EGFR domains, e.g. the gatekeeper mutation T790M, are thought to play a role in clinical resistance of approximately half the patients that experience disease progression during treatment with TKIs, and novel therapeutic agents are in development to circumvent this resistance mechanism. Tissue based assays, requiring repeat biopsy, are fundamentally unattractive, and detection of AR mutations in circulation would be an appealing alternative. Here we present data demonstrating the feasibility of detection of activating and AR EGFR mutations with a targeted resequencing panel, using a combined single-step exosomal RNA (exoRNA) and cell-free DNA (cfDNA) isolation to maximize sensitivity.
Methods:
Plasma from more than 40 lung cancer patients was collected at the time of clinical resistance to EGFR TKI therapy. The plasma samples are complemented by EGFR-genotyping on time-matched tissue from a repeat biopsy. We applied our proprietary column-based method to co-isolate both exoRNA and cfDNA from patient plasma, and analyzed the mutations with a custom procedure for next generation sequencing (NGS). The targeted resequencing panel covers the most important mutation hotspots in NSCLC relevant genes including EGFR mutations on exon 19, 20 and 21. A custom library preparation method and bioinformatics pipeline is used to efficiently call rare mutations in a qualitative and quantitative manner.
Results:
Our data demonstrate the ability to detect low copy numbers of activating and AR mutations in plasma of lung cancer patients by combining the mutation signal from exoRNA and cfDNA and using a focused NGS gene panel. The mutation signal in plasma is highly concordant with data obtained from repeat biopsies, showing the feasibility of the approach. Moreover, EGFR mutations of patients with intrathoracic disease (M0/M1a) are readily detected in the combined exoRNA/cfDNA isolation, in contrast to methods relying only on the isolation of cfDNA.
Conclusion:
Detection of both activating and AR mutations to EGFR therapy in plasma is a feasible alternate to repeat biopsy and the combined isolation of exoRNA and cfDNA offers superior sensitivity. Especially in challenging cases, e.g. with intrathoracic disease, the advantage of combined plasma exoRNA/cfDNA isolation substantially improves the sensitivity over approaches that utilize only cfDNA.
