ncounter Leukemia Fusion Gene Expression Assay Molecules That Count Product Highlights ncounter Leukemia Fusion Gene Expression Assay Overview

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1 ncounter Leukemia Fusion Gene Expression Assay Product Highlights Simultaneous detection and quantification of 25 fusion gene isoforms and 23 additional mrnas related to leukemia Compatible with a variety of specimen types, including FFPE, peripheral blood, bone marrow, and cell lines 15 minutes hands-on time to analyze 12 samples and no specialized skills required Identify specific fusion gene isoforms for a fraction of the cost of FISH or PCR ncounter Leukemia Fusion Gene Expression Assay Overview The ncounter Leukemia Fusion Gene Expression Assay Kit allows researchers to profile a comprehensive set of fusion genes which result from balanced translocations in different leukemia sub-types. In addition to leukemia fusion genes, the kit includes probes for 11 wild-type genes involved in the different translocations, 12 leukemia-related biomarkers, and 2 normalization genes. The 50-plex assay-kit facilitates cutting-edge research by leveraging the advantages of the ncounter analysis system to provide robust analysis of a variety of specimen types, including FFPE, with less hands-on time than any other method available. The ncounter Leukemia Fusion Gene Expression Assay Kit is offered for research use only (RUO) and is ideal for characterization of retrospectively or prospectively collected specimens, cell lines, and other sample types where a comprehensive profile of fusion gene isoforms and leukemia biomarkers may lead to important biological insights. Unique to the Leukemia Fusion Gene Expression Assay is the Junction Probe design which enables detection of fusion gene isoforms with high specificity. NanoString s standard probe design using border probes for the detection of fusion genes can only distinguish between variants with different downstream exons. In order to differentiate fusion genes with the same downstream exon, the leukemia fusion gene assay utilizes Junction Probes which target the unique sequence spanning the fusion junction of the two exons. In Figure 1, the design of the Junction Probes is illustrated on the major and minor BCR-ABL fusion transcripts (Capture Probes and Reporter Probes are denoted CP and RP respectively). NanoString has optimized the probe design to achieve high sensitivity and specificity by sequence modification. Molecules That Count Translational Research Gene Expression mirna Expression Copy Number Variation 1

2 CP-e1 e1 CP-b2 b2 CP-b3 b3 BCR ABL RP-e1 RP-b2 RP-b3 Figure 1: Leukemia Junction Probes target the unique sequence spanning the fusion junction. ncounter Leukemia Fusion Gene Expression Assay Kit Performance Each ncounter Leukemia Fusion Gene Assay generates 50 data points per sample which characterize 25 fusion genes, 11 potential translocation partner genes, 12 leukemia-related biomarkers, and 2 normalization genes. Figure 2 shows a complete data set for a single sample in which the assay identified the correct fusion gene isoform and also generated additional information on the expression levels of potential fusion partners and biomarkers. To demonstrate the reproducibility of data generated via the ncounter Leukemia Fusion Gene Assay, we processed total RNA isolated from bone marrow derived from a leukemia patient prior to treatment. 500ng of total RNA was used per (technical) replicate. Raw data was normalized to internal positive spike controls present in each reaction to account for minor differences in hybridization and purification efficiencies. Counts for individual probes in technical replicates within a cartridge were highly correlated between sample preparations (R 2 > 0.99, Figure 3). Fusion Genes Translocation Partner Genes Biomarker Genes TEL-AML1 (e5e3) TEL-AML1 (e5e2) PML-RARA (bcr2) PML-RARA (bcr3) MLL-AF4 (e11e4) MLL-AF4 (e11e5) MLL-AF4 (e10e4) MLL-AF4 (e10e5) MLL-AF4 (e9e4) MLL-AF4 (e9e5) MLL-AF4 (e8e7) E2A-PBX1 (e13e2) E2A-PBX1(e13-ins... CBFß-MYH11 (e5e7) CBFß-MYH11 (e5e8) CBFß-MYH11 (e5e12) AML-ETO BCR-ABL (e1a3) BCR-ABL (b2a3) BCR-ABL (b3a3) BCR-ABL (e19) BCR-ABL (e1) BCR-ABL (b3) BCR-ABL (b2) BCR ABL1 AFF1 (AF4) MLL MYH11 CBFß PBX1 E2A (TCF3) ETV6 (TEL) RARA PML TP53 RB1 NRAS EVI1 MN1 ERG MLLT11 PRAME BAALC FLT3 WT1 SOCS2 TBP GUSB Counts Figure 2: A complete ncounter Leukemia Fusion Gene Profile shows the sample positive for BCR-ABL b2 and provides additional information on gene expression for other biomarkers and reference genes. Figure 3: Counts for mrna transcripts contained in the leukemia fusion gene assay in total RNA from leukemic bone marrow were highly correlated between technical replicates. As shown in Figure 1, the Junction Probe design allows for detection of fusion gene isoforms in leukemia sub-types with high specificity. To demonstrate the specificity of the Junction Probe design, the multiplexed probeset was hybridized to individual synthetic RNA targets and then analyzed. Each probe was evaluated based on the percentage of off-target counts that it generated. Table 1 shows that all BCR-ABL isoform probes exhibited less than 1 percent 2

