An NGS workflow to detect down to 0.1% allelic frequency in cfDNA
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This document details the development and validation of Oncomine cfDNA assays for noninvasive detection of low-abundance tumor mutations from blood samples, achieving a sensitivity of over 80% and specificity exceeding 98% at a 0.1% limit of detection. Three distinct panels were created for lung, colon, and breast cancers, each targeting specific mutation hotspots. These assays support comprehensive oncology research by enabling detection of rare mutations that may inform tumor progression and therapeutic resistance.
An NGS workflow to detect down to 0.1% allelic frequency in cfDNA
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Assay Genes Selected Hotspots Oncomine Lung cfDNA assay ALK, BRAF, EGFR, ERBB2, KRAS, MAP2K1, MET, NRAS, PIK3CA, ROS1, TP53 EGFR: T790M, C797S, L858R, Exon 19 del KRAS: G12X, G13X, Q61X BRAF: V600E ALK: Exon 21-25 PIK3CA: E545K, H1047R, E542K Oncomine Colon cfDNA assay AKT1, BRAF, CTNNB1, EGFR, ERBB2, FBXW7, GNAS, KRAS, MAP2K1, NRAS, PIK3CA, SMAD4, TP53, and APC KRAS/NRAS: G12/G13/Q61 BRAF: V600E PIK3CA: E545K, H1047R TP53: R175H R273H/C/L Recurrent deleterious APC mutations (including p.R876*, p.R1114*, p.Q1378*, and p.R1450*) SMAD4: R361C/H CTNNB1: S45F, T41A Oncomine Breast cfDNA assay AKT1, EGFR, ERBB2, ERBB3, ESR1, FBXW7, KRAS, PIK3CA, SF3B1, TP53 PIK3CA: E545K and H1047R AKT1: E17K ESR1: Mutations associated with anti-estrogen resistance TP53: Mutations associated with loss of function ERBB2: Mutations associated with sensitivity to antiERBB2 therapies Dalia Dhingra, Richard Chien, Jian Gu, Yanchun Li, Kunal Banjara, Dumitru Brinza, Ruchi Chaudhary, Kelli Bramlett, Thermo Fisher Scientific, 180 Oyster Point Blvd., South San Francisco, CA 94131 ABSTRACT Noninvasive detection of rare mutations in blood could allow tumor monitoring for research purposes. Research studies have suggested that cfDNA contains DNA from tumor cells with somatic mutations that could inform on tumor progression and therapeutic resistance. Here, we demonstrate a complete workflow from a single tube of blood through data analysis for research samples down to a 0.1% allelic frequency. The low abundance tumor mutations found in cfDNA requires sensitive and accurate mutation detection. We have developed two panels that utilize an amplification- based assay that generates tagged DNA copies, which allows detection of low abundance tumor mutations found in cfDNA. The two panels allow multiplex interrogation of primary driver and resistance mutations specific to ctDNA from breast and colon cancer. The Oncomine™ Colon cfDNA panel targets 236 hotspots within 14 genes while the Oncomine Breast cfDNA panel covers 157 hotspot mutations in 10 genes. This workflow was validated from matched single blood tubes, Streck and K2EDTA. Additionally, the utility for cancer research was demonstrated with concordance studies using matched FFPE and plasma from oncology samples. To further characterize these panels we have developed an oncology control for cfDNA with nucleosome fragment sizing and minimal sonication damage. This engineered control contains SNPs and indels at 0.1% allelic frequencies, orthogonally confirmed with TaqMan® based Rare Mutation assays. With this control, the Oncomine Breast cfDNA panel had over 81% sensitivity and 99.9% specificity. The Oncomine Colon cfDNA panel had over 85% sensitivity and 100% specificity. The Oncomine Breast cfDNA panel and Oncomine Colon cfDNA panel integrated into a complete