Precision Medicine andPrecision Medicine and Next Generation Sequencing: Next Generation Sequencing:

Practical ApplicationsPractical Applications

John Pfeifer, MD, PhDJohn Pfeifer, MD, PhDWashington University School of MedicineWashington University School of Medicine

Department of PathologyDepartment of Pathology

PreliminarPreliminary ADASP y ADASP ProgramProgram

(Oct. (Oct. 2007)2007)

Association of Directors of Anatomic and Surgical Pathology Saturday, March 1, 2008

Hyatt Regency Hotel HH Agate A-C

0730- 1730, MT Denver, Colorado

Proposed Speaker 0730- 0800 Continental Breakfast THREATS and OPPORTUNITI ES in ACADEMIC PATHOLOGY 0800-0805 Welcome and Introduction Dr Otis 0805-0835 Generational expectations, X, Y, Z Dr Wick 0835-0905 Medical School Curricula Changes/ Deficiencies Dr De Young Residency Training 0905-0935 Gaps in Resident Training Dr Talbert 0935-1005 Discussion and Break membership 1005-1025 Opportunities for Resident Training I mprovement Dr Farver Threats to Academic Attractiveness 1025-1055 Why Enter Academics Dr Brownlee 1055-1125 Solutions and Uncertainties Dr Moskaluk 1125-1200 Discussion membership 1200-1330 LUNCH (HH Mineral B) Pathology Business Innoivations and Competition 1330-1400 Laboratory Niche and Competitive Advantages Bernie Ness 1400-1430 Opportunities for Counter Competition Dr Worsham 1430-1450 Collaborative Opportunities and Solutions Dr Myers Technological Advances and Threats to Pathology 1450-1520 New Imaging Advances Dr Deryk 1520-1550 Competition from Molecular Pathology Dr Pfeifer 1550-1610 Discussion and Break 1610-1630 Molecular Pathology Opportunities Dr 1630-1700 Discussion membership 1700-1715 Business Meeting Dr Otis 1730-1930 Reception and Adjourn (Douglas Pavillion D) membership

PreliminarPreliminary ADASP y ADASP ProgramProgram

(Jan. (Jan. 2008)2008)

Association of Directors of Anatomic and Surgical Pathology Saturday, March 1, 2008

Hyatt Regency Hotel HH Agate A-C

0730- 1730, MT Denver, Colorado

Proposed Speaker 0730- 0800 Continental Breakfast THREATS and OPPORTUNITI ES in ACADEMIC PATHOLOGY 0800-0805 Welcome and Introduction Dr Otis 0805-0835 Generational expectations, X, Y, Z Dr Wick 0835-0905 Medical School Curricula Changes/ Deficiencies Dr De Young Residency Training 0905-0935 Gaps in Resident Training Dr Talbert 0935-1005 Discussion and Break membership 1005-1025 Opportunities for Resident Training I mprovement Dr Farver Threats to Academic Attractiveness 1025-1055 Why Enter Academics Dr Brownlee 1055-1125 Solutions and Uncertainties Dr Moskaluk 1125-1200 Discussion membership 1200-1330 LUNCH (HH Mineral B) Pathology Business Innoivations and Competition 1330-1400 Laboratory Niche and Competitive Advantages Bernie Ness 1400-1430 Opportunities for Counter Competition Dr Worsham 1430-1450 Collaborative Opportunities and Solutions Dr Myers Technological Advances and Threats to Pathology 1450-1520 New Imaging Advances Dr Deryk 1520-1550 Competition from Molecular Pathology Dr Pfeifer 1550-1610 Discussion and Break 1610-1630 Molecular Pathology Opportunities Dr 1630-1700 Discussion membership 1700-1715 Business Meeting Dr Otis 1730-1930 Reception and Adjourn (Douglas Pavillion D) membership

Many of the perceived challenges mix Many of the perceived challenges mix together diagnostic, prognostic, and together diagnostic, prognostic, and

therapeutic implications, which fosters therapeutic implications, which fosters uncertainty and anxietyuncertainty and anxiety

““We have recently entered a transition We have recently entered a transition period in which specific genetic knowledge period in which specific genetic knowledge is becoming critical to the delivery of is becoming critical to the delivery of effective health care for everyone.”effective health care for everyone.”

Guttmacher AE, et al. Guttmacher AE, et al. N Engl J N Engl J MedMed 2002;347:1512-1520 2002;347:1512-1520

“… “… the impending revolution in molecular the impending revolution in molecular and computational biology is comparable and computational biology is comparable with previous periods of sweeping change in with previous periods of sweeping change in diagnostic pathology.”diagnostic pathology.”

Finn WG. Finn WG. J Mol DiagnJ Mol Diagn 2007;9:431- 2007;9:431-436436

Patient CarePatient Care

Growing complexity of recognized genetic Growing complexity of recognized genetic aberrations characteristic of specific diseases aberrations characteristic of specific diseases offers opportunities to develop new clinical offers opportunities to develop new clinical molecular testing paradigmsmolecular testing paradigms

Growing complexity of molecular testing Growing complexity of molecular testing paradigms offers opportunities for defining paradigms offers opportunities for defining indications for testing and test interpretationindications for testing and test interpretation

Growing demand creates opportunities for Growing demand creates opportunities for possible new revenue streams possible new revenue streams

Research opportunitiesResearch opportunities

““Considering the fact that cell function Considering the fact that cell function is controlled by a complex network of is controlled by a complex network of functionally active signaling pathways, functionally active signaling pathways, it is unlikely that expression analysis it is unlikely that expression analysis of a single or small number of proteins of a single or small number of proteins will precisely predict the clinical will precisely predict the clinical outcome of an individual tumor.”outcome of an individual tumor.”

Dietel M, et al. Dietel M, et al. Virchows ArchVirchows Arch 2006;448:744-755 2006;448:744-755

Opportunities for Education:Opportunities for Education:Residents and FellowsResidents and Fellows

Community hospital pathologists were recently asked Community hospital pathologists were recently asked to identify the skills and knowledge they expect of a to identify the skills and knowledge they expect of a newly minted pathologist:newly minted pathologist:

Since… Since… many groups are doing at least PCR, FISH, ISH, and many groups are doing at least PCR, FISH, ISH, and

cytogeneticscytogenetics clinicians are demanding molecular testingclinicians are demanding molecular testing

Groups depend on young pathologists to…Groups depend on young pathologists to… bring these techniques with them from the universitybring these techniques with them from the university know the indications for testingknow the indications for testing interpret the resultsinterpret the results

Horowitz RE. Hum Pathol 2006;37:969-973

Pathology

Microarrays

Cytogenetics

Next Gen Sequencin

g

SangerSequencin

g

Biomedical

Informatics

Comprehensive Cancer Set

•Have an established role in patient care for diagnosis, prognosis, or therapy•Comparable performance with FFPE and frozen samples•Analysis of small biopsy & cytology specimens•Detect variants with as little as 20% tumor cellularity•1000x fold sequence read depth•25-30 fold sample multiplexing to reduce cost•Comprehensive informatics and interpretive reporting•Currently reimbursed by Sanger methodology

Requirements

Practical Applications:Practical Applications:The first thing practicing The first thing practicing

pathologists need to know is that pathologists need to know is that there is no need for anxietythere is no need for anxiety

Only about 6% of cases need molecular testingOnly about 6% of cases need molecular testing In most cases tested, morphology is required to In most cases tested, morphology is required to

establish the need for molecular analysisestablish the need for molecular analysis In most cases tested, morphology is required to identify In most cases tested, morphology is required to identify

tissue to be evaluated tissue to be evaluated Even in cases tested, molecular analysis can not Even in cases tested, molecular analysis can not

currently be employed to establish stage (depth of currently be employed to establish stage (depth of invasion) or is untenable as a routine method for invasion) or is untenable as a routine method for establishing stage (status of all lymph nodes)establishing stage (status of all lymph nodes)

Even in cases tested, molecular analysis can not be Even in cases tested, molecular analysis can not be used to evaluate the margins of excisionused to evaluate the margins of excision

Need more Pathologists than are availableNeed more Pathologists than are available

CAP, AMP, ACMG, and so on, have committees CAP, AMP, ACMG, and so on, have committees working in the personalized medicine landscapeworking in the personalized medicine landscape

TRIG and other non-institutional working groups TRIG and other non-institutional working groups are developing teaching materialsare developing teaching materials

The CAP checklist for NGS was released The CAP checklist for NGS was released summer 2012; the ACMG guidelines for NGS summer 2012; the ACMG guidelines for NGS were just released for commentwere just released for comment

Published guidelines are being Published guidelines are being developed that address the developed that address the required fund of knowledgerequired fund of knowledge

The American Board of Pathology The American Board of Pathology has yet to provide guidancehas yet to provide guidance

http://www.abpath.org/MOCBofI.pdf

Section III Cognitive Expertise of the ABP’s “Maintenance of Certification Booklet of Information” states that: E. The ABP recognizes the breadth of pathology practice.1. The primary examinations (AP, CP, AP/CP) will be modular and the diplomats will be able to select modules that are as relevant as possible to individual practice settings.2. The subspecialty MOC examinations in Hematology, Molecular Genetic Pathology, Neuropathology, and Pediatric Pathology will be modular. The remainder of the subspecialty examination will consist of 150 questions covering the general practice of the subspecialty.3. For both primary and subspecialty exams, all modules will be graded together as one examination.4. See “MOC Presentation” on the MOC section of the ABP Web site for a proposed list of modules. These are subject to change prior to administration of the first examination in 2014.F. The exact nature of the modules to be provided is under development…

PathologyAssessment

Accessioning

DNA Isolation

Tissue Sample

Submission

DNA Sample

Submission

Library Submissio

n

DataSubmissio

n

LibraryPreparatio

n

Sequencing

BioinformaticAnalysis

Tier 1•Base Calling•Alignment•Variant Calling

Tier 2•Annotation•Knowledgebase

Tier 3•Clinical Report

Clinical Samples

Practicing pathologists who do Practicing pathologists who do genomic pathology need some level genomic pathology need some level of technical and genetic expertise of technical and genetic expertise

