Targeted NGS for Precision Cancer Care
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This slidedeck details two comprehensive informatics solutions — the Biomedical Genomics Workbench and Ingenuity Knowledge Base Variant Analysis platforms. We show the intuitive user interface of CLC Cancer Research Workbench and demonstrate how the rich biological content from Ingenuity Knowledge Base helps you rapidly identify critical variants in your samples.
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Targeted NGS for Precision Cancer Care
- 1. Sample to Insight Mutational analysis using QIAGEN’s GeneRead panels and Sample-to-Insight NGS solutions Raed Samara, PhD 1 Part 3 Data Analysis Interpretation
- 2. Sample to Insight Legal disclaimer NGS part 3: Targeted NGS for Cancer Research 2 • QIAGEN products shown here are intended for molecular biology applications. These products are not intended for the diagnosis, prevention or treatment of a disease. • 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 www.QIAGEN.com or can be requested from QIAGEN Technical Services or your local distributor.
- 3. Sample to Insight Precision medicine: right drug, right patient, right time and dose ‘One size fits all’ does not work NGS part 3: Targeted NGS for Cancer Research 3
- 4. Sample to Insight Mutations AGCTCGTTGCTCAGCTC Reference genome AGCTCGTTGCTCAGCGTTC Insertion AGCTC---GCTCAGCTC Deletion Indels Copy number variations T G CA T G A C DNA variants for precision medicine RS NGS part 3: Targeted NGS for Cancer Research 4
- 5. Sample to Insight Mutations AGCTCGTTGCTCAGCTC Reference genome AGCTCGTTGCTCAGCGTTC Insertion AGCTC---GCTCAGCTC Deletion Indels Copy number variations Actionable DNA Variant BRAF V600E EGFR E746-750 + Kinase domain mutation HER2 Disease Melanoma Lung adenocarcinomas IDC-Breast cancer Therapy Vemurafenib (PLX4032) Erlotinib/ Gefitinib Trastuzumab Actionable DNA variants for precision medicine Only a handful of mutations are actionable RS NGS part 3: Targeted NGS for Cancer Research 5
- 6. Sample to Insight Actionable DNA variants for precision medicine How many? RS NGS part 3: Targeted NGS for Cancer Research 6
- 7. Sample to Insight • How many mutations to test for? • How to test for these mutations? o Sequential testing o Parallel testing EGFR (L858R) KRAS (G12C) + Response rates of >70% in patients with non-small cell lung cancer treated with either erlotinib or gefitinib Poor response rate in patients with non-small cell lung cancer treated with either erlotinib or gefitinib Adapted from Govindan et al., Cell 150 (2012) Precision medicine for lung cancer Current lung cancer biomarker landscape KRAS 25% EGFR 23% EML4-ALK 6% BRAF 3%PIKC3A 3% MET 2% ERBB2 1% MAP2K1 0.4% NRAS 0.2% Unknown 37% RS NGS part 3: Targeted NGS for Cancer Research 7
- 8. Sample to Insight Actionable DNA variants for precision medicine Targeted DNA sequencing delivers accurate information required for precision medicine Attribute/ Parameter Information level Cost per sample Coverage achieved DNA input No. of samples multiplexed Whole Genome Sequencing 3 x 109 bps $5000 30x 1 µg 1^ Whole Exome Sequencing 5 x 107 bps $2000 100x 100 – 200 ng 2* Targeted DNA Sequencing 6 x 104 bps# $200 1000x 10 ng 96* Benefits of Targeted DNA Sequencing: Clinical utility requires targeted analysis More relevant data More cost effective Detect low-frequency mutations Lower DNA requirements Higher multiplexing capabilities RS NGS part 3: Targeted NGS for Cancer Research 8
- 9. Sample to Insight Why choose targeted DNA sequencing? • Well-defined content • Small target size • More reads per sample • Examine variants that matter • Multiplex many samples to save money • Detect low frequency variants Features Benefits Targeted DNA sequencing limits the genes or targets to be sequenced RS NGS part 3: Targeted NGS for Cancer Research 9
