Oncology Insights Enabled by Knowledge Base-Guided Panel Design and the Seamless Workflow of the GeneReader NGS System

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1 White Paper Oncology Insights Enabled by Knowledge Base-Guided Panel Design and the Seamless Workflow of the GeneReader NGS System Abstract: This paper describes QIAGEN s philosophy and process for developing integrated NGS assays tailored to the needs of the clinical research laboratory. A case study for this process is the QIAGEN Actionable Insights Tumor Panel. The panel is optimized to provide sequencing insights relevant for clinical research, and to be run on the GeneReader NGS System as part of a completely integrated workflow. As such, QIAGEN has reduced the complexity of NGS production such that small and medium-sized labs are now empowered to take full advantage of NGS for their routine use. QIAGEN is uniquely positioned to produce these solutions by leveraging its core strengths in sample preparation and post-sequencing analytics and now, sequencing. Introduction Next-generation sequencing (NGS) has revolutionized the speed of genetic and genomic discovery, and advanced our understanding of important insights into molecular mechanisms of diseases. However, three major hurdles remain and still prevent NGS from being broadly adopted in the community especially for labs new to this technology: 1. lack of standardized defined, relevant and meaningful content; 2. lack of a streamlined and integrated workflow; 3. lack of expertise required for processing complex bioinformatics data and interpreting the results. As such, NGS largely remains only accessible to a few academic centers with sophisticated infrastructure and plentiful resources. QIAGEN has developed the GeneReader NGS System, the first complete Sample to Insight NGS solution designed for any research lab to easily and reliably deliver targeted nextgeneration sequencing results. to the needs of the clinical research laboratory. QIAGEN s bioinformatics platform combines the largest collection of expert-curated scientific literature, including drug label data, leading professional guidelines and late-stage clinical trials. This platform is unified with the GeneReader NGS workflow to distill sequencing data into customizable reports that are easy to create and understand. QIAGEN s GeneReader NGS System is the promise of NGS fulfilled. Contents Introduction 1 Purpose-built panel 2 Connected workflow 4 Delivering insights 5 Summary 6 Using the QIAGEN Actionable Insights Tumour Panel as a case study, this paper presents QIAGEN s process and philosophy for developing integrated NGS assays tailored

2 Purpose-built panel, integrating design, analysis and interpretation The QIAGEN Knowledge Base is an industry-leading database that includes relevant genotypic and phenotypic data for clinical research curated from scientific literature, approved drug labels, professional association practice guidelines and active clinical trials. QIAGEN s team of highly qualified Ph.D. and MD staff are dedicated to continuously conducting exhaustive curation of new primary literature and review of clinical cases to ensure the database contains the most up-to-date information. The QIAGEN Knowledge Base is used to define QIAGEN s GeneReader NGS targeted panel content ensuring the most appropriate content is included in each panel. QIAGEN Clinical Insight (QCI ) is a comprehensive, scalable tool through which laboratories can access information contained in the QIAGEN Knowledge Base to analyze, interpret and report on genomic variants identified using NGS. For a given case it is possible to retrieve a specific list of genomic loci from the Knowledge Base with associated known allelic frequencies, thus creating a necessary and sufficient set of variants for targeted genetic interrogation. The same QIAGEN Knowledge Base is used in both panel design and result interpretation, thus linking sample processing to insight generation, and integrating the overall NGS workflow. These seamless tools allow a lab to now enrich for, sequence, analyze and interpret the same set of data with ease and confidence. As a first example of this powerful pipeline and design philosophy, we created the GeneRead Actionable Insights Tumor Panel using this comprehensive and continuously updated database (Figure 1, list of genes). The resulting design is a unique set of variants with an unparalleled level of direct relevance to human disease, giving you confidence in obtaining meaningful results. KRAS, NRAS, KIT, BRAF, PDGFRA, ALK, EGFR, ERBB2, PIK3CA, ERBB3, ESR1, RAF1 This panel content has also been validated extensively with well-recognized US and European experts who have been conducting cancer testing using NGS who universally agreed with our design philosophy and approach. When compared to the Illumina TruSight Tumor 26 or Thermo Fisher Cancer HotSpot panels, the QIAGEN panel contains variants that are unique to our design (Figure 2). Genes Number of variants unique to QIAGEN Actionable Insights Tumor Panel Not covered by Thermo AmpliSeq HotSpot Panel V2 Not covered by Illumina TruSight Tumor 26 Panel Figure 2. List of genes in the Actionable Insights Tumor Panel. Not covered by either EGFR PI3KCA KIT ALK KRAS 12 BRAF ERBB PDGFRA Total One example of a variant unique to QIAGEN s panel design is ERBB2 (Her2) S301F, which has been identified in 1-2% of breast and lung cancers, as well as in ovarian and bladder cancers (1, 2). This mutation has been shown to cause constitutive activation of ERBB2 (2, 3). The QIAGEN Clinical Insight (QCI) software clearly identifies this variant, and provides linkage to critical insights that can be used to guide clinical research on this gene and research sample (Figure 3). In addition to tumor somatic testing, this design philosophy can also be applied to other indications and applications, including but not limited to hematological or inherited cancers and infectious diseases. The same process is expected to yield equally relevant designs, with the power to deliver meaningful insights. Figure 1. List of genes in the Actionable Insights Tumor Panel. 2

