CRAVAT and MuPIT Interacve: Web Tools for Cancer Mutaon Analysis

Rachel Karchin, Ph.D. Department of Biomedical Engineering Instute for Computaonal Medicine Johns Hopkins University

The Cancer Genome Atlas’ 2nd Annual Scienfic Symposium November 27-28, 2012 Need for computaonal tools to analyze large-scale cancer mutaon data

2 Goal is to provide an end-to-end mutaon analysis workflow

List of mutaons from tumor sequencing

Idenfy type of Idenfy known Map to transcripts change (missense, variants and nonsense, silent) mutaons

Analysis

Predict driver vs. Predict funconal Visualize Find significantly random impact of mutaons on mutated genes mutaons mutaons terary structure and pathways

3 The majority of somac mutaons in tumor exomes are missense

Sjoblom et al 2006 Hunter et al 2005 Davies et al 2005 Stephens et al 2005 Sjoblom et al 2006 Missense

Nonsense

Silent

Splice

Other Colorectal Breast

Greenman et al 2007 Jones et al 2008 Parsons et al 2008 McLendon et al 2008 Ding et al 2008

Parsons et al 2011 Jones et al 2010 TCGA 2010 Stransky et al 2011 Li et al 2011

4 hp://www.cravat.us

Tools for evaluang missense mutaons – CHASM: cancer driver analysis hp://mupit.icm.jhu.edu – VEST: Funconal effect analysis – Annotaons (1000g, ESP6500, COSMIC, GeneCards, PubMed)

Interacve visualizaon on 3D protein structure – Automac mapping onto available structures – Simple interacve interface – UniProtKB feature table annotaons provided – Publicaon quality figures 5 Outline

• Introducon to CRAVAT • Introducon to MuPIT • Future plans • Your input

6 CRAVAT Web Server A+)%$&0$5<%"/&()$ 0'&5$%<5&'$ hp://www.cravat.us ).B<.(*+(3$ ,-.(/01$%1#.$&0$ ,-.(/01$9(&:($ !"#$%&$%'"()*'+#%)$ *2"(3.$45+)).().6$ ;"'+"(%)$"(-$ (&().().6$)+7.(%8$ 5<%"/&()$

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7 List of mutaons from tumor Input sequencing • Paste into text box or upload text file

• Genomic Coordinates – Unique id for variant – Chromosome – 0-based start posion – 1-based end posion – Strand on which bases are reported – Reference base – Alternate base RECOMMENDED – sample ID (oponal)

• Transcript Coordinates – Unique id for variant – Transcript – Amino Acid Substuon – sample ID (oponal)

8 Map to transcripts

• Transcripts are scored by coverage of coding bases and agreement between RefSeq and Ensembl transcript definions. • A greedy algorithm selects the “best transcript” for each mutated posion

Scores

0.557 0.557 0.378 0.378

0.731 0.685 0.731 0.731 0.731 0.731 0.685 0.731 0.685 0.731 0.731 0.731

Douville et al. CRAVAT: Cancer-Related Analysis of VAriants Toolkit. 2012 (submied) 9 Idenfy known variants and mutaons

Occurrenc ESP6500 ESP6500 1000 es in Amino Amino allele allele HUGO Genomes COSMIC Occurrences in COSMIC by primary sites [exact Transcript acid acid dbSNP frequency frequency symbol allele [exact nucleode change] posion change (European (African frequency nucleod American) American) e change] RALYL NM_173848.5 270 M270I 0 0 0 1 upper_aerodigesve_tract(1) TRIM29 NM_012101.3 216 P216H 0 0 0 1 breast(1) ZNF317 NM_001190791.1 280 G280V 0 0 0 1 large_intesne(1) CD248 NM_020404.2 115 T115N rs140616335 0 0 0.0007 0 SRPX2 NM_014467.2 393 R393Q 0 0 0.0007 0 KDR NM_002253.2 539 G539R rs55716939 0 0.0022 0.0005 0 LGI2 NM_018176.3 322 R322H rs149130054 0.0005 0.0001 0 0 LILRB1 NM_006669.3 189 P189L 0 0.0001 0 0

dbSNP 1K Genomes ESP6500 COSMIC

10 Predict driver vs. Analysis random mutaons Supervised machine learning!

Training Set

http://euvolution.com/futurist-transhuman-news-blog/

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Carter et al.. Cancer-specific high-throughput annotaon of somac mutaons. Cancer Research. 2009 11 Where do the decision rules come from?

