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Somatic mutation webinar

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  • 1. qBiomarker™ Somatic Mutation PCR Arrays Cancer Mutation Analysis Made Easy Shankar Sellappan, Ph.D Global Product Manager Shankar.Sellappan@QIAGEN.com -1- Sample & Assay Technologies
  • 2. Legal Disclaimer • SABiosciences products 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. -2- Sample & Assay Technologies
  • 3. Welcome to SABiosciences: Who We Are • SABiosciences is now a Company -3- Sample & Assay Technologies
  • 4. Table of Contents • DNA Mutations Overview • What is a qBiomarker Somatic Mutation PCR Array? How to Analyze Samples for DNA Mutations • Workflow for qBiomarker Somatic Mutation PCR Arrays • How qBiomarker Somatic Mutation PCR Assays Work • Data Analysis • How to Order -4- Sample & Assay Technologies
  • 5. What is a Somatic DNA Mutation? Definition Alterations in DNA that can occur in any of the cells of the body except germ cells – NOT passed on to children Single Point Mutation or Multiple Mutations Possible within a Gene -5- Sample & Assay Technologies
  • 6. Somatic Mutations vs Single Nucleotide Polymorphisms (SNPs) • Somatic Mutations occur in cells that are NOT passed down to children • SNPs occur in Germline cells (that ARE passed down) • What is important in SNP analysis is the Allele Frequency • Which means how often it occurs Note: qBiomarker Somatic Mutation PCR Arrays & Assays only show the Presence of a Mutation Qualitative, not Quantitative Information Quantitative may come in future. -6- Sample & Assay Technologies
  • 7. Physiological Implications of DNA Mutations Changes in a single or multiple nucleotides within a gene, or across related genes in a pathway, may alter the resulting protein, either by activity level or functional ability, resulting in: Cancers Cardiovascular diseases (Arrhythmia) Blood/hematologic disorders -7- Sample & Assay Technologies
  • 8. Implications of DNA Mutations in Drug Development Drug compounds may behave differently in a similar profile of sample, based on Somatic Mutation profile. Ex. 30% of Breast Cancers overexpress HER2. –However, not all respond to HER2 therapy (i.e. Herceptin®) • This may indicate a different HER2 mutation occurring in samples, thereby yielding different outcomes. -8- Sample & Assay Technologies
  • 9. How do DNA Mutations Occur? • Copying Errors • as part of Replication Process • Estimated Error Rate of DNA Polymerase: 1 in 108 bases • Human Genome = 2.9 billion bases (2.9 x 109 bases) • DNA Damage • Resulting from Environmental Agents, such as: • Sunlight (UV) • Cigarette Smoke • Radiation -9- Sample & Assay Technologies
  • 10. What Kinds of DNA Mutations are There? • Point Mutation •A G • Frame Shift Mutation / Insertion / Duplication • ACGTACGTC • ACGATACGTC • ACGTACGTC • ACGTACGTC • ACGAGGTACGTC • ACGTACTACGTC • Deletion • ACGTACGTC From 1 – 100s of bases • ACGACGTC • Inversion • ACGTACGTC • ACTGACGTC - 10 - Sample & Assay Technologies
  • 11. How many DNA Mutations have been identified? • ~ 140,000 DNA Mutations Identified • Most DNA Mutations NOT Functionally Relevant at the Protein Level • Multiple Consortiums • COSMIC: Catalog of Somatic Mutations in Cancer: Sanger Institute • HGMD: The Human Gene Mutation Database at the Institute of Medical Genetics in Cardiff • dbRBC: Blood Group Antigen Gene Mutation Database: NCBI • EXPECTATIONS IMPORTANT POINT TO REMEMBER • Cells under study may contain one to a few mutations as assayed for on a pathway-focused qBiomarker Somatic Mutation PCR Array - 11 - Sample & Assay Technologies
  • 12. Table of Contents • DNA Mutations Overview • What is a qBiomarker Somatic Mutation PCR Array? How to Analyze Samples for DNA Mutations • Workflow for qBiomarker Somatic Mutation PCR Arrays • How qBiomarker Somatic Mutation PCR Assays Work • Data Analysis • How to Order - 12 - Sample & Assay Technologies