3 A streamlined report for each sample 3 Gain of function mutation 2 Somatic mutation not seen in general population allele frequency in sample identified Figure 3. QCI provides clear insights into this variant in the ERBB2 gene. Oncology Insights Enabled by Knowledge Base-Guided Panel Design and the Seamless Workflow of the GeneReader NGS System 3

4 The world s first fully connected workflow The existing NGS platforms suffer from fragmented workflows. Labs using these technologies have been left to identify and validate upstream sample preparation and downstream bioinformatics solutions. As such, there is an urgent need for a simpler, more cost-effective and efficient workflow for all labs including current NGS users and labs new to NGS in order to take full advantage of NGS technology. The GeneReader NGS System comprises an end-to-end solution for NGS Sample to Insight with all components designed and validated to be the world s first fully unified workflow optimized to deliver NGS insights, not sequences. To enable this, the GeneReader workflow starts with the proprietary GeneRead FFPE DNA Kit designed specifically to reduce artifacts known to occur in FFPE samples. As seen in Figure 5, when using old (3 20 years) FFPE samples, this kit successfully corrects more than 90% of all the C T artifacts as caused by cytosine deamination. The high-quality nucleic acid is then used to prepare the sequencing library with the uniquely selected panel content, optimized for the QIAGEN GeneReader workflow. Library preparation has been optimized and automated on the QIAcube platform, which has been shown to save up to 75% of hands-on time when compared to traditional manual methods. Clonal amplification is also optimized and automated on the newly designed GeneRead QIAcube. The NGS sequencing RAS agreement study therascreen PCR and Pyro Assays * >5% Ras variant allele frequency cut-off + (MT) (WT) Total GeneReader NGS System + (MT) (WT) Total % agreement between GeneReader NGS System and QIAGEN therascreen RGQ PCR & Pyro Assays * If KRAS is mutant by therascreen KRAS RGQ PCR Assay or by therascreen RAS Extension Pyro Assay, the condition is regarded as mutant (MT). For Actionable Insights Tumor Panel, a 5% allelic frequency cut-off was used to call variants for codons 12, 13, 59, 61, 117, 146 which are addressed by established QIAGEN therascreen assays. Figure 5. Actionable Insights Tumor Panel RAS Agreement Study. reaction is then performed on the GeneReader platform, which includes a revolutionary turntable design, enabling up to 4 flow cell runs in parallel or staggered fashion. This configuration allows for unparalleled batching flexibility and cost efficiency, and enables a lab to scale their operations freely. In summary, QIAGEN s completely integrated NGS workflow delivers high levels of standardization and automation from Sample to Insight. Patchwork processes are replaced by a single solution, giving users the efficiency of a unified workflow and the confidence of analytically validated results. QIAGEN s renowned reputation to quality extends as your trusted partner to deliver the sequencing insights you require. Standard sample prep Number of of RAS variants detected at % allele frequency cut-off >5% >2.5% >1% MiSeq Not Testable GeneReader GeneRead DNA FFPE sample prep + GeneReader Formalin is known to cause cytosine deaminiation and introduce C T artifacts 90% of false positives detected with standard sample prep were C T changes. The GeneRead DNA FFPE Kit effectively removes formalin-associated false positive artifacts Figure 4. The GeneRead FFPE DNA sample preparation kit successfully corrects FFPE artifacts. 4