workflow starting from a single tube of blood can advance oncology research with the ability to detect blood based cancer biomarkers present at 0.1%. ASSAYS CONCLUSIONS Oncomine cfDNA assays allow for a 0.1% limit of detection from 20 ng of cfDNA. We have validated over 80% sensitivity and above 98% specificity for all three Oncomine cfDNA assays at a 0.1% limit of detection. The sensitivity increases to over 99.9% at a 0.5% limit of detection. Variant results can be obtained within 2 days using the Ion Torrent NGS workflow starting from a single tube of blood. This workflow has been demonstrated with 10 mL blood samples as well as with matched FFPE and plasma samples. FOR RESEARCH USE ONLY. Not for use in diagnostic procedures. © 2016 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. An NGS workflow to detect down to 0.1% allelic frequency in cfDNA #5396 Thermo Fisher Scientific • 5781 Van Allen Way • Carlsbad, CA 92008 • thermofisher.com Assay Oncomine Lung cfDNA assay Oncomine Colon cfDNA assay Oncomine Breast cfDNA assay Input 6000 copies cfDNA control Limit of Detection 0.1% 0.5% 0.1% 0.5% 0.1% 0.5% Sensitivity 90% 99.9% 85.6% 100% 81.3% 100% Specificity 98.4% 98.3% 100% 100% 99.9% 99.9% Gene Variant Oncomine Lung cfDNA assay Oncomine Colon cfDNA assay Oncomine Breast cfDNA assay TaqMan dPCR KRAS p.G12D 0.20% 0.15% 0.20% 0.27% 0.17% 0.14% 0.14% 0.24% PIK3CA p.E545K 0.15% 0.26% 0.14% 0.02% 0.11% 0.16% 0.06% 0.19% EGFR p.E746_A750d elELREA 0.12% *N/A *N/A 0.10% 0.11% *N/A *N/A 0.12% WORKFLOW LIMIT OF DETECTION Figure 1: The NGS workflow starts with a single tube of blood and using Oncomine cfDNA assays, results in variant data within 2 days. The analysis is fully optimized for the Ion Torrent™ platform. Assay Oncomine Lung cfDNA assay Oncomine Colon cfDNA assay Oncomine Breast cfDNA assay Input Single K2EDTA tube of blood Number of samples 8 6 12 Average hotspot variants per sample 0 0 0.67 Maximum hotspot variants per sample 0 0 1 ONCOMINE cfDNA TECHNOLOGY WORKFLOW VALIDATION ORTHOGONAL VERIFICATION MATCHED SAMPLES Table 1: The Oncomine cfDNA assays are content optimized using the COSMIC and Oncomine databases. Each assay targets over 150 hotspots. Assay Sample Gene Variant FFPE Plasma Oncomine Lung cfDNA assay S1 EGFR p.L858R 71.42 2.66 S3 MET p.T1010I 43.87 51.75 KRAS p.G12C 34.62 0.28 S16 MET p.T1010I 50.42 47.44 KRAS p.G12D 3.98 0.22 S20 KRAS p.G13C 2.65 0.30 MET p.T1010I 2.21 Oncomine Colon cfDNA assay 442 PIK3CA p.E542K 13.95 APC p.R805Ter 15.19 0.2 APC p.R876Ter 0.4 TP53 p.R273C 0.05 PIK3CA p.H1047R 0.06 444 KRAS p.G12V 36.28 0.42 PIK3CA p.E545K 23.91 0.06 445 TP53 p.R175H 54.66 0.91 GNAS p.R201C 0.39 0.05 FBXW7 p.R465C 0.09 TP53 p.E286G 0.11 446 TP53 p.P250L 37.42 0.16 Figure 2: The Oncomine cfDNA technology utilizes tags. Table 2: The workflow was validated on purchased single K2EDTA tubes of blood. Any variants detected in these research samples are classified as false positives. No library had more than one false positive. Figure 3: Fragmented Acrometrix™ Oncology Hotspot Control was used to determine the sensitivity and specificity of the assays at 0.1% and 0.5% allelic frequencies. All assays have greater than 90% sensitivity at 0.1% with high specificity. Table 3: Digital PCR with TaqMan Rare Mutation assays orthogonally verified the allelic frequencies of the fragmentedAcrometrix Oncology Hotspot Control. Table 4: A selection of matched FFPE and plasma samples used for validation of our assays. The assays can use either FFPE or plasma as input with analysis workflows optimized for each type of sample.