Cancer Research & the Challenges of FFPE Samples – An Introduction
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A cascade of complex genetic and epigenetic changes regulate tumor formation and progression. Gene expression analyses can shed light on these changes at a molecular level and identify the key genes and associated pathways involved in cancer. Often the samples used in cancer research are FFPE samples, which pose a significant challenge in terms of nucleic acid quality. The quality of nucleic acids extracted from FFPE samples depends on a number of factors, including how the samples were handled before, during and after fixation and embedding. Dr. Vishwadeepak Tripathi describes the variability of sample purification from FFPE samples – in particular, samples to be used in cancer research. What are the challenges and solutions, and what quality control approach can ensure credible results? This webinar will focus on sample purification and the quality control of FFPE samples and compare different automated purification procedures.
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Cancer Research & the Challenges of FFPE Samples – An Introduction
- 1. Sample to Insight 1 Dr. Vishwadeepak Tripathi Vishwadeepak.Tripathi@qiagen.com Cancer Research & the Challenges of FFPE Samples – An Introduction
- 2. Sample to Insight Legal disclaimer 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 Cancer Research & the Challenges of FFPE Samples 3 An Introduction Focus on sample purification Quality Control of FFPE samples Comparison of different automated purification procedures Mutation analysis using PyroSequencing Technology ... Stay tuned – there is more to come!
- 4. Sample to Insight Outline 4 The reproducibility challenge & its impact1 Each step of the workflow counts 2 3 Wrap-up 4 5 The importance of Quality Control Things you can and cannot influence
- 5. Sample to Insight The reproducibility challenge & its impact 5 Insight Published results My results ? Increase peace of mind by improving reliability of research results ?
- 6. Sample to Insight The reproducibility challenge & its impact 6 Insight The more complex the sample or analysis workflow – the bigger the challenge My results Published results !
- 7. Sample to Insight Reproducibility crisis = credibility crisis? 7 http://www.nature.com/news/1-500-scientists-lift-the-lid-on-reproducibility-1.19970 http://www.nature.com/news/let-s-think-about-cognitive-bias-1.18520 How much work published in your field is reproducbile?Have you failed to reproduce an experiment?
- 8. Sample to Insight Low reproducibility is costly in the end 8 http://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.1002165 “Low reproducibility rates within life science research undermine cumulative knowledge production and contribute to both delays and costs of therapeutic drug development. An analysis of past studies indicates that the cumulative (total) prevalence of irreproducible preclinical research exceeds 50%, resulting in approximately US$28 Billion/year spent on preclinical research that is not reproducible—in the United States alone.” Freedman LP et al. (2015) The Economics of Reproducibility in Preclinical Research. PLoS Biol The more complex the sample or analysis workflow – the bigger the challenge
- 9. Sample to Insight Outline 9 The reproducibility challenge & its impact1 Each step of the workflow counts 2 3 Wrap-up 4 5 The importance of Quality Control Things you can and cannot influence
- 10. Sample to Insight Why FFPE samples? – Because this is what you get in your lab 10 Facts FFPE has become a standard method for long-term preservation of tissue biopsies Low chances to change pathology practices for research needs alone Very large number of unprocessed FFPE samples are archived in tissue banks and biorepositories Samples are highly valuable, often well-characterized with histological, pathological and follow-up clinical data Sometimes there is no other choice than FFPE Overcoming challenges you can control is critical to ensure credible results
