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Sequencing Cancer Genomes

Sequencing Cancer Genomes. John A Pack. Overview. Introduction DNA Sequencing Circos Plot IDH1 Sequencing Genomes Examples of Sequenced Cancer Genomes Sequencing Disagreements Sequencing Proponents Small Scale Projects Impact Conclusions Future Research.

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Sequencing Cancer Genomes

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  1. Sequencing Cancer Genomes John A Pack

  2. Overview • Introduction • DNA Sequencing • Circos Plot • IDH1 • Sequencing Genomes • Examples of Sequenced Cancer Genomes • Sequencing Disagreements • Sequencing Proponents • Small Scale Projects • Impact • Conclusions • Future Research

  3. Leading Causes of Death in US • Heart Disease: 631,636 • Cancer: 559,888 • Stroke (cerebrovascular diseases): 137,119 • Chronic lower respiratory disease: 124,583 • Accidents (unintentional injuries): 121,599 • Diabetes: 72,449 Data for 2006 obtained from Centers for Disease Control and Prevention (CDC) (http://www.cdc.gov/nchs/fastats/lcod.htm)

  4. Genetics and Genomics Timeline http://www.nature.com/nature/journal/v422/n6934/full/nature01626.html

  5. What is DNA Sequencing • DNA • Made up of 3 billion chemical building blocks (A, T, C, and G) • DNA Sequencing • Process of determining the exact order of the building blocks that make up the DNA of the 24 different human chromosomes • Revealed the estimated 20,000-25,000 human genes within our DNA as well as the regions controlling them http://www.scq.ubc.ca/a-monks-flourishing-garden-the-basics-of-molecular-biology-explained/

  6. DNA Sequencing Process http://www.ornl.gov/sci/techresources/Human_Genome/graphics/DNASeq.Process.pdf

  7. Circos Plot • Circos is a software package for visualizing data and information • Used for identification and analysis of similarities and differences arising from comparisons of genomes Ledford, Heidi. “The Cancer Genome Challenge”.

  8. http://mkweb.bcgsc.ca/circos/

  9. http://mkweb.bcgsc.ca/circos/

  10. http://mkweb.bcgsc.ca/circos/

  11. Previous Discovery • Mutation in the gene IDH1 found in 2006 study of 35 colorectal cancers • Not expected to be of importance • Changed only a lowly housekeeping enzyme involved in metabolism • 13,000 other genes sequenced from each of 300 more samples http://waynesword.palomar.edu/molecu1.htm

  12. IDH1 Mutation Surfaced Again • 12% of samples of glioblastomamultiforme (type of brain cancer) • 8% of actue myeloid leukaemia samples http://www.mir.wustl.edu/neurorad/internal.asp?NavID=92 http://www.cancerhelp.org.uk/type/aml/about/the-blood-and-acute-myeloid-leukaemia

  13. Studying IDH1 Mutation • Studies showed the mutation changed the activity of isocirtratedehydrogenase • Caused a cancer-promoting metabolite to accumulate in cells • Pharmaceutical companies hunting for a drug to stop the process • IDH1 mutation is the inconspicuous needle found in a veritable haystack of cancer-associated mutations thanks to high powered genome sequencing. http://unitedcaremedical.com/services.shtml

  14. Sequencing Genomes • Labs around the world are teaming up to sequence DNA from thousands of tumors as well as healthy cells from the same person • Nearly 75 cancer genomes have at least begun to be to sequenced and published • By the end of 2010 researchers expect to have over 100 cancer genomes fully sequenced http://www.fhcrc.org/science/pacr/internships/index.html

  15. Difficulties in Researching • Further the research goes the larger the “haystack” • Comparison of tumor cell to healthy cell reveals dozens of single-letter changes, or point mutations • Comparison also reveals repeated, deleted, swapped, or inverted sequences http://activerain.com/blogs/davidhitt http://nl.ijs.si/et/talks/esslli02/metadata.html

  16. Difficulties • “The Difficulty is going to be figuring out hot to use the information to help people rather than to just catalogue lots and lots of mutations.” – Bert Voglestein, John Hopkins University • Clinically tumors can look the same but most differ genetically http://archive.sciencewatch.com/sept-oct2003/sw_sept-oct2003_page1.htm