3 NanoString Technologies Product Data Sheet cross-hybridization, indicating that the Junction Probe design for the ncounter Leukemia Fusion Gene Expression Assay can accurately distinguish between isoforms within a fusion gene family with great specificity. Similar experiments were performed to verify less than 1 percent cross-hybridization between isoforms for all other fusion gene families in the panel. The ncounter Leukemia Fusion Gene Assay gives consistently reliable results on RNA derived from a variety of sources. Table 2 shows data generated via the ncounter Fusion Gene Expression Assay for different fusion gene isoforms from total RNA isolated from a variety of sample types. Counts shown are normalized to positive controls included in the assay and typical background levels in these assays are ~20 counts. Each fusion gene shown in the table was correctly called by the ncounter Leukemia Fusion Gene Expression Assay. Biological variation in expression levels of fusion genes is evidenced by the wide range of counts seen for different fusion variants in samples derived from different patients. All patient samples are from untreated individuals. The performance and workflow advantages offered by the ncounter Leukemia Fusion Gene Expression Assay Kit make it an ideal tool for advancing research in the area of leukemia fusion gene variants. Visit our website to learn more ( Table 1: Low cross-hybridization between fusion genes of the BCR-ABL family demonstrates the high specificity of the junction probe design for the detection of specific isoforms. BCR-ABL Targets ncounter Probes e1 e1a3 b3 b2 b3a3 b2a3 e19 e1 100% % e1a3-100% b3-0.1% 100% 0.4% b2 0.1% % b3a % - - b2a % 100% - e % Table 2: Counts generated for different fusion gene isoforms via the ncounter Leukemia Fusion Gene Expression Assay in 500 ng of total RNA from various sample types (typical background is ~20 counts). ncounter Analysis System Sample Type Fusion Gene Isoform Counts The ncounter Analysis System from NanoString offers a simple, cost-effective way to profile hundreds of mrnas, micrornas, or CNVs simultaneously with high sensitivity and precision. The digital detection of target molecules and high levels of multiplexing eliminate the compromise between data quality and data quantity, bringing better sensitivity, reproducibility, and linearity to your results. It is ideal for studying defined gene sets across a large sample set, e.g., gene expression signature validation, pathway analysis, CNV validation studies, and mirna transcriptome analysis. The system utilizes a novel digital technology that is based on direct multiplexed measurement of gene expression and offers high levels of precision and sensitivity. The technology uses molecular barcodes and single molecule imaging to detect and count hundreds of unique transcripts in a single reaction. Unlike other methods, the protocol does not include any amplification steps that might introduce bias to the results. After hybridization with the Leukemia Fusion Gene Assay CodeSet, samples are transferred to the ncounter Prep Station where excess probes are removed and probe / target complexes are aligned and immobilized in the ncounter Cartridge. Cartridges are then placed on the ncounter Digital Analyzer for data collection. Each mrna transcript of interest is identified by the barcode generated by six ordered fluorescent spots present on the Reporter Probe. The Reporter Probes on the surface of the cartridge are then counted and tabulated for each target. FFPE Bone Marrow Fresh/Frozen Bone Marrow Peripheral Blood Cell Lines BCR-ABL b2 391 BCR-ABL e PML-RARA bcr3 145 AML-ETO e5e BCR-ABL b BCR-ABL b2 812 BCR-ABL b3a3 988 BCR-ABL e BCR-ABL e1a CBFb-MYH11 e5e7 94 E2A-PBX1 e13e PML-RARA bcr2 535 PML-RARA bcr1 502 PML-RARA bcr TEL-AML1 e5e2 116 AML-ETO e5e BCR-ABL b BCR-ABL b AML-ETO e5e BCR-ABL b3 328 BCR-ABL b BCR-ABL e1 370 BCR-ABL b E2A-PBX1 e13e MLL-AF4 e9e PML-RARA bcr1 61 TEL-AML1 e5e2 237 Molecules That Count 3

