Exosomes and other extracellular vesicles (commonly referred to as EVs) have generated a lot of attention for their potential applications in both diagnostics and therapeutics. The contents of these vesicles are the subject of intense research, and the relatively recent discovery of RNA inside EVs has raised interest in the biological function of these RNAs as well as their potential as biomarkers for cancer and other diseases. Traditional ultracentrifugation-based protocols to isolate EVs are labor-intensive and subject to significant variability. Various attempts to develop methods with robust, reproducible performance have not yet been completely successful. Here, we report the development and characterization of a spin column-based method for the isolation of total RNA from EVs in serum and plasma. This method isolates highly pure RNA of equal or higher quantity compared to ultracentrifugation, with high specificity for vesicular over non-vesicular RNA. The spin columns have a capacity to handle up to 4 mL sample volume, enabling detection of low-abundance transcripts in serum and plasma. We conclude that the method is an improvement over traditional methods in providing a faster, more standardized way to achieve reliable high quality RNA preparations from EVs in biofluids such as serum and plasma. The first kit utilizing this new method has recently been made available by Qiagen as “exoRNeasy Serum/Plasma Maxi Kit”.
BackgroundA major limitation of circulating tumor DNA (ctDNA) for somatic mutation detection has been the low level of ctDNA found in a subset of cancer patients. We investigated whether using a combined isolation of exosomal RNA (exoRNA) and cell-free DNA (cfDNA) could improve blood-based liquid biopsy for EGFR mutation detection in non-small-cell lung cancer (NSCLC) patients.Patients and methodsMatched pretreatment tumor and plasma were collected from 84 patients enrolled in TIGER-X (NCT01526928), a phase 1/2 study of rociletinib in mutant EGFR NSCLC patients. The combined isolated exoRNA and cfDNA (exoNA) was analyzed blinded for mutations using a targeted next-generation sequencing panel (EXO1000) and compared with existing data from the same samples using analysis of ctDNA by BEAMing.ResultsFor exoNA, the sensitivity was 98% for detection of activating EGFR mutations and 90% for EGFR T790M. The corresponding sensitivities for ctDNA by BEAMing were 82% for activating mutations and 84% for T790M. In a subgroup of patients with intrathoracic metastatic disease (M0/M1a; n = 21), the sensitivity increased from 26% to 74% for activating mutations (P = 0.003) and from 19% to 31% for T790M (P = 0.5) when using exoNA for detection.ConclusionsCombining exoRNA and ctDNA increased the sensitivity for EGFR mutation detection in plasma, with the largest improvement seen in the subgroup of M0/M1a disease patients known to have low levels of ctDNA and poses challenges for mutation detection on ctDNA alone.Clinical TrialsNCT01526928
In our recent article [1], we demonstrated the improved sensitivity of using combined exosomal RNA/DNA and cfDNA for liquid biopsies detecting EGFR mutations in plasma from patients with NSCLC. In reviewing our article, Bracht et al. [2] identified an editorial mistake in the main text of the article, in which we reference a supplementary analysis. The sentence on p. 704 should correctly have read:. .. exoNA was able to correctly identify non-responders to rociletinib, potentially allowing to switch these patients to a different treatment, while ensuring that 100% of responding patients continue on treatment (100% Negative Predictive Value (NPV), 45% specificity; see supplementary material S12, available at Annals of Oncology online). The calculations, numbers and the descriptions of the rationale behind development of the test both in the main text and in the supplement are all correct in the original article and remain unchanged.
Introduction: The EML4-ALK (echinoderm microtubule-associated protein-like 4/anaplastic lymphoma kinase) translocation represents a predictive driver mutation in non-small cell lung cancer (NSCLC). As EML4-ALK is both associated with resistance to EGFR inhibitors and druggability with FDA approved ALK kinase inhibitors, molecular profiling of the respective fusion transcripts is an important prerequisite for therapy. Ongoing clinical trials and development of new ALK inhibitors for personalized treatment also emphasize the need for development of accompanying diagnostics. Today's determination of EML4-ALK fusions is based on biopsies and fine-needle aspirates. These techniques are constrained by surgical complications, availability of tissue and sample heterogeneity. To overcome these challenges, the mutational analysis of fusion transcripts in plasma is expected to be a valuable benefit for non-surgical and longitudinal monitoring of EML4-ALK positive NSCLC patients. Experimental procedures: Using our proprietary column-based method to isolate vesicle-derived RNA from a few ml of patient plasma from NSCLC patients, we were able to consistently and reproducibly isolate sufficient amounts of high quality exoRNA. The purified exoRNA was subjected to RT-qPCR screening for EML4-ALK fusion transcripts, with assays designed to independently detect fusion transcripts of EML4-ALK variants v1, v2 and v3. During development of the qPCR, assay performance was characterized on synthetic nucleic acid oligos spiked into plasma of healthy human donors. The diagnostic assay was finally validated on time-matched tissue and plasma samples