applications of next generation sequencing in solid …handouts.uscap.org/an2015/companion meeting...
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AMP COMPANION MEETING SYMPOSIUM AT USCAP 2015NEXT-GENERATION OF PATHOLOGY: ROLE OF
PATHOLOGIST IN NGS-BASED PERSONALIZED MEDICINE
APPLICATIONS OF NEXT GENERATION SEQUENCING IN SOLID TUMORS - PATHOLOGIST PROSPECTIVE
John D. Pfeifer, MD, PhDDepartment of PathologyWashington University School of Medicine
Disclosures
Vendor-Client relationships with:
• Illumina
• Agilent
• Life Technologies
• Affymetrix
Co-founder: PierianDx
Academic affiliation:•Washington University School of Medicine is an academic, tertiary care, urban medical center•GPS is a reference lab owned by the Department of Pathology that performs a range of molecular testing, including NGS
NGS performed at GPS@WUSTL is just one component of molecular testing performed by the lab
•Because it is by definition massively parallel, it provides the opportunity to evaluate panels of genes (in addition to single genes or small sets of genes)
•Other methods can also be used to evaluate panels of genes, but NGS provides more flexibility to detect a broader range of mutation types in a larger target region
•Can get reimbursed to do the testing, just like any test, if it is focused on clinically actionable information, and is not done on an experimental or investigational basis
One the one hand, NGS is a technique for DNA sequence analysis
Formalin fixation does not affect NGS variant calls
• Concordance between FFPE andfrozen tissue >99.99% for all positions
• 98.6% concordance for SNVs calls between FFPE and frozen tissue
• 100% Concordance between Array and NGS
• Variants unique to FFPE/frozen tissue represent tumor heterogeneity
Reference: Spencer D, et al. J Mol Diagn 2013;15:623-633
NGS also works from cytology samples
Comparison of Variant Calls
Reference: Karnes H, et al. Cancer Cytopathol 2014;122:104-113
When used merely as a sequencing method,NGS has diagnostic utility
Reference: Sehn JK, et al. Am J Surg Pathol 2014;38:534-541
Nonsynonymous, Nonpolymorphic Variants Identified by NGS:Oropharyngeal tumor: TP53 p.Y126* (c.378C>A)Oropharyngeal tumor TP53 p.P58R (c.173C>G) (germline)Oropharyngeal tumor NOTCH1 p.I2550V (c.7648A>G) (germline)Lung tumor CEBPA p.N307K (c.921C>G)Lung tumor TP53 p.N239S (c.716A>G)Lung tumor TP53 p.P58R (c.173C>G) (germline)Lung tumor NOTCH1 p.I2550V (c.7648A>G) (germline)
63 yo woman with prior history of oropharyngeal SCC, now with new lung SCC
ABL1ALKAPCASXL1ATM
BRAFCEBPACTNNB1DNMT3AEGFR
ERBB2ESR1FGFR4FLT3IDH1
IDH2JAK2KITKRASMAP2K2
MAPK1METMLLMPLMYC
MYD88NOTCH1NPM1NRASPDGFRA
PIK3CAPTENPTPN11RB1RET
RUNX1TET2TP53VHLWT1
ABL1ALKAPCASXL1ATM
BRAFCEBPACTNNB1DNMT3AEGFR
ERBB2ESR1FGFR4FLT3IDH1
IDH2JAK2KITKRASMAP2K2
MAPK1METMLLMPLMYC
MYD88NOTCH1NPM1NRASPDGFRA
PIK3CAPTENPTPN11RB1RET
RUNX1TET2TP53VHLWT1
ABCB1ABCC2ABL2AKT1AKT2AKT3ATRXBRCA1BRCA2CBLCDACDH1CDKN2ACDKN2B
CHD7CHIC2CREBBPCRLF2CSF1RCYP19A1CYP2A6CYP2B6CYP2C19CYP2C9CYP2D6 DDR1DDR2DDX3X
DPYD ERBB3ERBB4ERGESR2EZH2FBXW7FGFR1FGFR2FGFR3FLT1FLT4FSTL5GNA11
GNAQGNASGSTP1H3F3HNF1AHRASIKZF1IL2RAIL2RBIL2RGINPP4BJAK1JAK3KDM6A
KDRLAMA2LCKLTKMAP2K1MAP2K4MAP3K1MED13MLH1MST1RMTORNELL2NF1PDGFRB
PHF6PIK3R1PSMB1PSMB2PSMB5PSMD1PSMD2PTCH1RAF1RARARARBRARGROS1RPS6KB1
RXRARXRBRXRGSHHSHOC2SLC22A1SLC22A2SLC31A1SLC34A2SLC45A3SLCO1B1SMAD4SMARCA4SMARCB1
SMOSNCAIPSOS1SPRED1SRCSTK11SUFUTAS2R38TRRAPTYK2 UGT1A1YES1ZMYM3
ABCB1ABCC2ABL2AKT1AKT2AKT3ATRXBRCA1BRCA2CBLCDACDH1CDKN2ACDKN2B
CHD7CHIC2CREBBPCRLF2CSF1RCYP19A1CYP2A6CYP2B6CYP2C19CYP2C9CYP2D6 DDR1DDR2DDX3X
DPYD ERBB3ERBB4ERGESR2EZH2FBXW7FGFR1FGFR2FGFR3FLT1FLT4FSTL5GNA11
GNAQGNASGSTP1H3F3HNF1AHRASIKZF1IL2RAIL2RBIL2RGINPP4BJAK1JAK3KDM6A
KDRLAMA2LCKLTKMAP2K1MAP2K4MAP3K1MED13MLH1MST1RMTORNELL2NF1PDGFRB
PHF6PIK3R1PSMB1PSMB2PSMB5PSMD1PSMD2PTCH1RAF1RARARARBRARGROS1RPS6KB1
RXRARXRBRXRGSHHSHOC2SLC22A1SLC22A2SLC31A1SLC34A2SLC45A3SLCO1B1SMAD4SMARCA4SMARCB1
SMOSNCAIPSOS1SPRED1SRCSTK11SUFUTAS2R38TRRAPTYK2 UGT1A1YES1ZMYM3
Genes Reported Clinically (n = 40)
Clinical Research Genes (n = 111)
One the other hand, NGS can be a clinical testComprehensive Cancer Gene Set
Case Study: Patient with thymic carcinoma
58 yo woman diagnosed with widely metastatic thymic carcinoma
She had failed multiple rounds of conventional chemotherapy
Rapidly progressing mediastinal and liver disease
Comprehensive Oncology testing ordered in Spring 2012
NGS showed a KIT mutation Three bp deletion