- 10. Sample to Insight Targeted DNA sequencing (TDS) Shrink the genome Sample Insight The principle of targeted enrichment is to sequence millions of small DNA fragments that represent the region of interest, simultaneously gDNA Variants Report KRAS G12D EGFR T790M IDH1 R132H KRAS EGFR IDH1 RS NGS part 3: Targeted NGS for Cancer Research 10 Sample isolation Targeted enrichment Library construction NGS run Data analysis Interpretation
- 11. Sample to Insight Sample-to-Insight: Integrated universal targeted NGS workflow NGS part 3: Targeted NGS for Cancer Research 11 To overcome NGS challenges Sample Insight Turnaround time, and limited amounts of DNA Library yield Coming soon Data processing & Variant calling Isolation of high- quality DNA samples Quantifying amplifiable (not total) amounts of DNA Clinical & Biological interpretation of data RS Sample isolation Targeted enrichment Library construction NGS run Data analysis Interpretation Sample QC Library QC Variant confirmation
- 12. Sample to Insight Why choose multiplex PCR-based targeted enrichment? • Offers specificity that beats capture-based approaches • Offers uniformity that beats capture-based approaches • Use sequencing capacity on regions targeted by the panel, with minimal off- target sequencing • Achieve more uniform enrichment for more sequencing efficiency Features Benefits It delivers unmatched specificity and uniformity (compared to capture-based methods) RS NGS part 3: Targeted NGS for Cancer Research 12
- 13. Sample to Insight GeneRead DNAseq Panels V2 Multiplex PCR-based enrichment of gene(s) or genomic region(s) Builds on our 10-year experience in designing PCR assays GeneRead DNAseq Panel V2 GeneRead DNAseq PCR Kit V2 Collection of primer pairs dispended into primer pools/tubes. Primers correspond to the targeted region(s) (Gene 1 and Gene 2, as example) Primer pair; amplicon RS NGS part 3: Targeted NGS for Cancer Research 13
- 14. Sample to Insight Gene 1 Gene 2 Primer design algorithm GeneRead DNAseq Panels V2 Multiplex PCR-based enrichment of gene(s) or genomic region(s) PCR Chemistry Primer separation algorithm GeneRead DNAseq Panel V2 (sets of primer pools/tubes) GeneRead DNAseq PCR Kit V2 RS NGS part 3: Targeted NGS for Cancer Research 14
- 15. Sample to Insight GeneRead DNAseq Panels V2 Product description Sample Insight ACCAGTGAC TATAGCTAG GTCCTATTG CCGGTGTAC Variants Report Ref: AAGTCT Case: AACTCT GeneRead DNAseq Panel V2 GeneRead DNAseq PCR Kit V2 DNA Amplicons Library Reads GeneRead DNAseq panels enrich genomic targets by generating amplicons (small ds DNA fragments) through PCR RS NGS part 3: Targeted NGS for Cancer Research 15 Sample isolation Targeted enrichment Library construction NGS run Data analysis Interpretation
- 16. Sample to Insight Clinically- and biologically-focused panels Cataloged panels; bench-tested Type Panel name Solid Tumor Clinically Relevant Tumor Tumor Actionable Mutations Hematologic malignancies Myeloid Neoplasms Tissue Specific Breast Cancer Colorectal Cancer Liver Cancer Lung Cancer Ovarian Cancer Prostate Cancer Gastric Cancer Cardiomyopathy Gene(s)-specific BRCA1 and BRCA2 Comprehensive Cancer Predisposition Comprehensive Cancer Carrier testing Cancer Genome Census ClinVar Largest collection of pre-designed panels suitable for a wide range of needs RS NGS part 3: Targeted NGS for Cancer Research 16
- 17. Sample to Insight Application-specific amplicon design Builds on >10 years in assay development G12 Deep (redundant) tiling amplicon design Dense overlap amplicon design Sparse overlap amplicon design Primers Amplicons RS NGS part 3: Targeted NGS for Cancer Research 17
