Title: Precision Medicine and Next Generation Sequencing: Practical Applications
1Precision Medicine and Next Generation
Sequencing Practical Applications
- John Pfeifer, MD, PhD
- Washington University School of Medicine
- Department of Pathology
2Preliminary ADASP Program(Oct. 2007)
3Preliminary ADASP Program(Jan. 2008)
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5Many 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
6Patient 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
7Research 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
8Opportunities 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
9Pathology
Cytogenetics
Microarrays
Next Gen Sequencing
Sanger Sequencing
Biomedical Informatics
10GPS 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
11Practical 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
12Published 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
13The 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
14Clinical 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
15Practicing 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
16Practicing 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
17Detection 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
18Pathologists 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)
19Practicing 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
20Practicing 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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22A 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