  • 13. What is a Somatic Mutation PCR Array? Somatic Mutation PCR Array • Definition: Collection of mutation specific assays for multiple mutations in a single gene, as well as mutations present in genes in downstream signaling activities • Example: EGFR Pathway Somatic Mutation PCR Array • EGFR Mutations: 29 Different mutations present in the EGFR gene • AKT1 Mutations: 1 • BRAF Mutations: 12 • KRAS Mutations: 18 • HRAS Mutations: 11 • NRAS Mutations: 12 • MEK1 Mutations: 4 • PI3K Mutations: 7 • PTEN Mutations: 6 - 13 - Sample & Assay Technologies
  • 14. Why Study Somatic Mutations in a Pathway Array? EGFR Mutations AKT1 Mutations BRAF Mutations KRAS Mutations HRAS Mutations NRAS Mutations MEK1 Mutations PI3K Mutations PTEN Mutations - 14 - Sample & Assay Technologies
  • 15. What is the Layout of a qB Somatic Mutation PCR Array? Greek Symbol = Gene # = Mutation • For Normalization • Assays detect nonvariable region (not ARMSbased design) • Designed to control differences in sample input and quality - 15 - Sample & Assay Technologies
  • 16. Array Layout: Example: EGFR Pathway - 16 - Sample & Assay Technologies
  • 17. qBiomarker Cancer Somatic Mutation PCR Arrays Available Pathways • EGFR pathway (lung cancer, colorectal cancer, glioblastoma multiforme) • ERBB2 (HER-2/neu) pathway (breast cancer, lung, gastric cancers) • PDGFR pathway (GIST, glioblastoma, prostate cancer) • FLT3 pathway (acute meyloid leukemia (AML)) • RTK I & II (96-well) or RTK-HT (384-well) • KIT pathway (leukemia, gastrointestinal stromal, testicular germ cell tumors) • MET pathway (kidney, liver, stomach, breast and brain cancers) • FGFR pathway (leukemia and multiple myeloma) • Upcoming: •PI3K pathway (one of the most commonly activated pathways in cancer) • P53-Rb pathways (associated with resistance to multiple chemo-therapeutic drugs) • β-Catenin/CTNNB1-APC pathway (colon, prostate, uterine cancers) - 17 - Sample & Assay Technologies
  • 18. Table of Contents • DNA Mutations Overview • What is a qBiomarker Somatic Mutation PCR Array? How to Analyze Samples for DNA Mutations • Workflow for qBiomarker Somatic Mutation PCR Arrays • How qBiomarker Somatic Mutation PCR Assays Work • Data Analysis • How to Order - 18 - Sample & Assay Technologies
  • 19. Analysis Methods for DNA Mutations Method Benefits Drawbacks Point Mutation Analysis Exact Time Consuming Quantify DNA Targets Real-time PCR Fast Reliable High-throughput Mass Spectrometry Exact Time Consuming Need Instrument/Training High Resolution Melt Inexpensive Fast Simple Base change identity not obvious Microarray Large Coverage Sensitivity Protocol complex Need Instrument/Training Next-Generation Sequencing Exact Time Consuming Need Instrument/Training - 19 - Sample & Assay Technologies
  • 20. Sensitivity of Mutation ID Technologies / Principles Measurement of Sensitivity: In a population of 100 total cells, sensitivity of: • RT-PCR (PCR Arrays): 1-2% • 1-2 mutant DNA + 98-99 wild-type DNA • Pyromark Sequencing: 5% • 5 mutant DNA + 95 wild-type DNA • Method of DNA sequencing based on the “sequencing by synthesis principle” • Sequenom/Microarray: 10% • 10 mutant DNA + 90 wild-type DNA - 20 - Sample & Assay Technologies
  • 21. qBiomarker Somatic Mutation Assay Detection Limit WGA = Whole Genome Amplification Assay detection limit test for qBiomarker Braf V600E assay. A series of 10ng genomic DNA samples, which contain genomic DNA from A375 cell line mixed with WT genomic DNA at different ratios, were tested on qBiomarker Somatic Mutation Assay for Braf V600E with or without whole genome amplification. Mutation detection limit for this assay is determined to be ~1%. - 21 - Sample & Assay Technologies
  • 22. Table of Contents • DNA Mutations Overview • What is a qBiomarker Somatic Mutation PCR Array? How to Analyze Samples for DNA Mutations • Workflow for qBiomarker Somatic Mutation PCR Arrays • How qBiomarker Somatic Mutation PCR Assays Work • Data Analysis • How to Order - 22 - Sample & Assay Technologies
  • 23. DNA Sample Requirement • If less than 5 ng DNA available from Fresh frozen samples, Whole Genome Amplification recommended. NOTE: WGA NOT recommended for FFPE samples - 23 - Sample & Assay Technologies