- 18. Sample to Insight Customized panels Custom and Mix-n-Match 1 No competitor offers bench-tested gene designs 2 Fastest on the market What is the list of your targets? RS NGS part 3: Targeted NGS for Cancer Research 18 GeneRead Mix-n-Match GeneRead Custom • Access to 570 bench-tested gene designs1 • Fixed specifications • Turnaround time = 3 days2 • Bioinformatically target any gene(s) or genomic region(s) within the human genome • Flexible specifications • Turnaround time = 14 days2
- 19. Sample to Insight The power of Mix-n-Match pool of validated genes Consistent performance 0.0% 10.0% 20.0% 30.0% 40.0% 50.0% 60.0% 70.0% 80.0% 90.0% 100.0% Experimental Coverage Uniformity MET NRAS TP53PDGFRA PIK3CA PTEN RB1 RET RS NGS part 3: Targeted NGS for Cancer Research 19
- 20. Sample to Insight Why choose GeneRead panels workflow for targeted enrichment? • Small target sizes • Uses Multiplex PCR • Achieve a high degree of coverage depth • Low DNA input material • Quick turnaround time • No physical removal of primer sequences • Platform-independence • Pool amplicons before library construction • Integrated workflow • Multiplex many samples to save money • Achieve higher specificity and uniformity than other approaches1 • Make more confident calls to detect low frequency variants 4 • Sequence low-yielding samples such as FFPE and cfDNA samples • Go from sample to insight quickly • Reduce false positives and negatives2 • Achieve 100% specificity and 100% sensitivity3 • Use one panel for your different sequencers • Generate one library per sample to save money • Sequence low-quality samples Features Benefits GeneRead DNAseq panels address many NGS challenges 1. Mamanova et al. 2010. Nature Methods, 7(2):111 2. Satya and DiCarlo. 2014. BMC Genomics, 15:1073 3. Heydt et al. 2015. BMC Cancer, 15:291 4. Helen Fernandes, AMP 2014 RS NGS part 3: Targeted NGS for Cancer Research 20
- 21. Sample to Insight Why choose GeneRead panels workflow for targeted enrichment? Achieve better variant calling accuracy to reduce false negatives RS NGS part 3: Targeted NGS for Cancer Research 21
- 22. Sample to Insight Why choose GeneRead panels workflow for targeted enrichment? Achieve 100% specificity and 100% sensitivity through better variant calling accuracy The sensitivity of the AmpliSeq panel on the MiSeq™ (Illumina) was only 93%. Using this panel, four of the exon 11 mutations (p.M552_K558del, p.M552_V559del, K550_K558del, c.1648-5_1672del) could not be detected as these mutations were at the amplicon boundaries and primer binding sites. RS NGS part 3: Targeted NGS for Cancer Research 22
- 23. Sample to Insight Why choose GeneRead panels workflow for targeted enrichment? Integrated workflow to optimally-sequence low-quality samples The GeneRead workflow enables the optimal sequencing of low-yielding and low quality FFPE samples Data presented by Helen Fernandes (Cornell University) at AMP 2014 AmpliSeq workflow GeneRead workflow Group # of samples DNA Quant Library quality & quantity DNA Quality Library quality & quantity Qubit Agilent QuantiMIZE qPCR 1 30 2 3 3 6 X X Sequenced optimally Not sequenced optimally RS NGS part 3: Targeted NGS for Cancer Research 23 3
- 24. Sample to Insight Compatibility with major sequencing platforms Same panel for different platforms IlluminaQIAGEN Ion GeneRead® DNAseq Panels V2 HiSeq 2500 MiSeq NextSeq PGM Proton Confirm variants on different platforms using the same panel RS NGS part 3: Targeted NGS for Cancer Research 24
- 25. Sample to Insight Sample-to-Insight: Integrated universal targeted NGS workflow To overcome NGS challenges Sample Insight Turnaround time, and limited amounts of DNA Library yield Coming soon Data processing & Variant calling Isolation of high- quality DNA samples Quantifying amplifiable (not total) amounts of DNA Clinical & Biological interpretation of data RS NGS part 3: Targeted NGS for Cancer Research 25 1 Sample isolation Targeted enrichment Library construction NGS run Data analysis Interpretation Sample QC Library QC Variant confirmation