derived from NSCLC patients. Summary: Our data demonstrates the ability to detect rare predictive mutations in exosomal and other vesicular-derived RNA transcripts isolated from patient plasma. Here, NSCLC samples were characterized by monitoring their expression profile of EML4-ALK variants. The qPCR assays specifically detected the three major fusion transcript variants representing more than 70% of all EML4-ALK positive cases. The assay displayed high selectivity over wild type background and data showed correlation with tissue analysis. Iterative modeling of the assay significantly improved specificity and sensitivity towards its application as a diagnostic test. Conclusions: Liquid biopsies represent a low-risk and comprehensive method to screen for predictive cancer markers in plasma of NSCLC patients during longitudinal monitoring. The efficient isolation of exosomal/vesicle-derived RNA via spin column enables subsequent analysis of rare fusion transcripts for diagnostic development. As a proof of concept, we implemented a qPCR-based assay to determine EML4-ALK translocations in plasma from NSCLC patients, indicating the feasibility of developing a diagnostic test, which could facilitate effective personalized treatment. Citation Format: Kay Brinkmann, Daniel Enderle, Tina Koestler, Stefan Bentink, Jennifer Emenegger, Alexandra Spiel, Romy Mueller, Vincent O'Neill, Johan Skog, Mikkel Noerholm. Plasma-based diagnostics for detection of EML4-ALK fusion transcripts in NSCLC patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 545. doi:10.1158/1538-7445.AM2015-545
Background: After initial responses to tyrosine kinase inhibitors (TKIs), NSCLC patients (pts) harboring EGFR activating mutations inevitably progress, with the “gatekeeper” EGFR T790M resistance mutation accounting for approximately 60% of cases of acquired resistance (AR) to TKIs. EGFR activating and T790M resistance mutations can be found in plasma on both circulating free tumor DNA (ctDNA) and on RNA contained within exosomes. While ctDNA is thought to be primarily released by dying cells, exosome RNA is actively released by many living cells (Jahr et al. Cancer Res 2001; Thery et al. Nat Rev Immunol 2009). Some pts, particularly those with either early stage or intra-thoracic disease, do not seem to release mutations on ctDNA into circulation that is detectable by any current method. Here we present data demonstrating the detection of activating and AR EGFR mutations using a combined single-step exosomal RNA (exoRNA) and ctDNA approach to maximize sensitivity and demonstrate the ability to detect mutations using exosomal RNA on pts previously described as negative by ctDNA methods alone. Methods: Matched pretreatment tumor tissue and plasma were collected from 81 NSCLC pts enrolled in TIGER-X, a Ph1/2 study of rociletinib in previously treated mutant EGFR pts with advanced NSCLC. Among the 81 pts (all enrolled before Dec 2014), 56 cases were randomly chosen from the clinical patient population (including 35 cases ≥10 mutant copies/mL and 21 cases <10 mutant copies/mL) and an additional 25 cases were previously determined to have low T790M levels in plasma (<10 copies/mL) using ctDNA-based approaches. We applied a column-based method (EXO52) to co-isolate both exoRNA and ctDNA from up to 6 mL of patient plasma, where available, and analyzed the mutations with a custom procedure for next generation sequencing (EXO1000). The targeted EXO1000 sequencing panel covers EGFR mutations on exon 19, 20 and 21. A custom library preparation method and bioinformatics pipeline was used to identify rare mutations in a qualitative and quantitative manner. Results: For the 56 cases randomly chosen from the clinical patient population, 54 had valid tumor tissue results. The positive percent agreement (PPA) between plasma and tumor was 96% (52/54) for activating mutations and 86% (42/49) for T790M with tumor as the reference method. For most cases analyzed, the combined mutation signal from exoRNA and ctDNA was greater than the signal from ctDNA alone. Furthermore, we detect mutations in the circulation of some pts who were previously called negative by analysis of ctDNA alone, suggesting improved sensitivity from addition of exoRNA to the analysis. In the subset of pts with low or undetectable levels of ctDNA and valid tumor results (N = 45), we detect activating mutations in 38 of 45 cases (PPA 84%) compared to 27 of 45 (PPA 60%) by ctDNA alone, as well as T790M in 22 of 35 evaluable cases (PPA 63%) compared to 19 of 35 in ctDNA alone (PPA 54%). Conclusions: Our data demonstrate the ability to detect low copy numbers of activating and AR mutations in plasma of lung cancer pts by combining the mutation signal from exoRNA and ctDNA isolated by EXO52 and using the EXO1000 targeted NGS gene panel. By combining these two analytes, a higher sensitivity of mutation detection may be possible compared to analysis by ctDNA alone. Citation Format: Anne K. Krug, Chris Karlovich, Tina Koestler, Kay Brinkmann, Alexandra Spiel, Jennifer Emenegger, Mikkel Noerholm, Vince O'Neill, Lecia V. Sequist, Jean-Charles Soria, Jonathan W. Goldman, D. Ross Camidge, Heather A. Wakelee, Shirish M. Gadgeel, Elaina Mann, Shannon Matheny, Lindsey Rolfe, Mitch Raponi, Daniel Enderle, Johan Skog. Plasma EGFR mutation detection using a combined exosomal RNA and circulating tumor DNA approach in patients with acquired resistance to first-generation EGFR-TKIs. [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 B136.
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