AMP COMPANION MEETING SYMPOSIUM AT USCAP 2015 NEXT-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, PhD Department of Pathology Washington 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
One the one hand, NGS is a technique for DNA sequence analysis 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
Formalin fixation does not affect NGS variant calls Concordance between FFPE and frozen 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 63 yo woman with prior history of oropharyngeal SCC, now with new lung SCC 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) Reference: Sehn JK, et al. Am J Surg Pathol 2014;38:534-541
ABL1 ALK APC ASXL1 ATM BRAF CEBPA CTNNB1 DNMT3A EGFR ERBB2 ESR1 FGFR4 FLT3 IDH1 IDH2 JAK2 KIT KRAS MAP2K2 MAPK1 MET MLL MPL MYC MYD88 NOTCH1 NPM1 NRAS PDGFRA PIK3CA PTEN PTPN11 RB1 RET RUNX1 TET2 TP53 VHL WT1 ABL1 ALK APC ASXL1 ATM BRAF CEBPA CTNNB1 DNMT3A EGFR ERBB2 ESR1 FGFR4 FLT3 IDH1 IDH2 JAK2 KIT KRAS MAP2K2 MAPK1 MET MLL MPL MYC MYD88 NOTCH1 NPM1 NRAS PDGFRA PIK3CA PTEN PTPN11 RB1 RET RUNX1 TET2 TP53 VHL WT1 ABCB1 ABCC2 ABL2 AKT1 AKT2 AKT3 ATRX BRCA1 BRCA2 CBL CDA CDH1 CDKN2A CDKN2B CHD7 CHIC2 CREBBP CRLF2 CSF1R CYP19A1 CYP2A6 CYP2B6 CYP2C19 CYP2C9 CYP2D6 DDR1 DDR2 DDX3X DPYD ERBB3 ERBB4 ERG ESR2 EZH2 FBXW7 FGFR1 FGFR2 FGFR3 FLT1 FLT4 FSTL5 GNA11 GNAQ GNAS GSTP1 H3F3 HNF1A HRAS IKZF1 IL2RA IL2RB IL2RG INPP4B JAK1 JAK3 KDM6A KDR LAMA2 LCK LTK MAP2K1 MAP2K4 MAP3K1 MED13 MLH1 MST1R MTOR NELL2 NF1 PDGFRB PHF6 PIK3R1 PSMB1 PSMB2 PSMB5 PSMD1 PSMD2 PTCH1 RAF1 RARA RARB RARG ROS1 RPS6KB1 RXRA RXRB RXRG SHH SHOC2 SLC22A1 SLC22A2 SLC31A1 SLC34A2 SLC45A3 SLCO1B1 SMAD4 SMARCA4 SMARCB1 SMO SNCAIP SOS1 SPRED1 SRC STK11 SUFU TAS2R38 TRRAP TYK2 UGT1A1 YES1 ZMYM3 ABCB1 ABCC2 ABL2 AKT1 AKT2 AKT3 ATRX BRCA1 BRCA2 CBL CDA CDH1 CDKN2A CDKN2B CHD7 CHIC2 CREBBP CRLF2 CSF1R CYP19A1 CYP2A6 CYP2B6 CYP2C19 CYP2C9 CYP2D6 DDR1 DDR2 DDX3X DPYD ERBB3 ERBB4 ERG ESR2 EZH2 FBXW7 FGFR1 FGFR2 FGFR3 FLT1 FLT4 FSTL5 GNA11 GNAQ GNAS GSTP1 H3F3 HNF1A HRAS IKZF1 IL2RA IL2RB IL2RG INPP4B JAK1 JAK3 KDM6A KDR LAMA2 LCK LTK MAP2K1 MAP2K4 MAP3K1 MED13 MLH1 MST1R MTOR NELL2 NF1 PDGFRB PHF6 PIK3R1 PSMB1 PSMB2 PSMB5 PSMD1 PSMD2 PTCH1 RAF1 RARA RARB RARG ROS1 RPS6KB1 RXRA RXRB RXRG SHH SHOC2 SLC22A1 SLC22A2 SLC31A1 SLC34A2 SLC45A3 SLCO1B1 SMAD4 SMARCA4 SMARCB1 SMO SNCAIP SOS1 SPRED1 SRC STK11 SUFU TAS2R38 TRRAP TYK2 UGT1A1 YES1 ZMYM3 Genes Reported Clinically (n = 40) Clinical Research Genes (n = 111) One the other hand, NGS can be a clinical test Comprehensive 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
Case Study: Results of NGS 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: 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 highgrade 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 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-toconsumer (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 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