- 26. Sample to Insight Sample-to-Insight: Integrated universal targeted NGS workflow Bioinformatics solutions Sample Insight Upstream Analysis ‘Primary’ ‘Secondary’ ‘Tertiary’ NGS part 3: Targeted NGS for Cancer Research 26 Sample Prep Assay Data Sequence- Level Statistics Biology of Interest (Genes, Variants, etc.) Annotation & Comparative (Statistical Analysis) Annotation & Biological Interpretation
- 27. Sample to Insight CLC Biomedical: Flexible workflows tailored for end-users Lock key parameters of workflow to standardize processing Identify Variants Example Sample Prep Plug-in for GeneRead DNAseq targeted panels NGS part 3: Targeted NGS for Cancer Research 27 Assay Data Sequence- Level Statistics Biology of Interest Annotation & Comparative Analysis Annotation & Comparative Interpretation
- 28. Sample to Insight IVA: Rapid prioritisation and annotation of variants Examine a single sample or compare multiple samples NGS part 3: Targeted NGS for Cancer Research 28
- 29. Sample to Insight Examine evidence and references • Variant Findings • ACMG Classification Rules • Variant Views NGS part 3: Targeted NGS for Cancer Research 29
- 30. Sample to Insight Why choose targeted DNA sequencing? Detect known & discover novel variants AM & CR Disease- specific Comprehensive Detection Discovery Multiplexing Target size Custom & Mix-n-Match PanelsApplicationsSpecifications Clinical research Translational & Discovery research Whole Exome Seq Whole Genome Seq Targeted DNA sequencing: robust detection, limited discovery AM: actionable mutations panel; CR: clinically relevant panel RS NGS part 3: Targeted NGS for Cancer Research 30
- 31. Sample to Insight Novel mutation discovery by targeted sequencing Gene panels can facilitate the discovery of novel mutations Notably, this study facilitated the identification of BRCA2 Thr9976, which is the strongest genetic association in lung cancer reported so far. For a smoker carrying this variant (2% of the population), the risk of developing lung cancer is approximately doubled, which may have implications for identifying high-risk ever-smoking subjects for lung cancer screening. Additionally, future study of the effects of PARP inhibition in smokers with lung cancer carrying BRCA2 Thr9976 may be warranted. RS NGS part 3: Targeted NGS for Cancer Research 31
- 32. Sample to Insight Clinical value of targeted sequencing with targeted panels More is not necessarily better; in some cases, less is better RS NGS part 3: Targeted NGS for Cancer Research 32
- 33. Sample to Insight GeneRead Targeted Panels for NGS: Application Liquid biopsy to identify mechanisms of drug resistance in cancer • EGFR-mutant lung cancer is a subtype of non–small cell lung cancer (NSCLC) that exhibits sensitivity to EGFR TKIs such as erlotinib and gefitinib1 • However, acquired resistance develops after a median of 9–14 months1 • The most common mechanism of TKI resistance is a second- site mutation (T790M) in the EGFR kinase domain, which can be detected in >50% of biopsies done after resistance develops2 1Mok, T.S. et al. N. Engl. J. Med. 361, 947–957 (2009) 2Sequist, L.V. et al. Sci. Transl. Med. 3, 75ra26 (2011) Landscape of resistance mutations in NSCLC 2Sequist, L.V. et al. Sci. Transl. Med. 3, 75ra26 (2011) RS NGS part 3: Targeted NGS for Cancer Research 33
- 34. Sample to Insight GeneRead Targeted Panels for NGS: Application Liquid biopsy to Identify mechanisms of drug resistance in cancer 34 Power of liquid biopsy: Predict resistance before a solid biopsy is available • AZD9291 is an oral, irreversible, mutant-selective EGFR TKI developed to have potency against tumors bearing EGFR activating mutations (for example, L858R or exon 19 deletion) in the presence of the T790M mutation • Can we identify potential mechanisms of resistance to AZD9291 before the availability of resistance biopsy specimens? • Use GeneRead targeted NGS panel to screen cell-free plasma DNA (cfDNA) for potential resistance mutations by comparing pretreatment and post-disease progression plasma specimens from a phase I clinical trial o cfDNA serves as a liquid biopsy to monitor disease progression by examining the landscape of mutations before a resistance solid biopsy is available May 2015 RS