resulting in p.D579del KIT mutations have not
been extensively reported in thymic carcinomas
Particular mutation predicted to respond to TK inhibitors
NGS Data
Sanger Confirmation
Case Study: Results of NGS
Case Study: Patient treated with imatinib
Tumor response seen in one month post treatment CT
Disease has since been stable for over two years (and counting)
Reference: Hagemann IS, et al. J Thorac Oncol 2014;9:12-16
Clinical “NGS” is not one thing
Analytically: Different platforms for “NGS” Different assay designs for “NGS” Different annotation and interpretation schemes for
an identified variant
Conceptually: NGS as a methodology NGS as a clinical test (which centers on intended use)
Different Platforms
NGS instruments utilize different chemical and physical properties to infer nucleic acid sequences…
• Illumina markets the HiSeq series, the NextSeq, and the MiSeq series (of which the MiSeqDx is the first FDA-cleared IVD NGS system); fluorescence from incorporated bases during strand elongation
• Life Technologies markets the Ion Proton, Ion PGM, and Ion Chef systems; semiconductor technology to measure pH changes during strand elongation
• And others under development (e.g., Oxford Nanopore markets the GridION and MinION systems; conductance changes resulting from strand migration through a pore)
… and each has it’s own intrinsic strengths, weaknesses, and sequencing artifacts.
Different platforms yield different results
Reference: Boland JF, et al. Hum Genet 2013;132:1153-1163
Different assay designs
Even using the same NGS platform, the two general approaches to library preparation markedly impact assay results…
Amplification-based assays (generally limited to target regions of 50 kb or so; well suited to detection of SNVs and small indels; lower DNA inputs are required)
Hybrid capture-based assays (well suited to target regions of all sizes up to the whole exome; well suited to detection of SNVs, indels, CNVs, and SVs; higher DNA inputs are required)
Illustration of the intended use issue: GPS “Comprehensive Cancer Gene Set” versus…
Original GPS (v1) panel had 25 genes; v2 panel has 40 genes; v3 panels is disease specific and generally about 10-40 genes, each with an established roll in patient care for Dx, Px, or Tx
Illumina TruSight Cancer is 94 genes by hybrid capture Foundation One is 315 genes by hybrid capture Illumina TruSight Tumor is hotspots from 26 genes by amplification AmpliSeq Cancer Panel is hotspots from 46 genes by amplification
Utility of NGS“Information Theory”
One way to measure information…
Define the NGS test as a random variable and measure uncertainty over the outcome of the test as the Shannon entropy:
where P(g|D=d) is the probability of a specific NGS test result g for a particular cancer type d, and G is the set of all genes tested.
Reference: Shannon CE: A Mathematical Theory of Communication. Bell System Technical Journal 1948, 27 (Part 1):379-423.
…and it turns out genomic sequencing is quite informative
NGS is most informative for colorectal cancer (2.722 bits of information/case) followed by high-grade glioma (2.236 bits), lung cancer (2.133 bits), sarcoma (0.965 bits) and pancreatic cancer (0.907 bits).
In the most informative tumor types, NGS testing has entropy similar to surgical pathology examination (modeled at 2–3 bits).
Reference: Hagemann IS, et al. Diagnostic Yield of Targeted Next-Generation Sequencing in Various Cancer Types: An Information-Theoretic Approach. Submitted.
Pathologists should recognize their central educational role in the setting of genetic testing 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%, but 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 1997;336:823-7
Pathologists need to recognize their central educational role in the setting of genetic testing extends beyond
their clinical colleagues
They are also a source of professional information regarding DTC testing and so-called “recreational genomics” which may cause: Needless worry Poor medical decisions Discrimination Potential for litigation (eg, right to know, relatedness)
Reference: Kaiser J. Science 2007;318:1843
February 19, 2015: FDA News Release. “The U.S. Food and Drug Administration today authorized for marketing 23andMe’s Bloom Syndrome carrier test, a direct-to-consumer (DTC) genetic test to determine whether a healthy person has a variant in a gene that could lead to their offspring inheriting the serious disorder…”