  • 24. How do qBiomarker Somatic Mutation Arrays Work? Fresh/frozen samples: 200 – 500 ng gDNA FFPE samples: 200 – 500 ng gDNA Fresh/frozen samples: 5 – 10 ng gDNA - 24 - Sample & Assay Technologies
  • 25. Table of Contents • DNA Mutations Overview • What is a qBiomarker Somatic Mutation PCR Array? How to Analyze Samples for DNA Mutations • Workflow for qBiomarker Somatic Mutation PCR Arrays • How qBiomarker Somatic Mutation PCR Assays Work • Data Analysis • How to Order - 25 - Sample & Assay Technologies
  • 26. How do qBiomarker Somatic Mutation Assays Work? . Hydrolysis Probe Assays + ARMS - 26 - Sample & Assay Technologies
  • 27. Definition: Hydrolysis Probes & Assays . Relies on the 5’ – 3’ exonuclease activity of the Taq activity of Taq Polymerase The Exonuclease Activity degrades a hybridized non-extendable DNA probe during the extension step of the PCR. DNA Probe: Hybridizes to a region within the amplicon and is dual-labeled with a reporter dye and quenching dye. - 27 - Sample & Assay Technologies
  • 28. Definition: Allele Specific Amplification with ARMS® ARMS: Amplification Refractory Mutation System Strategy in which Primers Discriminate Among Templates Differing by a Single Nucleotide Oligos with a mismatched 3’-residue WILL NOT FUNCTION as Primers in a PCR Reaction But with the 3’-residue matching at the exact spot of the mutation - 28 - Sample & Assay Technologies
  • 29. Primer Elongation & Probe Activation with ARMS® Common Region F F - 29 - Q Q Sample & Assay Technologies
  • 30. Key to qBiomarker Somatic Mutation Assay Design Additional mismatch used Mutation Position & Additional Mutation – 1-3 bp near mutation site (location is proprietary) Increases specificity - 30 - Sample & Assay Technologies
  • 31. Allele Specific Amplification with ARMS® ARMS: A Closer Look } Will Work ARMS Mutation Primer Template: Mutation TCAGTAAA AGTCAGTT Mutation Locus: C is correct for WT Why this works: Internal Mismatches: Enzyme Requirement Less Stringent } Won’t Work Add’l Mutation: Location Varies ARMS Mutation Primer Template: Wild-Type TCAGTAAA AGTCAGTC ARMS Mutation Mutation Locus: C is correct for WT - 31 - Sample & Assay Technologies
  • 32. Why ARMS + Hydrolysis Probe? Why not just Hydrolysis Probe? Hydrolysis Probes Measures just target strand abundance ARMS (Amplification Refractory Mutation System) Preferentially amplifies the mutant alleles – We are trying to detect as low as 1-2% somatic mutation in a wild-type DNA background - ARMS – designed primers will preferentially amplify the mutant sequence - 32 - Sample & Assay Technologies
  • 33. qBiomarker Somatic Mutation Array Detection of BRAF, EGFR, and KRAS Mutations • Samples: Asian, Female, non-smoker origin; Lung Adenocarcinoma • Expected: 30% Mutation in EGFR EGFR BRAF KRAS ∆Ct FFPE Samples Mutations Notice how each FFPE sample harbors only 1 or two mutations So we are NOT looking for spikes everywhere - 33 - Sample & Assay Technologies
  • 34. qBiomarker Somatic Mutation Assay Validation Pyrosequencing Confirmation of KRAS & EGFR Mutations - 34 - Sample & Assay Technologies
  • 35. Table of Contents • DNA Mutations Overview • What is a qBiomarker Somatic Mutation PCR Array? How to Analyze Samples for DNA Mutations • Workflow for qBiomarker Somatic Mutation PCR Arrays • How qBiomarker Somatic Mutation PCR Assays Work • Data Analysis • How to Order - 35 - Sample & Assay Technologies
  • 36. Somatic Mutation PCR Array Data Analysis • Purpose: Qualitative assessment of samples • Is the DNA Mutation Present and if so, which one? • Data Analysis Methods depends on: • 1. Availability of wild-type sample • 2. Sample type/quality • Cell lines • Fresh or frozen samples • FFPE samples • 3. How many samples? • NOTE: Compare Presence of Same Mutant Assay Across Samples • NOTE: Each sample harbors only a single or at most few mutations - 36 - Sample & Assay Technologies
