John A Pack. Introduction DNA Sequencing Circos Plot IDH1 Sequencing Genomes Examples of Sequenced Cancer Genomes Sequencing Disagreements.

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

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)

Genetics and Genomics Timeline

http://www.nature.com/nature/journal/v422/n6934/full/nature01626.html

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/

DNA Sequencing Process

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

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”.

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

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

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

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

IDH1 Mutation Surfaced Again

12% of samples of glioblastoma multiforme (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

Studying IDH1 Mutation

Studies showed the mutation changed the activity of isocirtrate dehydrogenase 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

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

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://nl.ijs.si/et/talks/esslli02/metadata.html

http://activerain.com/blogs/davidhitt

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

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

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

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

Ledford, Heidi. “The Cancer Genome Challenge”.

Contributing Countries

UNITED STATES OF AMERICA

BRITAIN

More than 6 types of cancer being sequenced

Ovarian Cancer Brain Cancer

Glioblastoma Multiforme (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.state.gov/p/eur/ci/uk/

http://www.medicstravel.co.uk/countryhospitals/usacanada/usa_and_canada.htm

Contributing Countries

FRANCEAUSTRALIA

Breast Cancer HER2 overepxpressing

Liver Cancer Alcohol-associated

Renal-cell carcinoma European Union

Sponsored

Pancreatic Cancer Ductal

adenocarcinoma Ovarian Cancer

http://www.state.gov/p/eur/ci/fr/

http://www.state.gov/p/eap/ci/as/

Contributing Countries

CANADA CHINA Pancreatic Cancer

Ductal adenocarcinoma

Gastric Cancer

Germany• Pediatric Brain Cancer

– Medulloblastoma– Pilocytic Astrocytoma

• Oral Cancer– Gingivobuccal

India

http://www.thecommonwealth.org/YearbookHomeInternal/138389/

http://www.oiep.umd.edu/Training/chinaMap.html

http://geology.com/world/germany-satellite-image.shtml

http://www.state.gov/p/sca/ci/in/

Contributing Countries

ITALY JAPAN Rare Pancreatic

Cancers Enteropancreatic

endocrine Pancreatic exocrine

Liver Cancer Virus-

Associated

Spain• Chronic lymphocytic leukaemia

http://www.state.gov/p/eur/ci/it/ http://www.state.gov/p/eap/ci/ja/

http://www.state.gov/p/eur/ci/sp/

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.bfeedme.com/cancer-fighting-foods-spices/

http://www.icgc.org/

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

TCGA Network

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

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

“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

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

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/

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

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

Cancer Genomes Coming Fast

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 Glioblastoma multiforme

(Type of Brain Cancer)http://www.mir.wustl.edu/neurorad/internal.asp?NavID=92

http://www.mydochub.com/skin-cancer.php

http://www.asa3.org/ASA/topics/Youth%20page/index.html

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/

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”.

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

• Highlights:Patterns of mutation reflect damage by ultraviolet light

Ledford, Heidi. “The Cancer Genome

Challenge”.

Brain CancerCancer: Glioblastoma Multiforme 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

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

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

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

Array-Based Process

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

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

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/

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

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

Impact

Global Cancer is a world-wide disease

Cancer Patients New Technology New Treatment Processes

Researchers More grants to make new advances

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

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)

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/