- 11. Sample to Insight A complex workflow – which aspect can you control? 11 Sample Insight Sample collection Sample stabilization NA purification Assay & Analysis Data interpretation
- 12. Sample to Insight A complex workflow – which aspect can you control? 12 Sample Insight Sample collection Sample stabilization NA purification Assay & Analysis Data interpretation Some steps you cannot influence – but you have to deal with potential implications
- 13. Sample to Insight How were your samples derived and fixed? 13 Some aspects likely NOT under your control Amount of tissue material* Cancer cell content Excision conditions Fixation conditions * Pictures from Getz G et al., The BROAD Institute, ‘Mutation analysis in frozen and FFPE tumor samples’
- 14. Sample to Insight Fixation conditions? 14 Factors impacting fixation Fixative used? Buffered vs. unbuffered formalin? Fixation efficiency/rate of penetration Volume of formalin solution vs. thickness of tissue specimen Temperature Duration of fixation Storage conditions There is a fine line between under- and over-fixation
- 15. Sample to Insight The balance between under- and over-fixation 15 Fixation conditions influence nucleic acid integrity & downstream assay performance
- 16. Sample to Insight Fixation of sliced tissue improves RNA integrity 16 Impact of sample thickness on RNA integrity & RT-PCR performance Underfixation can lead to drop-outs in downstream RT-PCR
- 17. Sample to Insight Overfixation can negatively impact downstream assays 17 Effect of fixation duration on RNA integrity and RT-PCR performance Overfixation can lead to drop-outs in downstream RT-PCR
- 18. Sample to Insight Storage conditions do matter! 18 Storage conditions influence RNA integrity RT-PCR performance is influenced by RNA integrity
- 19. Sample to Insight Storage conditions do matter! 19 Storage conditions influence RNA integrity RT-PCR performance is influenced by RNA integrity
- 20. Sample to Insight Formalin fixed samples exhibit sequence alterations Am J Pathol. 1999 November; 155(5): 1467–1471. PMCID: PMC1866966 A High Frequency of Sequence Alterations Is Due to Formalin Fixation of Archival Specimens Cecilia Williams,* Fredrik Pontén,† Catherine Moberg,‡ Peter Söderkvist,§ Mathias Uhlén,* Jan Pontén,† Gisela Sitbon,‡ and Joakim Lundeberg* … In formalin-fixed material up to one mutation artifact per 500 bases was recorded. The chance of such artificial mutations in formalin-fixed material was inversely correlated with the number of cells used in the PCR—the fewer cells, the more artifacts. A total of 28 artificial mutations were recorded, of which 27 were C-T or G- A transitions…. 20
- 21. Sample to Insight Outline 21 The reproducibility challenge & its impact1 Each step of the workflow counts 2 3 Wrap-up 4 5 The importance of Quality Control Things you can and cannot influence
- 22. Sample to Insight The first step in your lab – nucleic acid purification? 22 Each step of the workflow counts Sample Insight Sample collection Sample stabilization NA purification Assay & Analysis Data interpretation Remove cross-links and retrieve ALL nucleic acids in your sample
- 23. Sample to Insight gDNA purification from FFPE material 23 Kit specifications Silica membrane based Up to 8 sections, each with a thickness of up to 10 µm and a surface area of up to 250 mm2 Purification of genomic DNA & mitochondrial DNA Elution volume 20–100 µl QIAcube protocol available Paraffin removal and sample lysis No need for overnight incubation Paraffin is dissolved in deparaffinization solution and removed Sample lysis under denaturing conditions with proteinase K (1 h, 56°C) Incubation at 90°C to reverse formalin crosslinking Optional RNase treatment step Will a 90°C incubation affect my downstream results?