  • 37. qBiomarker Somatic Mutation PCR Array Data Analysis Average Ct Method Recommended for experiments using: • FFPE samples • Large #s of samples (> 4) • Wide range of sample quality • Wild-type controls not necessary • Assumes that for a given locus, mutation occurs in small % of samples. average ∴Ct = Mutation Assay Background in Wild-Type Sample sample Compare Ct sample If Ct average to Ct << Ct average = ∆Ct Sample has Mutation - 37 - Sample & Assay Technologies
  • 38. Average Ct method examples . FFPE cell line samples Lung Cancer & Colon Cancer cell lines MDA-MB-231 as a positive control G464V Braf and G13D Kras mutation status gathered from COSMIC database and confirmed by: – DxS Kras PCR test – Braf Pyrosequencing assay . 9 FFPE lung cancer samples, 1 placenta FFPE sample Sample origin Adenocarcinoma Expected EGFR mutation rate: ~10% - 38 - Sample & Assay Technologies
  • 39. Average Ct: Example Data: Collaboration: β-Test Mutation Profiling of FFPE Samples on EGFR PCR Array l Cel cer Can ∆Ct es L in Conclusion of the β-test The two known mutations in MDA-MB-231 cell line FFPE sample were correctly called: 2 Kras and 1 EGFR mutation (~10%) called in 3 lung cancer FFPE samples. Mutation - 39 - Sample & Assay Technologies
  • 40. Average Ct: Example Data: In-House Data Mutation Profiling of FFPE Samples on EGFR PCR Array Tested 10 FFPE adenocarcinoma samples Expect high % (>30%) EGFR activating mutations (point mutation & deletions) Paez et al. Science. (2004) 304:1497-500 c ocae Aden ∆Ct a rinom Mutation - 40 - Sample & Assay Technologies
  • 41. qBiomarker Somatic Mutation PCR Array Data Analysis ∆∆Ct Method Recommended for experiments using: • Small #s of samples (< 4) • Basic Cell Line Experiments….Looking for Mutation in just a couple of cell line samples • Results affected by sample quality (Wild-type and Test Samples) • Wild-type controls NECESSARY • Wild-type controls MUST be of high-quality • Considers Copy # Changes • NORMALIZES (∆Ct) • Caveats • Penalizes good quality samples (False Negatives) • Rewards bad quality samples (False Positives) - 41 - Sample & Assay Technologies
  • 42. qBiomarker Somatic Mutation PCR Array Data Analysis ∆∆Ct Method * A raw Ct cutoff for Ct (sample locus X) will be applied (usually 35); If sample Ct > cutoff Ct, the sample will NOT be called “mutant” for that locus Normalized Ct For Allele/Mutation Site = ∆Ct = CtMUT – CtCNA Mutation Assay CNA ∆Ct WT 40 28 12 Mutant 30 27 3 - 42 - ∆∆Ct = 9 Sample & Assay Technologies
  • 43. Canc er Cell L ines ∆∆C ∆∆ t Method: Example EGFR Pathway: Cancer Cell Lines (200 ng gDNA) Mutation - 43 - Sample & Assay Technologies
  • 44. qBiomarker Somatic Mutation PCR Array Data Analysis Two Methods: ∆∆Ct vs Average Ct: How to Choose Data Analysis Method Selection Summary • ∆∆Ct method • Must have wild-type control • Works well for projects using: • Fresh/frozen samples • Smaller (≤4) number of samples • i.e. sample quality similar • Average Ct method • Works well for projects using: • FFPE samples • Large number of samples • i.e. sample quality can be in a wide range • Without Wild-type controls - 44 - Sample & Assay Technologies
  • 45. Data Analysis: Excel Files: Pathway-Specific - 45 - Sample & Assay Technologies
  • 46. Table of Contents • DNA Mutations Overview • What is a qBiomarker Somatic Mutation PCR Array? How to Analyze Samples for DNA Mutations • Workflow for qBiomarker Somatic Mutation PCR Arrays • How qBiomarker Somatic Mutation PCR Assays Work • Data Analysis • How to Order - 46 - Sample & Assay Technologies
  • 47. qBiomarker Somatic Mutation: Product Availability 337021: qBiomarker Somatic Mutation PCR Arrays • Please visit http://sabiosciences.com/somaticmutationarray.php for the most up-todate list of Pathway/Disease Focused Somatic Mutation PCR Arrays 337011: qBiomarker Somatic Mutation PCR Mutation Assays 100 reactions •NOTE: Corresponding qBiomarker Probe Master Mixes are included with ALL ORDERS. - 47 - Sample & Assay Technologies
  • 48. Additional Resources E-Learning Center: Recorded Seminars - 48 - Sample & Assay Technologies
  • 49. qBiomarker™ Somatic Mutation PCR Arrays & Assays Shankar Sellappan, Ph.D. Global Product Manager Shankar.Sellappan@QIAGEN.com - 49 - Sample & Assay Technologies

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