4 ncounter Leukemia Fusion Gene Assay Target List Fusion Genes Disease Translocation Fusion Gene Family Target CML ALL AML AML / APL t(9;22) BCR-ABL b2 b3 b3a3 b2a3 e1a3 e19 t(9;22) BCR-ABL e1 t(4;11) MLL-AF4 e8e7 e9e5 e9e4 e10e5 e10e4 e11e5 e11e4 t(1;19) E2A-PBX1 e13-insertion-n27-e2 e13e2 t(12;21) TEL-AML1 e5e2 e5e3 t(8;21) AML-ETO e5e12 Inv(16) CBFß-MYH11 e5e12 (Type A) e5e8 (Type D) e5e7 (Type E) t(15;17) PML-RARA bcr1 bcr3 bcr2 Translocation Partners Biomarkers Fusion Gene Family Target Disease Target BCR-ABL BCR ABL1 SOCS2 WT1 MLL-AF4 MLL AF4 (AFF1) FLT3 BAALC E2A-PBX1 TEL-AML1 E2A (TCF3) PBX1 TEL (ETV6) AML, CML, ALL PRAME MLLT11 ERG CBFß-MYH11 CBFß MYH11 MN1 EVI1 PML-RARA PML RARA NRAS RB1 TP53 Normalization Genes GUSB TBP 4

5 Description Number of targets Recommended amount of starting material Sample types supported Sample volume Hybridization reaction volume Limit of detection Specifications 25 Leukemia Fusion Genes 11 Translocation Partners 12 Leukemia - Biomarkers 500ng purified total RNA RNA from a variety of sources including Bone Marrow, Peripheral Blood, FFPE and Cell Lines* 5 μl 30 μl 0.5 fm Spike titration linearity R Linear dynamic range ncounter Prep Station throughput ncounter Digital Analyzer throughput Controls 7 x 10 5 total counts 12 samples < 2.5 hours 12 samples / 4 hours (up to 72 samples per day unattended running in continuous mode) 6 positive assay controls 8 negative assay controls 2 mrna housekeeping controls *Human samples should be derived from untreated individuals. Post-treatment individuals will likely have expression levels below the limit of detection. Description Quantity / Use Part Number (P/N) ncounter Leukemia Fusion Gene Expression Kit 12 assays 24 assays 48 assays 96 assays FUS-LEU1-12 FUS-LEU1-24 FUS-LEU1-48 FUS-LEU1-96 ncounter Analysis System (includes the Prep Station and Digital Analyzer) 1 NCT-SYS-120 Additional ncounter Prep Station 1 NCT-PREP-120 Additional ncounter Digital Analyzer 1 NCT-DIGA-120 NanoString Technologies, Inc. Contact Us Sales Contacts 530 Fairview Ave N Suite 2000 Seattle, Washington United States: Tel: (888) Europe: Fax: (206) Japan: Other Regions: 2012 NanoString Technologies, Inc. All rights reserved. NanoString, NanoString Technologies, ncounter, Molecules That Count, nsolver, Plex2, ChIP-String and mirge are registered trademarks or trademarks of NanoString Technologies, Inc., ( NanoString ) in the United States and/or other countries. All other trademarks and or service marks not owned by NanoString that appear in this document are the property of their respective owners. The manufacture, use and or sale of NanoString product(s) may be subject to one or more patents or pending patent applications owned by NanoString or licensed to NanoString from Life Technologies Corporation and other third parties. For research use only. Not for use in diagnostic procedures. v

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