- 24. Sample to Insight 1 h incubation at 90°C allows for optimal recovery of DNA 24 Effect of incubation times and temperatures on DNA yield No need for an overnight ProtK digestion
- 25. Sample to Insight Lower Ct values due to higher amounts of usable DNA 25 Effect of incubation times and temperatures on downstream real-time PCR
- 26. Sample to Insight Sequence Artifacts in FFPE …We show that DNA molecules amplified by PCR from DNA extracted from animal bones and teeth that vary in age between 25 000 and over 50 000 years carry C→T and G →A substitutions. These substitutions can reach high proportions among the molecules amplified and are due to the occurrence of modified deoxycytidine residues in the template DNA. If the template DNA is treated with uracil N- glycosylase, these substitutions are dramatically reduced. They are thus likely to result from deamination of deoxycytidine residues. 26
- 27. Sample to Insight One more thing to be aware of – Preventing artifacts 27 All FFPE samples potentially carry random C>T mutation artifacts Cytosine bases within the DNA strand in FFPE samples are subject to deamination This chemical reaction turns a Cytosine into a Uracil base It is believed to occur randomly during formalin fixation and with aging The reasons for this chemical reaction is still unknown Generally low frequency of these artifacts In subsequent sequencing analysis this base appears as Thymine (SNP) Differentiate between artificial and true mutation
- 28. Sample to Insight Efficient removal of artificial C>T mutations 28 GeneRead DNA FFPE Kit True mutations are kept while artificial C>T mutations are reduced by >90% Improved Lysis Buffer for higher yields Enzymatic removal of C>T artifacts built-in DNA preparation DNA isolation according to the proven QIAamp technology Automated on the QIAcube
- 29. Sample to Insight Artifact removal does not negatively impact yields 0 1 2 3 4 5 6 µgdouble-strandedDNAextracted Standard GeneRead Single 10µm slices, varying surface area. Average yield 29 Yields equivalent or better with new method – optimized for low input
- 30. Sample to Insight Outline 30 The reproducibility challenge & its impact1 Every step of the workflow counts 2 3 Wrap-up 4 5 The importance of Quality Control Things you can and cannot influence
- 31. Sample to Insight Why is sample QC so important? 31 ? % success µg Assess samples’ quality according to relevant parameters and thresholds Downstream processing of high-quality samples only, to maximize success of assays Highest quality results are only achieved from quality samples, Only quality results are worth sharing. Analyze, interpret and share results with confidence Track samples’ quality along the workflow
- 32. Sample to Insight Is it worth the investment? 32 Cost of insufficient or inappropriate QC Cost of QC Instruments, consumables, training, time… ? The overall cost of not performing QC, i.e. risking processing poor quality samples along downstream applications, is often overlooked if calculated at all. • Can you exactly tell the cost/sample of your downstream experiment? • Time spent interpreting challenging results? • Price of your confidence and peace of mind? Price per sample Time Downstream application qPCR 1.5 $US 4-5 hrs Pyrosequencing 3.0 $US 7-8 hrs Sanger Sequencing 5-6 $US 4-6 hrs Next-Gen Sequencing >200 $US(5-plex) 2-3 working days A trade-off must be found between the cost of QC procedure and the cost of not doing QC
- 33. Sample to Insight Multiple studies investigating variation in FFPE sample processing 33 What impacts downstream results most?
- 34. Sample to Insight Which factor has the highest impact on DNA quantity measured? 34 Study design Samples 5 different rat tissue types 2–3 different blocks 5-6 x 3 sections, 10 µm thickness Assess variability caused by the samples themselves FFPE Samples Nanodrop QIAxpert Qubit QIAcube Manual w/ RNase digest w/o RNase digest DNA purification Automated using the QIAcube or manual processing QIAamp DNA FFPE Kit With and without RNase digest Assess variability introduced by operator (manual vs. automated) DNA quantification Using three different methods, 5 replicates/sample Assess variability caused by downstream quantification method In total 6000 data points!
- 35. Sample to Insight What has the highest influence on quantification of nucleic acids purified from FFPE samples? 35 %Contribution Quantification technology 0.2816 Purification method 0.2569 Tissue type 0.2288 RNase digest 0.1424 FFPE block 0.0542 FFPE section 0.0362 The chosen quantification technology matters most!
- 36. Sample to Insight Multiple studies investigating variation in FFPE sample processing 36 What impacts downstream results most?