  17. Distinguishing Mutation Data • Drivers – mutations that cause and accelerate cancers • Passengers – Accidental by-products and thwarted DNA-repair mechanisms • Distinguishing between the drivers and passengers is not always trivial http://www.modified.com/motorsports/0203scc_subaru_us_rally_team_petter_solberg/photo_02.html

  18. Finding Mutations • Mutations that pop up again and again • Identify key pathways that are mutated at different points • Finding more questions than answers • How do researchers decide which mutations are worthy of follow up and functional analysis? http://www.answersingenesis.org/home/area/cfol/ch2-mutations.asp

  19. World Collaboration • The International Cancer Genome Consortium Pilot Project • 11 Countries to sequence DNA • 20 cancer types • 500 tumor samples for each • Cost to sequence each cancer type = US$20 Million http://www.jonessoda.com/wordpress/?m=200904

  20. Ledford, Heidi. “The Cancer Genome Challenge”.

  21. Contributing Countries United States of America Britain • More than 6 types of cancer being sequenced • Ovarian Cancer • Brain Cancer • GlioblastomaMultiforme (IDH1 Mutation found in 12%) • Lung Cancer • Adenocarcinoma • Acute Myeloid Leukaemia (IDH1 Mutation found in 8%) • Colon Cancer • Adenocarcinoma • Others • Breast Cancer • ER-, PR-, HER- • Breast Cancer • Lobular • Breast Cancer • ER+, HER- • European Union Sponsored http://www.medicstravel.co.uk/countryhospitals/usacanada/usa_and_canada.htm http://www.state.gov/p/eur/ci/uk/

  22. Contributing Countries France Australia • Breast Cancer • HER2 overepxpressing • Liver Cancer • Alcohol-associated • Renal-cell carcinoma • European Union Sponsored • Pancreatic Cancer • Ductaladenocarcinoma • Ovarian Cancer http://www.state.gov/p/eap/ci/as/ http://www.state.gov/p/eur/ci/fr/

  23. http://www.thecommonwealth.org/YearbookHomeInternal/138389/ Contributing Countries Canada China • Pancreatic Cancer • Ductaladenocarcinoma • Gastric Cancer http://www.oiep.umd.edu/Training/chinaMap.html India Germany • Pediatric Brain Cancer • Medulloblastoma • PilocyticAstrocytoma • Oral Cancer • Gingivobuccal http://geology.com/world/germany-satellite-image.shtml http://www.state.gov/p/sca/ci/in/

  24. Contributing Countries Italy Japan • Rare Pancreatic Cancers • Enteropancreatic endocrine • Pancreatic exocrine • Liver Cancer • Virus- Associated http://www.state.gov/p/eur/ci/it/ http://www.state.gov/p/eap/ci/ja/ Spain • Chronic lymphocytic leukaemia http://www.state.gov/p/eur/ci/sp/

  25. ICGC • The International Cancer Genome Consortium (ICGC), est. 2008, combined two older, large scale projects • The Cancer Genome Project • Over 100 partial genomes and roughly 15 whole genomes. Tends to tackle over 2,000 more in the next 5-7 years • The US National Institutes of Health’s Cancer Genome Atlas (TCGA) • Sequence up to 500 tumors for each of 20 cancers over next 5 years http://www.icgc.org/ http://www.bfeedme.com/cancer-fighting-foods-spices/

  26. TCGA Pilot Project • The two groups in the TCGA are collaborating to sequence a subset of tumor samples (about 100) from each cancer type • The most promising areas of the genome will then be sequenced in the remaining 400 samples

  27. TCGA Network http://cancergenome.nih.gov/wwd/pilot_program/process.asp?processStyle=image

  28. From the Study • Larger sample numbers could provide driver mutations like the one in IDH1 • Knowledge and study of these mutations could lead to developing new cancer therapies according to researchers http://www.cancercompass.com/cancer-guide/complementary-therapies/complementary-therapies.html

