Overview Next Generation Sequencing and Precision Medicine in Hematological Malignancies Sharathkumar Bhagavathi, MD University of Iowa Carver College of Medicine NGS as a genotyping platform in hematopathology Implementation of NGS Precision hematology Future directions William Beaumont Hospital_09_2017_DNA Symposium Genotyping in hematology Genotyping in hematology Diagnosis: JAK2, MPL, SF3B1 Prognosis: FLT3, NPM1, KIT Therapies: FLT3, IDH1, IDH2 At diagnosis For diagnosis For classification (WHO) For prognostication for targeted therapy During course of the disease For definition of remission For minimal residual disease To stop therapy To predict relapse MRD: NPM1, t(8;21), t(16;16) Methods Diagnostic challenge-discoveries in hematology Cytomorphology C Histology Immunophenotype Cytomorphology Cytogenetics FISH Molecular Grimwade et al. Blood 2016;127:29 41 1
Sequencing of AML NGS Workflow Library construction Clonal amplification Massive parallel sequencing Data analysis Ley TJ et al N Engl J Med 2013; 368:2059 2074 Workflow of RainDance ThunderBolts Myeloid Panel Assay Specimen preparation DNA extraction Library preparation Sequencing Data analysis Interpretation ~1 day* ~2 days* ~2 days* ~2 days* Sample type: Bone marrow and blood Bone marrow and blood: 200 L Manual DNA extraction using QIAmp DNA Blood Mini kit or Automated DNA extraction using QIAsymphony QC: Quantification of DNA using Trinean and Qubit. Normalization of DNA samples: 10 ng/ l Droplet formation using the RainDrop Source 1st PCR: droplet thermal cycling and amplification & 1st PCR SPRIselect bead purification 2nd PCR: addition of adaptors and index Sequences & 2nd PCR SPRIselect Bead Purification Quantification of samples using the Agilent Bioanalyzer prior to sequencing (QC) Final library and customized sequencing primer preparations Loading the MiSeq cartridge and paired end sequencing QC: determining the quality of sequencing using the Illumina Amplicon Viewer Alignment and variant Calling: NextGENE software (SoftGenetics) using hg19 as the reference genome. Determining the coverage of amplicons Filtering raw variant calling to generate final variant call report Review of variant call data by pathologists Categorizing variants using guidelines and recommendation from CAP, AMP and others Correlation of clinical, NGS, and other laboratory data by pathologists. RainDance Myeloid Panel RainDance Thunderbolts Myeloid panel TAT: 2 to 3 weeks Input DNA: 40 ug 548 amplicons 12 samples per run 49 genes Reporting Recommendations for further patients evaluation based on NGS data Recommendations may include further testing, new therapy, or clinical trial enrollment Report to submitting physician RainDance/ BioRad *Estimation based on 12 samples/run NGS Workflow A. Partially digest primer sequences B. Ligate adapters to the amplicons C. Purify the unamplified libraries D. Amplify purified libraries E. Amplify targets F. Purify the amplified libraries G. Quantify the amplified library with the Qubit 2.0 Fluorometer H. Dilute amplicon libraries I. Combine amplicon libraries Illumina Cluster generation by bridge amplification Clonal Amplification Emulsion PCR https://youtu.be/wybzbxifuks Fil te r B ThermoFisher (Life Technology) Emulsion oil BC P1 PCR A mix adapter target seq. ISP adapter Libra ry Mix Adapters position on ISP IS 5 3 B P P1 target X Key A B Barcode Barcoded Library Sequence Paired reads provide very accurate read alignment and improves accuracy 2
Sequencing Sequencing Platforms in our Laboratory Illumina ThermoFisher Illumina ThermoFisher reversible chain termination MiSeq NextSeq 500 Chef and S5XL Nucleotides flow sequentially over Ion semiconductor chip One sensor per well per sequencing reaction Direct detection of natural DNA extension Millions of sequencing reactions per chip Fast cycle time, real time detection Instrument MiSeq Next Seq S5XL Output (Gb) 5 20 120 10 15 Run time (hrs) 28 29 5 (+ 18) Reads per run (mln) 30 130 400 60 80 Exomes per run X100 0 4 2 Exomes per run X500 0 1 0 1 RainDance Myeloid Panel UIHC AML/MDS 30 Gene Panel Pathway / Classification Genes 1. Nucleophosmin NPM1 2. Myeloid Transcription Factors RUNX1, CEBPA, GATA2 3. Activated Signaling FLT3, KIT, CBL, JAK2, KRAS, NRAS, HRAS, BRAF 4. Tumor Suppressors TP53, PHF6, NF1, PTEN, PTPN11, WT1 5. Epigenetic Regulation/ Chromatin Modifiers DNMT3A, TET2, IDH1, IDH2, MLL, ASXL1, EZH2 6. Spliceosome ZRSR2, SF3B1, SRSF2, U2AF1 7. DNA Replication SETBP1 16 Data analyses NGS data analyses work flow Malignant cell DNA Mutations Indels RNA Fusion genes Expression analyses Mutations (cdna) Software tools & analyses Cosmic ClinVar Polypen SIFT dbsnp IARC Mutation taster IARC Report generation 3