- 35. Sample to Insight GeneRead Targeted Panels for NGS: Application Liquid biopsy to Identify mechanisms of drug resistance in cancer Liquid biopsy approach Collect 10-20 cc blood (pre-treatment (Baseline) and post progression) Prepare plasma Isolate cfDNA using the QIAmp Circulating Nucleic Acid Kit Enrich targets (20 genes) using the GeneRead DNAseq Lung Cancer panel Construct libraries Ultra-deep sequencing (30,000x) on a HiSeq (2 × 100 bp) RS NGS part 3: Targeted NGS for Cancer Research 35
- 36. Sample to Insight GeneRead Targeted Panels for NGS: Application Liquid biopsy to Identify mechanisms of drug resistance in cancer Liquid biopsy: Proof of concept Targeted NGS identified an acquired T→A mutation (green) in 1.3% of reads, encoding an EGFR C797S mutation. RS NGS part 3: Targeted NGS for Cancer Research 36
- 37. Sample to Insight GeneRead Targeted Panels for NGS: Application Liquid biopsy to Identify mechanisms of drug resistance in cancer Liquid biopsy: EGFR C797S mutation in progression/resistance to AZD9291 (TKI) NGS of baseline and progression tumor biopsies (top) confirmed the acquired C797S mutation detected with plasma NGS (bottom). Plasma NGS detects the same T→A C797S mutation (green) found in the tumor and additionally detects a second G→C mutation encoding the C797S mutation (blue). RS NGS part 3: Targeted NGS for Cancer Research 37
- 38. Sample to Insight Thank you for attending today’s webinar! Contact QIAGEN Call: 1-800-426-8157 Email: techservice-na@qiagen.com BRCsupport@QIAGEN.com Questions? Thank You For Attending NGS part 3: Targeted NGS for Cancer Research 38
Notes de l'éditeur
- The concept of one size fits all for disease management is not effective. With this approach, some patients show positive effects, while others show no effects or adverse effects. The concept of precision medicine of delivering the right drug, to the right patient, at the right time and dose is more effective. It builds on the idea of utilizing genetic information for disease management.
- There are several DNA variants that can be used for precision medicine. Three are shown here.
- Out of all the DNA variants, only a handful are actionable, which means we can use the information from these variants for disease management. For example, the presence of the V600E mutation in BRAF is seen in a large number of melanoma patients. While this variant causes the development and progression of the disease, it’s presence is favorable for melanoma patients because the variant (mutation) can be targeted by an approved therapy (Vemurafenib).
- How many actionable mutations are there? Only a handful. Shown here is a typical process of filtering variants to build a list of actionable mutations. Starting with millions of common variants, we end up with 10s-100s of actionable variants.
- As an example, in lung cancer, we see the following mutations accumulate in lung cancer patients. We can use the information on the type of mutations for disease management. For instance, patients who harbor the EGFR L858R mutation respond very well to tyrosine kinase inhibitors, while those harboring an additional mutation in KRAS, their responses to the same class of drugs drop.
- There are three main sequencing levels which can be used to detect actionable mutations. The table here shows how these three levels differ on several parameter. Targeted DNA sequencing and analysis is extensively used in clinical research.
