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Bioinformatics for the Practicing PathologistGregory C. Critchfield, MD, MS

Walter Noll, MD

© College of American Pathologists 2004. Materials are used with the permission of Gregory C. Critchfield, MD, MS, FCAP.

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Disclosure

• Dr. Critchfield is an officer, employee and shareholder of Myriad Genetics, Inc.

• Dr. Noll is an employee and shareholder of Myriad Genetics, Inc.

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Course Objectives

• List Web-based bioinformatics tools and resources used in the evaluation of molecular pathology diagnostic data

• Apply bioinformatics tools and techniques in a case of hereditary cancer

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Agenda

12:00 – 12:05Summary and Closing

10:30Opening/ Introductions

11:20 – 12:00Beyond Paper: Electronic MedicalRecord

11:15-11:20Break10:40 – 11:15Bioinformatics Case Presentation10:35-10:40Introduction to Bioinformatics

TimeTopic

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Human Genome

• 3.12 billion base pairs• 30,000 – 40,000 genes estimated

• Few genes are well characterized

• The Bioinformatics Challenge: Characterize genes and gene-gene interactions in disease pathways• Then use information clinically: diagnostics and

therapeutics

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DNA Diagnostics

• Goal: Discover useful information from human DNA

• Bioinformatics: Discipline using computers and statistics to extract meaningful information from DNA

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Current Health CareReactive

Current Health CareCurrent Health CareReactiveReactive

New ParadigmProactive

New ParadigmNew ParadigmProactiveProactive

Predictive Medicine

Improved Response & Improved Response & SurvivalSurvival

Diagnosis Diagnosis Genetic Risk ProfileGenetic Risk Profile

Treatment Treatment Preventive ActionPreventive Action

MonitorinMonitoring g Earlier DiagnosisEarlier Diagnosis

Customize TreatmentCustomize Treatment

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Hereditary Breast and Ovarian Cancer

• 7% of breast cancer cases and 10% of ovarian cancer cases are associated with an autosomal dominant pattern of inheritance

• BRCA1 and BRCA2 mutations account for 84% of hereditary breast cancer and the majority of hereditary ovarian cancer

• Most BRCA mutations are unique, but important “founder” mutations are present in some populations

• A small number of cases of hereditary breast cancer can be attributed to mutations in TP53 (Li-Fraumeni) and PTEN (Cowden)

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Case Presentation• 35 year-old woman with newly diagnosed

Stage I breast cancer

• Diagnosis?• Should HBOC be considered in this patient?

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Case Presentation (cont.)

• Premenopausal invasive ductal breast cancer, Stage I

• What additional information would be helpful?

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Case Summary

• Premenopausal invasive ductal breast cancer, Stage I

• Family history– No history of premenopausal breast or

ovarian cancer in extended family

• What is the risk that the patient has a BRCA1or BRCA2 mutation?

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BRCA1/BRCA2

• Discovered in 1994 and 1995 by Skolnick team—Myriad and other scientists

• Responsible for most hereditary breast/ovarian cancer syndrome cases

• Tumor suppressor genes– “Caretakers”– DNA double strand break repair; preservation of

chromosome structure [Venkitaraman AR. Cell 2002;108:171-182]

• Protein truncating mutations obliterate function of either gene

• Over 3,200 mutations described in BIC database: http://www.research.nhgri.nih.gov/bic/

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Statistics for Mutation Carriers

• Lifetime probability of breast cancer > 80%*• Lifetime probability of ovarian cancer*

– BRCA1 mutation: 54%– BRCA2 mutation: 23%

• Probability of second breast cancer: 65%• Probability of ovarian cancer with previous breast

cancer: 10x increase for mutation carriers

* King MC et al. Science 2003; 302:574-5. Breast and ovarian cancer risks due toinherited mutations in BRCA1 and BRCA2.

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Clinical Utility

• BRCA1/2 mutation highly accurate predictor of HBOC

• Effective cancer-preventive measures exist– Enhanced surveillance: mammography, MRI– Oral contraceptives reduce the risk of ovarian cancer but may increase the risk of

breast cancer (Narod SR. Nature Rev Cancer 2002;2:113-123.)– Tamoxifen reduces risk of contralateral breast cancer 50% (Narod SR et al. Lancet

2000;356:1876-1881.)– Prophylactic oophorectomy reduces risk of ovarian cancer 96% and breast cancer 50%

(Kauf et al. New Engl J Med 2002;346:1609-1615, Rebbeck et al. New Engl J Med 2002;346:1616-1622.)

– Prophylactic mastectomy reduces risk of breast cancer by at least 90% (Hartman et al. J Natl Cancer Inst 2001;93:1633-1637, Meijers-Heijboer et al. New Engl J Med 2001;345:159-164.)

