Development and Clinical Utility of a Blood-Based Test Service for the Rapid Identification of Actionable Mutations in Non–Small Cell Lung Carcinoma

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Presentation transcript:

Development and Clinical Utility of a Blood-Based Test Service for the Rapid Identification of Actionable Mutations in Non–Small Cell Lung Carcinoma Hestia Mellert, Trudi Foreman, Leisa Jackson, Dianna Maar, Scott Thurston, Kristina Koch, Amanda Weaver, Samantha Cooper, Nicholas Dupuis, Ubaradka G. Sathyanarayana, Jakkie Greer, Westen Hahn, Dawne Shelton, Paula Stonemetz, Gary A. Pestano The Journal of Molecular Diagnostics Volume 19, Issue 3, Pages 404-416 (May 2017) DOI: 10.1016/j.jmoldx.2016.11.004 Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Precision testing. A: Assays for genetic variants were evaluated by Droplet Digital (dd) PCR (Bio-Rad, Pleasanton, CA) at Horizon Discovery (Cambridge, UK) with a prequalified standard of known percent minor variant frequencies (%MVF). The same standards were evaluated at Biodesix, Inc. (Boulder, CO) using the QX200 ddPCR system (Bio-Rad). EML4-ALK was not evaluated in the study. B: Intrarun studies for each EGFR and KRAS variant were run with three plasma samples from donors with cancer or, in the case of L858R, using analytical cell line standards (Horizon Discovery). C: Intrarun studies for the EML4-ALK multiplexed assay was run with three concentrations of analytical RNA standard. D: Inter-run studies of EGFR and KRAS were performed as in B. E: Interday testing of the EML-ALK assay was performed as in C. Data are expressed as means ± SD. n = 3 (B and C, independent runs; D and E, consecutive days of testing). Med., medium. The Journal of Molecular Diagnostics 2017 19, 404-416DOI: (10.1016/j.jmoldx.2016.11.004) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Robustness testing. Analytical positive control was spiked into normal human plasma, extracted, and tested by Droplet Digital PCR (Bio-Rad, Pleasanton, CA) over 21 consecutive business days. Both mutant (MUT) and wild-type (WT) copies are reported. A: EGFR variants ΔE746-A750, L858R, and T790M. B: KRAS variants G12C, G12D, and G12V. C: EML4-ALK fusion copies and control gene copies detected using the EML4-ALK multiplex for the detection of variants 1, 2, and 3. The Journal of Molecular Diagnostics 2017 19, 404-416DOI: (10.1016/j.jmoldx.2016.11.004) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Sensitivity and specificity clinical validation studies. Validation samples with reference result and Droplet Digital (dd) PCR (Bio-Rad, Pleasanton, CA) result, along with associated sensitivity, specificity, and concordance calculations (left) and an example of a two-dimensional plot of results not detected and detected from each validation set (right) are shown for EGFR variants ΔE746-A750 (A), L858R (B), and T790M (C); KRAS variants G12C (D), G12D (E), and G12V (F); and EML4-ALK multiplex for the detection of variants 1, 2, and 3 (G). ∗Prevalence of EML4-ALK variants 1, 2, and 3 represents that 78.4% of the total ALK mutations were used to calculate sensitivity and concordance. WT, wild type. The Journal of Molecular Diagnostics 2017 19, 404-416DOI: (10.1016/j.jmoldx.2016.11.004) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Assay utilization by ordering physicians. A: Workflow overview of the CLIA-certified laboratory test (Biodesix, Inc., Boulder, CO). The test process is initiated when whole blood is drawn into two blood-collection tubes (BCT; Streck, La Vista, NE) and the collection kit arrives at the centralized CLIA laboratory. Patient samples are accessioned into the Laboratory Information Management System (LIMS) and processed through parallel workflows to isolate either cDNA or RNA. Following nucleic acid extraction (and cDNA synthesis for RNA), samples are processed using the QX200 Droplet Digital (dd) PCR system (Bio-Rad, Pleasanton, CA). Droplet count evaluation is conducted using QuantaSoft version 1.7.4.0917 (Bio-Rad), and test result reports are generated from the secure LIMS. B: The percentages of orders submitted from physicians affiliated with community, academic, and government-run centers. Undefined represents those centers for which information was not provided with the sample. C: The percentage of test result reports with an individual variant, that variant with at least one other variant, and the variant as one of a full set of seven. cfDNA, circulating free DNA. The Journal of Molecular Diagnostics 2017 19, 404-416DOI: (10.1016/j.jmoldx.2016.11.004) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions