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Title: Precision Medicine and Next Generation Sequencing: Practical Applications


1
Precision Medicine and Next Generation
Sequencing Practical Applications
  • John Pfeifer, MD, PhD
  • Washington University School of Medicine
  • Department of Pathology

2
Preliminary ADASP Program(Oct. 2007)
3
Preliminary ADASP Program(Jan. 2008)
4
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5
Many of the perceived challenges mix together
diagnostic, prognostic, and therapeutic
implications, which fosters uncertainty and
anxiety
  • We have recently entered a transition period in
    which specific genetic knowledge is becoming
    critical to the delivery of effective health care
    for everyone.
  • Guttmacher AE, et al. N Engl J Med
    20023471512-1520
  • the impending revolution in molecular and
    computational biology is comparable with previous
    periods of sweeping change in diagnostic
    pathology.
  • Finn WG. J Mol Diagn 20079431-436

6
Patient Care
  • Growing complexity of recognized genetic
    aberrations characteristic of specific diseases
    offers opportunities to develop new clinical
    molecular testing paradigms
  • Growing complexity of molecular testing paradigms
    offers opportunities for defining indications for
    testing and test interpretation
  • Growing demand creates opportunities for possible
    new revenue streams

7
Research opportunities
  • Considering the fact that cell function is
    controlled by a complex network of functionally
    active signaling pathways, it is unlikely that
    expression analysis of a single or small number
    of proteins will precisely predict the clinical
    outcome of an individual tumor.
  • Dietel M, et al. Virchows Arch 2006448744-755

8
Opportunities for EducationResidents and Fellows
  • Community hospital pathologists were recently
    asked to identify the skills and knowledge they
    expect of a newly minted pathologist
  • Since
  • many groups are doing at least PCR, FISH, ISH,
    and cytogenetics
  • clinicians are demanding molecular testing
  • Groups depend on young pathologists to
  • bring these techniques with them from the
    university
  • know the indications for testing
  • interpret the results

Horowitz RE. Hum Pathol 200637969-973
9
Pathology
Cytogenetics
Microarrays
Next Gen Sequencing
Sanger Sequencing
Biomedical Informatics
10
GPS Comprehensive Oncology Gene Set
Comprehensive Cancer Set
Requirements
  • 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

11
Practical ApplicationsThe first thing
practicing pathologists need to know is that
there is no need for anxiety
  • Only about 6 of cases need molecular testing
  • In most cases tested, morphology is required to
    establish the need for molecular analysis
  • In most cases tested, morphology is required to
    identify tissue to be evaluated
  • Even in cases tested, molecular analysis can not
    currently be employed to establish stage (depth
    of invasion) or is untenable as a routine method
    for establishing stage (status of all lymph
    nodes)
  • Even in cases tested, molecular analysis can not
    be used to evaluate the margins of excision
  • Need more Pathologists than are available

12
Published guidelines are being developed that
address the required fund of knowledge
  • CAP, AMP, ACMG, and so on, have committees
    working in the personalized medicine landscape
  • TRIG and other non-institutional working groups
    are developing teaching materials
  • The CAP checklist for NGS was released summer
    2012 the ACMG guidelines for NGS were just
    released for comment

13
The American Board of Pathology has yet to
provide guidance
Section III Cognitive Expertise of the ABPs
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
http//www.abpath.org/MOCBofI.pdf
14
Clinical Samples
Tissue Sample Submission
DNA Sample Submission
Library Submission
Data Submission
Pathology Assessment Accessioning DNA Isolation
Library Preparation
Sequencing
  • Bioinformatic
  • Analysis
  • Tier 1
  • Base Calling
  • Alignment
  • Variant Calling
  • Tier 2
  • Annotation
  • Knowledgebase
  • Tier 3
  • Clinical Report

15
Practicing pathologists who do genomic pathology
need some level of technical and genetic
expertise
  • Familiarity with different platforms
    (specifically those marketed by Illumina and Life
    Technologies)
  • Differences in turn around time
  • Differences in error rate
  • Differences in capacity
  • Understanding of the differences in
    hybridization-based approaches versus
    amplification-based approaches
  • Use of panels, versus exomes, versus genomes
  • Subspecialty training in molecular pathology

16
Practicing pathologists who do genomic pathology
also need some level of bioinformatic expertise
  • Implications of depth of coverage for detecting
    germline mutations versus acquired mutations
  • Optimization and clinical validation of
    bioinformatic pipelines
  • Differences in bioinformatic approaches for SNVs,
    indels, CNVs, and translocations
  • Impact of nucleic acid quantity and quality on
    Bayesian metrics
  • Different -omic techniques

Wood LD, et al. Science 20073181109-1113 Intern
ational HapMap Consortium. Nature
2007449851-861 Korbel JO, et al. Science
2007318420-426 West RB, et al. Lab Invest
200787967-970
17
Detection of indels varies by software tool 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

red not detected greendetected
Reference Spencer DH et al. J Mol Diagn
20131581-93
18
Pathologists will need to be familiar with a
range of -omics techniques
  • Genomics (DNA)
  • Transcripomics (mRNA)
  • Interferomics (iRNA)
  • Epigenomics
  • Tumor cells (oncomics) versus stroma (stromics)
  • Proteins versus nucleic acids (eg, proteomics and
    metabolomics)

19
Practicing pathologists who do genomic pathology
will need to understand their central educational
role to their clinical colleagues
  • In a model system evaluating the use of APC
    testing,
  • Only 83 of patients had valid reasons for
    testing (clinical features of familial
    adenomatous polyposis or were at risk for the
    disease)
  • The appropriate strategy for pre symptomatic
    testing was used in 79, only 19 received
    genetic counseling before the test, and only 17
    provided written informed consent
  • In 32 of the cases the physicians misinterpreted
    the test results

Giardiello FM, et al. N Engl J Med 1997336823-7
20
Practicing pathologists who do genomic pathology
will need to understand their central educational
role extends beyond their clinical colleagues to
include
  • As a source of professional information
    regarding so called recreational genomics which
    may cause
  • Needless worry
  • Poor medical decisions
  • Discrimination
  • Potential for litigation (eg, right to know,
    relatedness)

Kaiser J. Science 20073181843
21
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22
A concluding note of caution
  • the four letters of the genetic code
  • are H, Y, P, and E, and medical providers
  • must realize that the molecular biology
  • business is as adept at promoting its
  • wares as is any other.
  • Jones S. http//www.milbank.org/reports/000712gene
    tics.html
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