5 Delivering insights that matter most to you With a focus on delivering insights, we investigated the ability of the QIAGEN GeneReader NGS System to interrogate a set of FFPE colorectal tumor samples in a laboratory setting. GeneReader NGS System results were compared to alternative technologies including therascreen real-time PCR assays and Illumina platforms. Data from the GeneReader NGS System is 100% concordant with the results of the therascreen (PCR) and Pyrosequencing RUO assays (Figure 6), while data from the GeneReader NGS system is concordant with results obtained on an Illumina MiSeq sequencer (Figure 7). For read alignment and variant calling, we used QCI Analyze. This provides visualization of the sequencing results as well as a summary of the data. Quality assessment is also supported, both at the overall sequencing run level and for the analytic validity of individual variants to reduce false positive and negative results. With QCI Analyze, you can easily judge the quality of the results, and assess any variants of interest in detail. Moreover, QCI Analyze is fully unified with the GeneReader NGS System from defining the components of your workflow to reviewing the analytic validity of your variants and basic reporting. This unification makes secondary analysis a streamlined and easy process for the users while a streamlined interface to QCI Interpret allows effortless downstream interpretation and reporting. Sample no. KRAS AA change RAS Variant Allele Frequency (%) therascreen PCR/ Pyro GeneReader + MiSeq 1 G12D G12D A59T G13D G12D Q61H A146P Q61H Q61H K117N G13D G12C G13D Q61H % positive agreement between GeneReader and MiSeq +: Mutant identified by therascreen RGQ; allele frequency not available. : Sample processed from different FFPE sections with potentially different tumor content and variant allele frequency. Figure 6. The GeneReader NGS System shows concordance with MiSeq. Note: All results were confirmed by Sanger sequencing and passed QIAGEN s sample quality step. QCI Interpret can be used to populate a customizable report for each test based on detected variants and curated content, with a summary of findings and direct links to data sources. With all relevant information right at the user s fingertips, reports can be created in a few mouse clicks with the highest confidence and efficiency. The QIAGEN GeneReader NGS System is the first truly integrated solution that is focused on delivering insights with maximal efficiency and minimal adoption hurdle. This system is very well suited for labs new to the NGS technology. Scott Steelman, Ph.D., Associate Director of Technology Labs, Broad Institute. Oncology Insights Enabled by Knowledge Base-Guided Panel Design and the Seamless Workflow of the GeneReader NGS System 5

6 KRAS, NRAS, KIT, BRAF, PDGFRA, ALK, EGFR, ERBB2, PIK3CA, ERBB3, ESR1, RAF1 Figure 7. QCI Analyze for secondary analysis. Summary The GeneReader NGS System is the first complete Sample to Insight NGS solution designed for any clinical research lab to deliver actionable sequencing results. Now all labs, regardless of experience, can join the NGS revolution and unlock the power of this technology with the Sample to Insight solution from QIAGEN. For the first time, one partner is able to provide a seamlessly integrated workflow offering ease of use and efficiency for NGS. With scalable batch sizes and continuous loading of multiple flow cells, the GeneReader NGS system is able to adapt and scale to match every lab s individual needs. The optimized and automated GeneReader NGS System removes hurdles and delivers insights in an efficient, cost-effective workflow; putting NGS within reach for everyone. Welcome to the GeneReader NGS System. Together we will deliver the promise of NGS, giving you the actionable insights you need and creating value for your lab. 6

7 References 1. Cancer Genome Atlas Research Network (2014) Comprehensive molecular profiling of lung adenocarcinoma. Nature 511, Chumsri, S. et al. (2015) Prolonged response to Trastuzumab in a patient with HER2-nonamplified breast cancer with elevated HER2 dimerization harboring an ERBB2 S310F mutation. J. Natl. Compr. Canc. Netw. 13, Herter-Sprie, G.S., Greulich, H., and Wong, K. K. (2013) Activating mutations in ERBB2 and their impact on diagnostics and treatment. Front. Oncol. 3, NCCN Guideline: assessed October Oncology Insights Enabled by Knowledge Base-Guided Panel Design and the Seamless Workflow of the GeneReader NGS System 7

8 For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at or can be requested from QIAGEN Technical Services or your local distributor. For research use only. Not for use in diagnostic procedures. Trademarks: QIAGEN, QIAcube, GeneRead, Pyrosequencing, Sample to Insight, QCI, therascreen (QIAGEN Group); GeneReader (Intelligent Bio-Systems, Inc.); Illumina, MiSeq (Illumina, Inc.) / QIAGEN, all rights reserved. PROM Ordering Technical Support support.qiagen.com Website