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Simultaneous detection of activating somatic DNA mutations and expressed fusion transcripts from lung tumor FFPE samples # 2422/9 A. McGarry Houghton1, Gavin Meredith2, Julia Kargl1,3, Jill McKay-Fleisch2, P. Martin Ross2, Anisha Kharkia2, Afshin Mashadi-Hossein2, Dae Kim2, Joseph Beechem2. 1Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N. Seattle, WA 98109; 2NanoString Technologies, Inc. 530 Fairview Ave. N. Seattle, WA 98109 Abstract SNV Panel multiplex detection of 5% alleles from FFPE Worldwide, lung cancer is the most commonly diagnosed form of cancer with a survival rate among the lowest. Combined, somatic mutations (in the form of SNVs and InDels) and gene fusions, account for the majority of interpretable and actionable genomic alterations. Importantly, this typically requires the analysis of DNA and RNA from limited amounts of FFPE-preserved specimens. Currently, these analyses typically require complex sample pre-processing for assay on separate platforms or separate complex library preparation methods for assessment by high throughput sequencing. To provide a unified and simpler alternative, NanoString’s molecular barcoding technology has been modularized to permit simultaneous digital measurement of cancerrelevant targets that span these two analyte classes. Novel ‘SNV’ probes enable sensitive and specific identification of DNA mutant allele sequences down to a level of detection of 5% from 5 ng of FFPE-extracted genomic DNA. Fusion transcripts are detected with 5’/3’ imbalance probes and toehold-mediated junction probes. This dual analyte workflow requires just a single 5-10 micron section of FFPE tissue and provides sample-to-answer results with approximately 5 minutes of hands-on time per sample after nucleic acid extraction. To demonstrate utility, 45 lung cancer samples were assayed simultaneously with an SNV panel that targets >100 solid tumor somatic mutations and a lung cancer fusion gene panel that provides general evidence of ALK, RET, and ROS1 gene fusion events along with specific detection of 35 unique fusion transcripts that correspond to known break-points. In this particular cohort, 19 samples were positive for activating KRAS SNVs (one of which was also positive for an activating STK11 variant), 3 were positive for activating EGFR mutations including two SNVs and an 18-base InDel, one was positive for an activating NRAS mutation and one was positive for an activating KIF5B16:RET12 fusion transcript. Positive mutation calls obtained with the SNV panel could only be confirmed by whole-exome sequencing (average depth of 100X) for 19 of 25 variants detected; however, ultradeep (average depth of 4400X) targeted sequencing revealed that the 5 additional panel-detected mutations were, in fact, present along with one apparent false positive call. With DNA from fresh frozen tissue and measured against the sequencing datasets, the SNV panel provided 100% sensitivity, specificity, accuracy and precision for all variants present at 5% or greater allele frequency. SNV detection calls were 95.7% concordant between tumor-matched FFPE and fresh frozen samples. The KIF5B_15:RET_12 fusion event was also confirmed by genomic DNA sequencing. Combined, these results show that these two important classes of activating mutations can be readily and efficiently assayed together on a NanoString nCounter® system (for research use only). FFPE gDNA: EGFR Reference Standard FFPE gDNA: KRAS Reference Standard 5% Sample ID 5% 5% Reporter tag Probe T Capture probe • DNA target fragment of interest • • • • Source Solid Tumor Fusion by Lung SNVs Gene Fusion Panel Diagnosis Detected assay NGT-ALK-006 PrecisionMed none NGT-ALK-008 PrecisionMed none B140001262 NWBioTrust none No specific ALK fusion transcript detected Interphase FISH Comments NSCLC, adeno ALK fusion positive POSITIVE for a missing signal of 5' ALK Positive by 3 methods ALK 3'/5' imbalance NSCLC, adeno ALK fusion positive POSITIVE for rearrangement of ALK at 2p23 Positive by 3 methods EML4_13:ALK_20 KIF5B_15:RET_12 NSCLC, adeno IHC results KIF5B_15:RET_12 transcript detected none none Sanger Seq. confirmed (see below) The Vantage 3D™ Lung Fusion Panel includes 63 probes: 24 for positional gene expression imbalance detection of ALK, RET, and ROS1 derived transcripts, 35 for specific fusion detection that also covers 2 NTRK1 variants. Fusion probe counts for two IHC- and FISH-verified ALK fusion positive FFPE samples obtained from PrecisionMed (Solana Beach, CA) and one unverified sample from NWBioTrust (Seattle, WA) are shown. The KIF5B_15:RET_12 fusion event was subsequently verified by Sanger sequencing of PCR-amplified genomic DNA (below). KIF5B_15:RET_12 fusion verified by Sanger sequencing Each SNV Probe is an oligonucleotidebased construct with: Probe S In order to estimate mutational burden, whole exome sequencing (WES, 111X average depth) was used to survey the mutations present in a cohort of lung cancer cases. Both fresh-frozen and FFPEpreserved tissue samples were available. Genomic DNA from fresh-frozen samples was used for WES library preparation and it was also used in the SNV panel assay for 42 of 45 tumor samples. EML4_13:ALK_20 transcript detected nCounter® Vantage 3D™ Probe Design for Specific SNV Detection • Imbalance probes detect presence of a fusion Specific fusion transcript detection 67% 5% Simultaneous, Sensitive Detection of SNVs & Fusion Transcripts via Vantage 3D™ Assays Verifies and Augments Sequencing Data Lung Cancer Gene Fusions detected from FFPE samples 5% Probe S with two ‘short’ arms basepairs contiguously with the DNA target strand Reporter tag uses existing NanoString optical fluorescent barcodes A single mismatch on one arm inhibits hybridization of SNV probe S Detection of an SNV results from counting the SNV assigned barcode A simple hybridization protocol precedes nCounter quantification Each assay use probes for the reference and the variant allele Simultaneous SNV and Fusion Detection on a Positive Control 5% SNV detection results Separately, 2 FFPE sections from each sample was used to isolate DNA and RNA using a Qiagen AllPrep DNA/RNA FFPE Kit. For these samples yields, determined by Qubit assay, ranged from 680-5900 ng (avg: 2300 ng) for gDNA and from 2.1-10.5 µg (avg: 5.3 µg) for total RNA per section. The combined 3D Biology™ SNV + Fusion detection assay workflow was used for all 45 FFPE tumors. Lung Tumor Gene B140001390 B140001383 B150001509 B140001157 B140000283 B150000039 B140000090 B140000885 B150001490 B140001139 B140000270 B150001359 B140000892 B140002445 KRAS KRAS EGFR KRAS KRAS EGFR KRAS KRAS KRAS KRAS KRAS KRAS KRAS KRAS B150001491 COSMIC ID of Base variant change(s) COSM516 COSM522 COSM6224 COSM522 COSM520 COSM6224 COSM521 COSM521 COSM520 COSM520 COSM554 COSM520 COSM521 COSM516 FreshFrozen Tumor SNV panel call Variant freq: Variant freq: FFPE Tumor Fusion Targeted NGS WES (111X SNV panel call Detected (4720X avg) avg) 34G>T 35G>C 2573T>G 35G>C 35G>T 2573T>G 35G>A 35G>A 35G>T 35G>T 183A>C 35G>T 35G>A 34G>T Detected TP Detected TP Detected TP Detected TP Detected TP Detected TP Detected TP Detected TP Detected TP Detected TP Detected TP Detected TP Detected TP Detected TP Detected TP Detected TP Detected TP Detected TP Detected TP Detected TP Detected TP Detected TP Detected TP Detected TP Detected TP Detected TP Detected TP Detected TP 51.2% 48.9% 48.7% 44.4% 43.1% 39.3% 38.3% 33.4% 32.8% 32.5% 31.1% 23.3% 20.8% 20.6% 53.2% 51.5% 47.9% 45.8% 38.7% 37.6% 36.0% 30.0% 21.4% 35.1% 34.9% 15.9% 19.5% 18.2% none none none none none none none none none none none none none none STK11 COSM1523962 465-2A>T Detected TP Detected TP 17.4% 9.8% none Detected TP Detected FP 9.7% Undetected Undetected Undetected none none Detected TP 15.7% 19.0% none KRAS CTNNB1 B140002436 EGFR B140001273 B140001176 B140001275 B140001269 B150001547 B140001377 B140001231 KRAS KRAS KRAS KRAS KRAS KRAS NRAS COSM555 COSM5662 183A>T Detected TP 110C>T Undetected TN 2240_2257 COSM12370 Detected TP del18 COSM520 35G>T Detected TP COSM521 35G>A Detected TP COSM516 34G>T Detected TP COSM527 37G>T Detected TP COSM516 34G>T Detected TP COSM516 34G>T Not Testable COSM573 38G>A Not Testable Detected TP Detected TP Detected TP Detected TP Detected TP Detected TP Detected TP None Not Testable Detected None None Detected Detected 11.2% 10.3% 9.9% 7.9% 5.8% 19.3% not tested Undetected none 5.3% none 11.5% none Undetected none Undetected none Undetected none 2.40% none None KIF5B:RET Detected None none Detected Samples that yielded positive SNV or fusion transcript not tested B140001262 none none NA detection calls with the nCounter assays are shown at 21 more not tested none none NA left. A subset of samples that tumors produced discordant mutational profiling data between the WES and nCounter® assays were used to prepare amplicon libraries for “Deep” targeted NGS sequencing. In all cases tested, the variants detected by the SNV assay from fresh/frozen tumors were verified to be ‘True Positives’ and all variants detected by WES were confirmed by the nCounter® Vantage 3D™ DNA SNV Solid Tumor Panel assay. Five of the 24 (~20%) verified mutations detected with the SNV panel failed to be detected by WES. SNV detection was >95% concordant between matched fresh/frozen and FFPE samples. One sample, B140001262, was verified to harbor a detected KIF5B:RET fusion (see above and sequencing trace at left). RNA and Protein Expression Profiling with Vantage 3D™ Assays 5% PanCancer Pathways RNA MUTEGFR MUTKRAS Vantage 3D™ Protein Solid Tumor PanCancer Immune RNA MUTEGFR MUTKRAS MUTEGFR MUTKRAS p=0.01 5% p=0.01 p=0.01 p=0.05 p=0.05 p=0.05 3D Biology™ Workflow: Simultaneous DNA SNV & Fusion Detection Extract and quantify nucleic acids Hybridize in parallel overnight (16 hr) Pool hybridizations & load nCounter cartridge Count on nCounter system Obtain detection calls Imbalance probes detect presence of multiple fusions 50% In addition to SNV and Fusion profiling with Vantage 3D™ Assays, the gene expression profiles of the 45 lung samples were determined using 770 RNA in the PanCancer Pathways Panel and 770 RNA in the PanCancer Immune Profiling Panel, plus 28 total and phosphoproteins in the Vantage 3D™ Protein Solid Tumor panel. This analysis is ongoing and initial results suggest that MUTEGFR tumors show a more clustered and distinct phenotype than MUTKRAS samples when compared to all other samples. Combined with the SNV and Fusion profiling, this 3D Biology™ approach allows for more comprehensive molecular profiling of these precious samples. SNV probe counts Input mass Panel 5 ng* DNA Fresh/ Frozen or nCounter DNA SNV Solid Tumor Panel Fusion probe counts RNA FFPE nCounter Lung Fusion 50-300 ng Panel Conclusions Specific fusion transcript detection 12 samples per cartridge * The SNV detection assay requires 18-20 cycles of pre-amplification by PCR. The Vantage 3D™ DNA SNV Solid Tumor Panel permits detection of multiple DNA mutations down to 5% minor allele frequency from 5ng of FFPE-extracted gDNA. These detection plots show the 104 variants assayed. Detection calls (colored boxes and text) are based on three criteria: mean log2(fold-change), above reference control samples ≥ 2 (red-dashed line), estimated p-value ≤ 0.01 (confidence interval = box width), and variant probes counts ≥ 200. For gDNA extracted from commercial FFPE control samples (HD300 and HD301) from Horizon Discovery, plc, every expected variant is detected. Allele frequencies are indicated as percentages. Results were generated with an nSolver™ Analysis Software 4.0 Advanced Analysis module (alpha version). • • • • • • As proof-of-principal for the 3D Biology™ workflow to simultaneously detect SNVs and fusion transcripts that are associated with lung cancer, DNA and RNA were extracted from a single 10 micron section of a commercially obtained SNV & Fusion positive FFPE-preserved sample (HD-784; Horizon Discovery). DNA and RNA were processed per the workflow at left on an nCounter® MAX system. Analysis of the nCounter digital count data indicates the presence of KRAS 38G>A and PIK3CA 3140A>G SNVs and ALK, RET, and ROS1 fusion transcripts. NanoString’s new SNV probe technology enables sensitive and specific detection of somatic (5% allele frequency) SNVs, MNVs, and InDels. The Vantage 3D™ DNA SNV Solid Tumor Panel assay is optimized for very small amounts (5 ng) of FFPE gDNA. The Vantage 3D™ SNV assay workflow is simple and requires minimal hands-on time. 3D Biology™ Enabled: Simultaneous detection of DNA and RNA variants and of RNA and protein expression profiling can be performed on nCounter® systems. The 3D Biology™ workflow is especially useful for augmenting and verifying somatic cancer-driver mutations profiled by discovery datasets. For more information, please visit 3d.nanostring.com Acknowledgements Data and results cited in this study were funded, in part, as follows: Fred Hutchinson Cancer Research Center, STTR Oncoscape Award, NIH/NCI. JK received support through grant EU-FP7-PEOPLE-2012-IOF 331255 FOR RESEARCH USE ONLY. Not for use in diagnostic procedures. www.nanostring.com | [email protected] | @nanostringtech © 2017 NanoString Technologies, Inc. All rights reserved. Patents pending. NanoString, NanoString Technologies, the NanoString logo, nCounter, nSolver, 3D Biology, and Vantage 3D are registered trademarks or trademarks of NanoString Technologies, Inc., in the United States and/or other countries. NanoString Technologies 530 Fairview Avenue North, Seattle, WA 98109 March 2017