amino acid substuon properes human polymorphism properes

predicted local protein structure

UniprotKB features regional amino acid composion http://euvolution.com/futurist-transhuman-news-blog/ vertebrate ortholog conservaon (genome alignments) superfamily homolog conservaon (deep protein alignments) amino acid compability with orthologs and homologs

86 features pre-computed exome-wide in SNVBox

Wong et al.. CHASM and SNVBox toolkit. Bioinformacs. 2011 12 Where does the training set come from ?

Driver missense mutaons: • Gene harbors at least 5 mutaons • Oncogene: >15% of NS mutaons occur at the same posion • Tumor suppressor: > 15% of NS mutaons are inacvang Bozic et al PNAS 2010 Jones et al Science 2010 • All missense mutaons in these genes are included Vogelstein et al. submitted

Random passenger missense mutaons: • Generated in silico with a generave model • Designed to match tumor ssue di-nucleode mutaon spectrum • In CHASM feature space, they do not look like high allele frequency SNPs

13 Tissue-specific classifiers in CRAVAT

• Select from pull down list

• Pre-constructed classifiers for tumor types under analysis by TCGA and ICGC

• ‘Other’ offers a generic classifier if ssue under study is not yet supported

14 Predict funconal Analysis impact of mutaons • Variant Effect Scoring Tool (VEST) – Idenfy funconal mutaons – Same supervised learning algorithm and features as CHASM – Different training set • 47000 disease missense mutaons from HGMD1 2012v2 • 45000 neutral missense variants from the ESP65002 (AF>1%)

1 Stenson P, Mort M, Ball E, Howells K, Phillips A, Thomas N, Cooper D, et al.: The human gene mutaon database: 2008 update. Genome Med 2009, 1:13. 2 Exome Variant Server, NHLBI GO Exome Sequencing Project (ESP), Seale, WA [hp://evs.gs.washington.edu/EVS/]. 15 Why VEST? !""#$%&'()#&*+'(%,$#

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Funconal Benign (Damaging) 17  Why VEST?

Funconal Benign (Damaging) Driver vs. Passenger and Damaging vs. Benign classifiers provide complementary benefits for mutaon analysis.

18 Find significantly Analysis mutated genes and pathways

• Gene annotaons – PubMed – GeneCards • Gene scores – Combined P-values of VEST or CHASM scores (Stouffer’s)

19 Submit

20 Job completed: Email Noficaon

21 CRAVAT Results

If MuPIT Interacve input file is requested • File with mutaons formaed for input to MuPIT Interacve 22 muPIT Interacve mupit.icm.jhu.edu

Input genomic coordinates of mutaons of interest

23 Table of protein structures onto which your mutaons can be mapped

A subset of mutaons did not map onto protein structures

Posion-specific annotaons available for each structure can be used to select most interesng structures. 24 Selecng a structure brings you to a details page for interacve viewing

Applet supports rotaon and zoom

Your mutaons Annotaons from UniProt feature table

25 Easy to use interface for customized figures Firehose breast cancer mutaons

Five mutaons cluster together on a structure of RUNX1 (in complex with CBFbeta)

26 Zoom-in for view of the mutaons (green) and a funconally annotated region (red)

Displayed Controls 27 Funconally annotated region with text display

28 29 Future Funconality

• Integraon of CRAVAT and MuPIT into a single end-to-end service • GANT - a new stasc to find significantly mutated genes and pathways • Exhausve mapping of genomic coordinates onto all protein coding transcripts defined at a locus

30 External integraons

Coming in 2013

31 Thank you for listening!

Please visit Poster #75 to talk with us about CRAVAT and MuPIT Please visit Poster #80 to learn about the Intogen-SM analysis pipeline from Lopez-Bigas Lab (Spain) 32 Acknowledgments

NIH R21 CA135866 NIH R21 CA152432 NIH 3U24CA143858-­-2S1 NSF CAREER DBI 0845275 33