- 37. Sample to Insight Background 37 Theory – denaturation of dsDNA Increasing temperature leads to denaturation of dsDNA to ssDNA Conversion factors for dsDNA: OD1 = 50 ng/µl and ssDNA: OD1 = 33 ng/µl Especially high temperature treatments have a denaturation effect on dsDNA ? Renaturation of DNA Applying high temperatures may lead to overestimation of gDNA quantities due to denaturation effects
- 38. Sample to Insight Experimental design Sample types: Human gDNA (~23 kb); input concentration of ~200 ng/µl Purified from Jurkat cells with QIAamp DNA Mini Kit Covaris-fragmented human gDNA (~150 bp); input concentration of ~180 ng/µl Samples were aliquoted and subjected to different temperatures for 10 min 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, 90°C, 98°C DNA samples Renaturation Concentration determination Temperature treatment DNA sample pool DNA sample 1.1 DNA sample 1.2 DNA sample 1.3 DNA sample 2.1 DNA sample 2.2 DNA sample 2.3 10 min, 30°C 10 min, 40°C … … gDNA or fragmented gDNA Temperature treatment 38
- 39. Sample to Insight Experimental design Subsequent to temperature treatment, three renaturation conditions were applied to each sample: ≥ 10 min on ice ramp (1°C/min) down to 20°C ramp (1°C/min) down to 20°C + 7 days @ room temp. Concentration determination: measurement in quintuplicates on: QIAxpert with DNA QIAamp App (UV/VIS-based) Nanodrop8000 with DNA-50 Method (UV/VIS-based) Qubit with dsDNA BR Assay (dye-based) Tecan reader with Picogreen Assay DNA samples Renaturation Concentration determination Temperature treatmentDNA sample 1.1 DNA sample 1.2 DNA sample 1.3 DNA sample 2.1 DNA sample 2.2 DNA sample 2.3 10 min, 30°C 10 min, 40°C … … Temperature treatment Renaturation ≥ 10 min on ice ramp to 20°C ramp to 20°C + 7 days ≥ 10 min on ice ramp to 20°C ramp to 20°C + 7 days Concentration determination … 39
- 40. Sample to Insight 40 Human gDNA – incubation on ice Temperature effects visible from 80°C onwards
- 41. Sample to Insight 41 Human gDNA – slow cool down to 20°C Slow cool down with little impact on renaturation efficiency
- 42. Sample to Insight Outline 42 The reproducibility challenge & its impact1 Every step of the workflow counts 2 3 Wrap-up 4 5 The importance of Quality Control Things you can and cannot influence
- 43. Sample to Insight Wrap-up 43 FFPE samples are a challenging starting material Some prerequisites for optimal results may be out of your sphere of influence Be aware of possible pitfalls Standardization and Quality Control are highly important Yes, we do provide solutions to help you overcome challenges associated with FFPE samples!
- 44. Sample to Insight QIAGEN Kits & Automated Solutions for your research 44 Sample Insight Sample collection Sample stabilization NA purification Assay & Analysis Data interpretation Instruments NA Purification • QIAcube • QIAsymphony SP/RGQ Quality Control • QIAxcel Advanced • QIAxpert Assay Setup • QIAgility • QIAsymphony AS Detection • Rotor-Gene Q • PyroMark Systems Kits DNA • QIAamp DNA FFPE Kit • GeneRead DNA FFPE Kit RNA • RNeasy FFPE Kits • miRNeasy FFPE Kit AllPrep • DNA/RNA FFPE Kit REPLI-g • FFPE Kits EpiTect Fast • FFPE Kit cDNA Synthesis • QuantiNova Reverse Transcription Kit • miScript RT Kit QuantiNova • PCR Kits • RT-PCR Kits qBiomarker • Somatic Mutation PCR Arrays RT2 • Arrays/Assays miScript • RT Kit • PCR Arrays/Assays • Ingenuity Pathway Analysis (IPA) • GeneGloba Data Analysis Center RNAlater Tissue Protect Tubes PAXgene • Blood RNA Tubes • Bone Marrow RNA • Tissue miRNA • Blood DNA Tubes • Tissue Containers
- 45. Sample to Insight 45 Questions? Thank You for Attending! All our solutions from Sample to Insight on: QIAGEN.com Dr. Vishwadeepak Tripathi Vishwadeepak.Tripathi@qiagen.com