Analysis Analysis BAM & VCF files Varseq Secure pipeline Variants N of one Clinical interpretation Report Analysis IGV Integrative Genomic Viewer Chromosome position Tumor seq Normal seq RefSeq Genes Amplicon design Clinical Sequencing at UIHC Molecular Oncology Menu DNA sequencing AmpliSeq targeted sequencing : CHPv2, MPCP, AML. Detection: SNV (variant frequency 5%), Small indels (variant frequency 10%) RNA sequencing: Archer Fusion PlexPanel: Sarcoma, CTL Panels Detect: Gene fusions SNV, small indels Large indels Gene Expression CNV Targeted mutation testing: ALK Fusion ASXL1 (MDS) Mutation Analysis B cell, IGH RAR, clonality by PCR (lymphoma) BCR ABL, t(9;22), RNA Quantitation (CML, ALL) BRAF mutation analysis by sequencing (melanoma, thyroid, colon, lung, HCL) BRAF Fusion (Pilocytic astrocytoma) CALR Mutation Analysis (MPN) CEBPA Mutation Analysis (AML) EGFR sequencing (Lung) FGFR3 Mutation Detection (bladder) FLT3 Mutation Detection (AML) HRAS Mutation Analysis (thyroid) IDH1 and IDH2 (AML, GBM, sarcoma) JAK2 V617F, ex12 Mutation Detection Assay (PCV) KIT Mutation Analysis by Sequencing (GIST, AML) KRAS Mutation Analysis (colon, thyroid, lung) MET ex14 Skipping Mutation Microsatellite Instability testing (CRC) MPL W515/S505 (MPN) MYD88 Mutation Detection (WM/LPL) NPM1 Mutation Detection (AML) NRAS Mutation Analysis (CRC, melanoma) PDGFRA (GIST) PIK3CA (Lung and breast) ROS1 fusion (NSCLC) TCRG RAR (T cell clonality) by PCR (lymphoma) Cancer mutation profiling: 50 gene hotspot panel 23 gene custom panel 30 gene leukemia specific panel 26 gene Sarcoma Fusion Panel 24 gene Melanoma hotspot panel 19 gene Lung Fusion Panel 4
Precision Medicine Precision Medicine Use of molecular characteristics (genetic, transcriptomics, proteomics) to prevent, diagnose and treat disease. Case History Case History 5/2009 Diagnosis of de novo AML M4 (AMML) Karyotype normal, chemotherapy & remission 9/2010 Relapse #1 discovered incidentally at 26 WGA of 6 th pregnancy Normal karyotype, NPM1 mutation positive (FLT3 normal) HiDAC chemotherapy with cytarabine during pregnancy & MUD HCT 12/17/2010 11/2016 Relapse #2 Karyotype with complex abnormalities [add(1)(p11.2), del(5)(p15), add(7)(q22), add(8)(q11.2), del(12)(q13), del(17)(p13), add(20)(p13)] Molecular: NPM1 (49%), TP53 P190L (51%), NRAS (35%), IDH1 R132H (47%) 11/9/2016 chemotherapy Bone marrows on 11/22/16 and 11/28/16 showed persistent leukemic blasts with additional karyotype abnormalities del(13)(q12q22) and add(19)(p13) 12/2016 Enrolled in clinical trial AG120 001 through Northwestern University One cycle with ivosidenib (IDH1 inhibitor) Complications (septic cardiomyopathy, seizures, etc) Removed from study Bone marrow 4/2017 50% cellular with <1% blasts; flow cytometry with 0.6% aberrant blasts, consistent with MRD positive clinical remission Case History, cont. 07/2017 Relapse #3 Karyotype with complex abnormalities & similar molecular results SEER Leukemia AML Started on IDH1 inhibitor (Ivosidenib) 5
Survival in heme malignancies Mutations in AML Leukemia Lymphoma Society Ley TJ, et al N Engl J Med 2013; 368:2059 2074 Targeted therapy Precision Hematology Ley TJ et al N Engl J Med 2013; 368:2059 2074 Haferlach, MLL Beat AML Ph like ALL Robert KG et al. N Engl J Med 2014;371:1005 1015 Jain N et al Blood 2017;129:572 581 Leukemia Lymphoma Society 6
Ph like ALL Minimal Residual Disease Robert KG et al. N Engl J Med 2014;371:1005 1015 Case history 65 yr/f with AML (04/2017) Cytogenetic & FISH: Normal Molecular: NPM1 (44%), TET2 (46%) Chemotherapy Bone marrows (04/17, 05/17, 06/17, 08/17): Remission Molecular (06/17): TET2 (6%) Chemo or BMT? Difficulties Complexities of molecular alterations and their interactions Quantifying NPM1 mutated transcripts for patients with NPM1 mutated AML Outcome may be better predicted by MRD monitoring than by extensive molecular profiling at diagnosis Whether or not MRD monitoring will actually improve outcomes in AML remains to be seen. MRD Detection of MRD by NPM1 Ivey A et al. N Engl J Med 2016;374:422-433 Ivey A et al. N Engl J Med 2016;374:422-433 7
Detection of MRD by NPM1 Droplet Digital PCR Ivey A et al. N Engl J Med 2016;374:422-433 Microfluidics technology allows the generation of thousands to millions of droplets Each droplet contains target DNA and/or background DNA in a random distribution along with primers, probes, and master mix Droplet Digital PCR Droplet fluorescence can be read with a flow cytometer Droplets are scored as positive or negative fluorescence above/below threshold Future Directions Chen WW, et al. BEAMing and Droplet Digital PCR Analysis of Mutant IDH1 mrna in Glioma Patient Serum and Cerebrospinal Fluid Extracellular Vesicles. Mol Ther Nucleic Acids. 2013 Jul 23;2:e109. NGS data analyses work flow Malignant cell Archer FusionPlex (NGS)? BRAF DNA Mutations Indels RNA Fusion genes Expression analyses Mutations (cdna) Software tools & analyses Report generation 2017 ArcherDX, Inc. 8
Archer FusionPlex Myeloid Kit Single molecule sequencing Helicos Pacific Biosciences Oxford Nanopore Technologies 10X Genomics Thank you 9