  29. “If there are lots of abnormalities of a particular gene, the most likely explanation is often that those mutations have been selected for by the cancers and therefore are cancer-causing.” Michael Stratton (Co-Director of the Cancer Genome Project) http://www.icr.ac.uk/research/research_profiles/2750.shtml

  30. Challenging • IDH1 was first overlooked on the basis of the colorectal cancer data alone • Search expanded to other cancers before importance was revealed • Some drivers are mutated at very low frequency (less than 1% of the cancers) • heavy sampling is needed to find these low frequency drivers • Sequencing 500 samples per cancer reveal mutations present in as few as 3% of the tumors, but may still have important biological lessons • Need to know in order to understand the overall genomic landscape of cancer

  31. Another Popular Approach • Look for mutations that cluster in a pathway • In an analysis of 24 pancreatic cancers • 12 identified signaling pathways had been altered • Very difficult approach • Pathways overlap and boundaries not clear • Many pathways that are obtained using data from different animals or cell types do not always match up with what’s found in human tissue http://mkweb.bcgsc.ca/circos/

  32. There is A Lot More to Do • Distinguishing between drivers and passengers gets increasingly harder as researchers are beginning to sequence entire tumor genomes • Only a fraction of the existing cancer genomes have been completely sequenced http://www.lbl.gov/Science-Articles/Archive/sabl/2007/Jan/breast-cancer-genome.html

  33. Protein and Non-Protein Coding Regions • Most cancer genome sequences are only covering the exome • Keep costs low • Directly codes for protein (easiest to interpret) • Importance of mutations found in the non-protein coding depths • More challenging • Scientists don’t know what function these regions usually serve • Majority of mutations http://pandasthumb.org/archives/2005/12/another-example.html

  34. Cancer Genomes Coming Fast http://www.asa3.org/ASA/topics/Youth%20page/index.html • Some Full Genome have been Sequenced • Small-cell lung carcinoma (Type of Lung Cancer) • Metastatic melanoma (Type of Skin Cancer) • Basal-like breast cancer (Type of Breast Cancer) • Only exome has been sequenced • Glioblastomamultiforme (Type of Brain Cancer) http://www.mydochub.com/skin-cancer.php http://www.mir.wustl.edu/neurorad/internal.asp?NavID=92

  35. Lung CancerCancer: Small-Cell Lung Carcinoma • Sequenced: full genome • Source: NCI-H209 cell line • Point mutations: 22,910 • Point mutations in gene regions: 134 • Genomic rearrangements: 58 • Copy-number changes: 334 • Highlights: Duplication of the CHD7 gene confirmed in two other small-cell lung carcinoma cell lines http://scienceblog.cancerresearchuk.org/2009/12/16/skin-and-lung-cancer-genomes-are-truly-groundbreaking/

  36. Skin CancerCancer: Metastatic Melanoma • Sequenced: full genome • Source: COLO-829 cell line • Point mutations: 33,345 • Point mutations in gene regions: 292 • Genomic rearrangements: 51 • Copy-number changes: 41 • Highlights: Patterns of mutation reflect damage by ultraviolet light Ledford, Heidi. “The Cancer Genome Challenge”.

  37. Breast CancerCancer: Basal-Like Breast Cancer • Sequenced: full genome • Source: • primary tumor • brain metastasis • tumors transplanted into mice • Point mutations: • 27,173 in primary • 51,710 in metastasis • 109,078 in transplant • Point mutations in gene regions: • 200 • 225 • 328 • Genomic rearrangements: 34 • Copy-number changes: • 155 • 101 • 97 Ledford, Heidi. “The Cancer Genome Challenge”. • Highlights: Patterns of mutation reflect damage by ultraviolet light

  38. Brain CancerCancer: GlioblastomaMultiforme • Sequenced: exome (no complete Circos plot) • Source: • 7 patient tumors • 15 tumors transplanted into mice • Genes containing at least one protein altering mutation: 685 • Genes containing at least one protein altering point mutation: 644 • Copy-number changes: 281 • Highlights: Mutations in the active site of IDH1 have been found in 12% of patients http://www.mir.wustl.edu/neurorad/internal.asp?NavID=92