- What are the benefits of targeted DNA sequencing? What to include in a targeted panel? There are many approaches. One approach, highlighted by Dr. Carl Morrison is to include genes for which actionable information exists. In other words, examine targets upon which you can act.
- With targeted DNA sequencing, one shrinks the genome to a number of targets that are crucial to the disease being examined. In this example, the user is interested in only the three genes shown.
- QIAGEN has built a sample-to-insight workflow to overcome some of the main challenges associated with NGS.
- The reason we chose a multiplex PCR approach for target enrichment is to take advantage of the high specificity and uniformity that PCR provides, as shown in this report. Here, the authors compared different enrichment approaches and show that the specificity and uniformity of PCR surpasses those of hybridization approaches.
- We developed the GeneRead DNAseq panels to enable target enrichment prior to NGS. The panels use a multiplex PCR approach to enrich specific genes or genomic regions. We also developed the accompanying PCR chemistry.
- We utilize proprietary primers design and separation algorithms to design to primers for the specific targets and separate the primers into an appropriate number of PCR pools.
- We developed and bench-optimized 15 cataloged panels for a wide range of applications. The content of these panels is drawn from several sources such as The Cancer Genome Atlas, ….. The content represents the largest collection……
- Not only are the panels unique in their applications, but they are unique in their designs. We utilize unique approaches for different applications: For example, shown here is the coverage of a specific locus in the KRAS gene (the G12 locus). As you can see, this locus is redundantly covered by the actionable mutations panel.
- We also offer 2 approaches to build your own unique panel that fits your own needs and requirements. The Mix-n-Match approach gives you access to our 570 wet-bench tested gene designs, which is a very unique offering in the market. In case your genes of interest are not part of Mix-n-Match, you can use our Custom offering to build a fully customized panel.
- To show you the power of Mix-n-Match, shown here are performance metrics, namely specificity and uniformity, of some of our gene designs. One can see that regardless of the panel the gene design sits in, you get a high degree of specificity and uniformity.
- A unique feature of our technology and workflow is that we do not physically digest primer sequences. Instead we trim off primer sequences in the data analysis step right before variant calling. This allows to better align amplicons and avoid ‘blind spots’ at the edges of amplicons. ‘Blind spots’ or ‘Edge effects’ is the phenomenon in which bases at the end of amplicons are not sequenced. This is a very well established effect, and it can be mitigated by keeping primer sequences.
- The panels that we have developed are platform-independent, and have been tested on both the MiSeq and PGM.
- QIAGEN informatics provides a complete sample to insight solution however each component of the workflow can be purchased separately to allow customers to mix and match. QIAGEN is already a market leader in sample prep with recognized reagent brands as well as liquid handling/robotics to streamline sample prep. Later this year, QIAGEN will introduce the GeneReader that will support panel sequencing however customers can choose to use their favorite sequencing platform as QIAGEN products fully support Illumina, ION Torrent, and Roche systems. CLCbio’s Genomic Workbench is an industry leader that can accept data from any of these platforms and generate a list of detected variants is the sequenced samples. The resultant variant call file (vcf) is then automatically sent to Ingenuity Variant Analysis for fast, accurate interpretation of the identified variants.
- The parameters can be changed to fit your own needs, and can be locked.
- Ingenuity Variant Analysis provides a comprehensive, easy-to-use solution to filter, annotate, and interpret the variants present in your samples.
- One of the applications is liquid biopsies to identify mechanisms of drug resistance in cancer. This application is being developed as the biopsy method of choice for the management of lung cancer.
- In this report, researchers from AstraZeneca wanted to explore the idea of utilizing liquid biopsies to predict resistance to therapies before a solid biopsy is available. They used the GeneRead panels to screen……clinical trial.
- Shown here is their approach and workflow.
- As proof of concept, they show here that they are able to detect the C797S mutation, even at very low frequency (they can achieve this because the achieved very high depths of coverage of 30,000x)