Familiarity with different platforms (specifically those marketed by Familiarity with different platforms (specifically those marketed by Illumina and Life Technologies)Illumina and Life Technologies) Differences in turn around timeDifferences in turn around time Differences in error rateDifferences in error rate Differences in capacityDifferences in capacity

Understanding of the differences in hybridization-based approaches Understanding of the differences in hybridization-based approaches versus amplification-based approachesversus amplification-based approaches

Use of panels, versus exomes, versus genomesUse of panels, versus exomes, versus genomes

Subspecialty training in molecular pathologySubspecialty training in molecular pathology

Practicing pathologists who do Practicing pathologists who do genomic pathology also need some genomic pathology also need some

level of bioinformatic expertise level of bioinformatic expertise

Implications of depth of coverage for detecting germline Implications of depth of coverage for detecting germline mutations versus acquired mutationsmutations versus acquired mutations

Optimization and clinical validation of bioinformatic pipelinesOptimization and clinical validation of bioinformatic pipelines Differences in bioinformatic approaches for SNVs, indels, Differences in bioinformatic approaches for SNVs, indels,

CNVs, and translocationsCNVs, and translocations Impact of nucleic acid quantity and quality on Bayesian Impact of nucleic acid quantity and quality on Bayesian

metricsmetrics Different -omic techniquesDifferent -omic techniques

Wood LD, et al. Wood LD, et al. ScienceScience 2007;318:1109-1113 2007;318:1109-1113International HapMap Consortium. International HapMap Consortium. NatureNature 2007;449:851-8612007;449:851-861Korbel JO, et al. Korbel JO, et al. ScienceScience 2007;318:420-426 2007;318:420-426West RB, et al. West RB, et al. Lab InvestLab Invest 2007;87:967-970 2007;87:967-970

Detection of indels varies by software Detection of indels varies by software tool: FLT3 ITD detection is an exampletool: FLT3 ITD detection is an example

• Tested a set of 24 cases with known FLT3 ITDs by WUCaMP28

• Pindel correctly identified ITDs in 23/24 cases

• Identified all ITDs (24/24) by de-novo assembly methods (detected allele frequencies of less than 4%)

• No false positive results

Reference: Spencer DH et al. J Mol Diagn 2013;15:81-93

red= not detectedgreen=detected

Pathologists will need to be Pathologists will need to be familiar with a range of -omics familiar with a range of -omics

techniquestechniques Genomics (DNA) Genomics (DNA) Transcripomics (mRNA)Transcripomics (mRNA) Interferomics (iRNA)Interferomics (iRNA) EpigenomicsEpigenomics Tumor cells (oncomics) versus stroma (stromics)Tumor cells (oncomics) versus stroma (stromics)

Proteins versus nucleic acids (eg, proteomics Proteins versus nucleic acids (eg, proteomics

and metabolomics)and metabolomics)

Practicing pathologists who do genomic Practicing pathologists who do genomic pathology will need to understand their pathology will need to understand their central educational role to their clinical central educational role to their clinical

colleagues colleagues In a model system evaluating the use of In a model system evaluating the use of

APC testing,APC testing, Only 83% of patients had valid reasons for testing Only 83% of patients had valid reasons for testing

(clinical features of familial adenomatous polyposis (clinical features of familial adenomatous polyposis or were at risk for the disease)or were at risk for the disease)

The appropriate strategy for pre symptomatic The appropriate strategy for pre symptomatic testing was used in 79%, only 19% received genetic testing was used in 79%, only 19% received genetic counseling before the test, and only 17% provided counseling before the test, and only 17% provided written informed consentwritten informed consent

In 32% of the cases the physicians misinterpreted In 32% of the cases the physicians misinterpreted the test resultsthe test results

Giardiello FM, et al. N Engl J Med 1997;336:823-7

Practicing pathologists who do genomic Practicing pathologists who do genomic pathology will need to understand their pathology will need to understand their central educational role extends beyond central educational role extends beyond

their clinical colleagues to include their clinical colleagues to include

As a source of professional information As a source of professional information regarding so called “recreational genomics” regarding so called “recreational genomics” which may causewhich may cause Needless worryNeedless worry Poor medical decisionsPoor medical decisions DiscriminationDiscrimination Potential for litigation (eg, right to know, Potential for litigation (eg, right to know,

relatedness)relatedness)

Kaiser J. Science 2007;318:1843

A concluding note of A concluding note of caution…caution…

“… “… the four letters of the genetic code the four letters of the genetic code are H, Y, P, and E, and medical providers are H, Y, P, and E, and medical providers must realize that the molecular biology must realize that the molecular biology business is as adept at promoting its business is as adept at promoting its wares as is any other.”wares as is any other.”

Jones S. http://www.milbank.org/reports/000712genetics.htmlJones S. http://www.milbank.org/reports/000712genetics.html