• BRCA1/2 testing and preventive measures are cost-effective

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Links to BRCA Information

• Genetests – http://www.genetests.com• Myriad website – http://www.myriadtests.com• BIC database – http://research.nhgri.nih.gov/bic/• Genbank – http://www.ncbi.nih.gov/Genbank/

What is the risk that this individual carries a BRCAmutation?

• Myriad BRCA1/2 mutation prevalence tables (http://www.myriadtests.com/provider/mutprevo.htm; Frank et al. J Clin Oncol 2002;20:1480-1490.)

• BRCAPro(http://astor.som.jhmi.edu/BayesMendel/brcapro.html; Berry et al. J Clin Oncol 2002;20:2701-2712.)

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BRCA1/BRCA2 Prevalence Table

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Case Summary

• Pretest probability of BRCA1/2 mutation is high (183/2415 = 7.6%)

• ASCO guidelines suggest counseling for genetic testing when– Mutation risk is high (7.6% vs. 0.2%, a 38x

increased risk)– Testing can be interpreted– Clinical actions can confer benefit to patient

• What is recommendation to clinician?

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Genetic Counseling• ASCO guidelines for genetic testing for cancer

susceptibility (J Clin Oncol 2003;21:2397-2406)• Patient undergoes counseling and has questions

– What will I do with the results?– What kind of test is it?– How long does it take?– Does insurance pay for it?– Is insurance discrimination a problem?

• Patient elects to have test

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BRACAnalysis®

• Comprehensive sequence analysis– Labeled primers– Forward and reverse– Capillary electrophoresis– BRCA1: 5,400 bp; 22 exons + adjacent intronic

splice seq– BRCA2: 10,200 bp; 26 exons + adjacent intronic

splice seq– Total: 82 amplicons– Computer-based review; visual

inspection/confirmation x 2– Repeat analysis of all positives

• Large rearrangement panel: 5 recurrent large rearrangements in BRCA1

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Blood Blood samplesample

DNADNAIsolationIsolation

82 separate PCR 82 separate PCR reactions to reactions to

amplify 49 exons of amplify 49 exons of BRCA1 and BRCA1 and

BRCA2BRCA2

700 individual 700 individual Sequencing Reactions Sequencing Reactions (17,600 base pairs x 2)(17,600 base pairs x 2)

G T A C G T A C

Analyze data, Analyze data, compare to wildcompare to wild--typetype

F R

Repeat ifRepeat ifmutationmutation

foundfoundReportReport

Capillary Capillary ElectrophoresisElectrophoresis

BRACAnalysis®BRCA1 and BRCA2 Sequence Analysis

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Types of Mutations

• Frameshift: produces downstream stop codon(deleterious)

• Nonsense: produces stop codon (deleterious)• Missense: changes amino acid (deleterious, benign

polymorphism, unknown significance)• Large genomic rearrangements: delete or duplicate

one or more exons (deleterious)

• 92% of all deleterious mutations are protein-truncating

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Waveforms from Sequencing: Nonsense Mutation

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Waveforms from Sequencing: Frameshift Mutation

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Waveforms from BRCA1 Large Rearrangement Panel Genotyping

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Patient’s Analysis

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Example Report

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Bioinformatics in Genetic Tests

• Estimate of prior probability of mutation• Specimen tracking and chemical analysis• Quality process control• Interpretation of sequencing traces• Interpretation of genotypic analysis• Mutation naming and prevalence analysis• Reporting

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Web Resources (selected)• Bioinformatics glossary:

http://www.genomicglossaries.com/content/Bioinformatics_gloss.asp

• Department of Energy Primers on DNA/Human Genome Project: http://www.ornl.gov/sci/techresources/Human_Genome/publicat/primer/index.shtml

• Bioinformatics information/jobs/etc.: http://www.bioplanet.com/index.php

• Bioinformatics resource: http://www.utyx.com/bioinformatics/• Links to bioinformatics information:

http://www.ii.uib.no/~inge/list.html#soc• University of Colorado bioinformatics page:

http://www.colorado.edu/chemistry/bioinfo/• Weizman Institute bioinformatics:

http://bip.weizmann.ac.il/index.html• Gene expression links: http://industry.ebi.ac.uk/~alan/MicroArray/• Myriad web site: http://www.myriad.com

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Pearls of Pathology

• In a cancer of early onset, think “hereditary condition”—even in absence of strong family history

• Predictive testing allows strategies to address hereditary cancer risk

• Tools exist to assess pre-test risk of mutations in hereditary cancer genes

• Mutation status has important implications for management of hereditary cancer risks