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K. Brinkmann
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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): K. Brinkmann
- 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.
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P3.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 235)
- Event: WCLC 2015
- Type: Poster
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:
- Coordinates: 9/09/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P3.04-001 - Exosomal RNA Based Liquid Biopsy Detection of EML4-ALK in Plasma from NSCLC Patients (ID 2591)
09:30 - 09:30 | Author(s): K. Brinkmann
- Abstract
Background:
Molecular profiling to direct targeted therapy has revolutionized cancer treatment. For instance, the tailored therapy of NSCLC patients carrying somatic EML4-ALK rearrangements with ALK inhibitors has shown to be associated with substantial clinical response. A prerequisite of this approach is highly sensitive and specific diagnostics to detect and monitor the prognostic biomarker. Today’s tissue-based diagnostics like FISH are limited by complications of biopsy and technical challenges. Therefore, biomarker assessment in plasma circulation would be a valuable alternative to tissue based testing and provide a simple new option for identifying and monitoring EML4-ALK positive NSCLC patients. We previously demonstrated the feasibility of detecting EML4-ALK fusion transcripts in 6 plasma samples from patients known to be positive by tissue FISH testing (the gold standard). Here we present more comprehensive performance characteristics of this diagnostic test analyzing the exosomal expression of EML4-ALK in plasma of NSCLC patients.
Methods:
We developed a diagnostic test to monitor the expression of EML4-ALK fusion transcripts in low-volume plasma samples of lung cancer patients. The Exosome Diagnostics ALK assay comprises column-based isolation of total vesicular RNA from 0.5 – 2.0 ml patient plasma, followed by discrete detection of EML4-ALK variants v1, v2 and v3 via qPCR. Assay quality is confirmed by inclusion of internal and external controls. Following validation on both synthetic and human samples, we monitored variant-specific expression of EML4-ALK in a cohort of more than 20 plasma samples from NSCLC patients. The data was analyzed for concordance with time-matched tissue and aligned with patient’s response data.
Results:
Applying our diagnostic test for EML4-ALK fusion variants, we were able to identify the predictive biomarker in exosomal RNA transcripts isolated from patient plasma. We determined the variant-specific expression profile of EML4-ALK fusion transcripts in a cohort of NSCLC patients with high sensitivity and specificity. We observed high concordance of the qPCR-based plasma results with FISH-based tissue information.
Conclusion:
Liquid biopsies represent a low-risk and viable approach to testing for predictive cancer markers in NSCLC patients. Here, we demonstrate the capability of our validated diagnostic test to determine expression of rare EML4-ALK fusion transcripts in plasma as a sensitive alternative to repeat biopsy. Monitoring discrete EML4-ALK fusion variants would enable effective personalized treatment and has clear clinical application.
