Precision Medicine and Next Generation Sequencing: Practical Applications

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Precision Medicine and Next Generation
Sequencing Practical Applications

• John Pfeifer, MD, PhD
• Washington University School of Medicine
• Department of Pathology

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Preliminary ADASP Program(Oct. 2007)
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Preliminary ADASP Program(Jan. 2008)
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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

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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

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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

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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
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Pathology
Cytogenetics
Microarrays
Next Gen Sequencing
Sanger Sequencing
Biomedical Informatics
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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

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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

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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

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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
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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

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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

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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
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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
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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)

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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
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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
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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