  39. Finding all mutations • Very important to find all, even in non-protein, regions • Maybe none of these mutations could pertain to the causation of cancer • Some could • Only way to find out is to systematically investigate them

  40. Researcher Disagreements • Some researchers Argue against fully sequencing genomes • Cost of projects outweighs the benefits • Prices will drop due to technology advances in next few years, why not wait? • In the mean time • Mutations that affect how many copies of a gene are found in a genome • Cheaper to assess • Provide more intuitive insight into biological processes http://www.shutterstock.com/pic-2585059/stock-photo-costs-outweigh-benefits.html

  41. Sequencing Proponents • Changes in genome copy number detection • Array-based technology • Fast and relatively inexpensive • Sequencing • Higher-resolution snapshot of regions • The higher resolution can provide • More precision in mapping boundaries • Ability to catch tiny duplications or deletions that an array may not detect

  42. Array-Based Process http://www.nature.com/scitable/content/diagram-of-the-microarray-based-comparative-genomic-41020

  43. Don’t Wait to Sequence • A lot of small scale hospitals are investing millions of dollars into cancer sequencing projects • (e.g.) St. Jude Children’s Research Hospital • Proponents don’t want to wait • The real work starts after the sequencing is over • Determining what these mutations are doing • Old-fashioned biology and experimental analysis http://www.stjude.org/stjude/v/index.jsp?vgnextoid=f2bfab46cb118010VgnVCM1000000e2015acRCRD http://www.the-aps.org/education/k-12misc/careers.htm

  44. US National Cancer Institute • Two 2-year projects • Develop high-throughput methods • Test how the mutations identified by the TCGA pilot project affect cell function • Aim to pull needles from the haystack and make since of them (like the IDH1 mutation) http://www.bfeedme.com/cancer-fighting-foods-spices/

  45. US National Cancer Institute Projects • Dana-Farber Cancer Center (Boston) • Systematically amplify and reduce the expression of genes of interest in cell cultures • Cold Spring Harbor Laboratory (New York) • Study cancer-associated mutations using tumors transplanted into mice http://www.gbaohn.org/files_for_update/Dana%20Farber-job.htm http://www.scivee.tv/user/7054

  46. Other Large Scale Projects • Asses effects of deleting each gene in the mouse genome • Learn more about the normal function of genes that are mutated in cancer http://thecoloringspot.com/animals/animals-set-7.html

  47. Impact • Global • Cancer is a world-wide disease • Cancer Patients • New Technology • New Treatment Processes • Researchers • More grants to make new advances

  48. Conclusions • Sequencing tumor DNA genomes can lead to finding cancer-causing gene mutations • Very challenging to pinpoint gene mutations that are cancer-causing • Very high sample numbers • Sampling and sequencing full cancer genomes is extremely expensive • Some opponents think the cost outweighs the benefits right now • A lot of people think the cost is worth it, because there is a lot more work to do after sequencing, so we should not wait for prices to come down

  49. Future Research • Better technology for making the sequencing equipment to bring costs down • New technology to detect mutations • Complete Full genome sequences for all cancers • Developing ways to stop or kill these mutations but leave the healthy cells unharmed • Nanotechnology (nanopharmaceuticals could have an impact here)

  50. References • Ledford, Heidi. “The Cancer Genome Challenge”. Nature Journal. Vol 464. 15 April 2010. p. 972-974. Macmillan Publishers Limited. 2010 • Human Genome Project Information. Facts About Genome Sequencing. Accessed: April 29, 2010. Last modified: September 19, 2008. http://www.ornl.gov/sci/techresources/Human_Genome/faq/seqfacts.shtml. • Krzywinski, M. et al. Circos: an Information Aesthetic for Comparative Genomics.Genome Res (2009) 19:1639-1645 • Francis S. Collins1, et al. “A vision for the future of genomics research”. Nature Publishing Group. 2010. Accessed April 30, 2010. http://www.nature.com/nature/journal/v422/n6934/full/nature01626.html • Circos Website. Acessed April 30, 2010. http://mkweb.bcgsc.ca/circos/

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