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MOLECULAR PATHOLOGY LABORATORY Next Generation Sequencing for High Yield AML and MDS Analysis Next Generation Sequencing for High Yield AML and MDS Analysis Aaron D. Bossler, MD, PhD Clinical Associate Professor Director, Molecular Pathology Laboratory Phone: 319-384-9566 aaron-bossler@uiowa.edu 1 Disclosure: Grant or research support received from: Iowa Department of Health, Roche Diagnostics and Cepheid, Inc.

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MOLECULAR PATHOLOGY LABORATORY Acknowledgements Anup Tilak, PhD Natasha Guseva, PhD Aaron Stence Jon Pruessner Connie Floerchinger Deqin Ma, MD, PhD Anthony Snow, MD

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MOLECULAR PATHOLOGY LABORATORY Overview Mutations

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MOLECULAR PATHOLOGY LABORATORY WHO 2008 AML Classification

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MOLECULAR PATHOLOGY LABORATORY Recurrent Mutations in AML NEJM.366.1079.2012.Patel

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MOLECULAR PATHOLOGY LABORATORY Recurrent Mutations in MDS Leuk.28.241.2014.Haferlach

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MOLECULAR PATHOLOGY LABORATORY Chips are biosensors with 1-11 million pH meters each Ion Torrent PGM Semiconductor Sequencing pH sensing semiconductor sequencing by synthesis 35400bp read lengths PCR-based target capture requires only 10ng of DNA Bar-coding to be able to run multiple specimens Fast sequencing times (< 1 day) Relatively inexpensive to operate Detection of substitutions and small insertion/deletion (

MOLECULAR PATHOLOGY LABORATORY Utility of Mutations in Epigenetic Regulators DNMT3A Mutations in 18-22% of AML, 29-34% in NK AML Prognosis: poorer outcomes especially with R882 mutation, more favorable outcome with hi dose daunorubicin IDH1 and IDH2 6-9% and 8-12% of AML, resp. Prognosis: IDH1 worse Overall and Event-free Survival in NK AML with favorable- (NPM1 mutated) or intermediate- risk disease IDH2 mostly worse outcome except for R140 mutation Clinical trials of mutant selective inhibitors are now beginning TET2 Mutations are found in MDS (>30%), MPN and AML (~10%) Prognosis: usually poorer outcomes ASXL1 Mutations in chronic myelomonocytic leukemia, myelodysplastic syndrome, and myeloproliferative neoplasms (20-25% of cases) 516% of older patients (those aged 60 years and older) with AML have ASXL1 mutations Associated with poor outcome in all studies reported to date

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MOLECULAR PATHOLOGY LABORATORY Survival Associations NEJM.366.1079.2012.Patel

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MOLECULAR PATHOLOGY LABORATORY Risk Classification of Patients with Intermediate-Risk AML ECOG E1900 trial 398 patients Showed improved outcomes using induction therapy or cytarabine and high dose daunorubicin in patients with newly diagnosed AML compared to low does daunorubicin Profiling was performed to identify genetic differences and prognosis

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MOLECULAR PATHOLOGY LABORATORY Risk Stratification Based on Genetics NEJM.366.1079.2012.Patel MLL-PTD: partial tandem duplication in MLL

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MOLECULAR PATHOLOGY LABORATORY 6. Spliceosome SF3B1, ZRSR2, SRSF2, U2AF1

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MOLECULAR PATHOLOGY LABORATORY RNA Splicing Int.J.Hematol.101.229.2015.Zoi

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MOLECULAR PATHOLOGY LABORATORY Spliceosome (ZRSR2, SF3B1, SRSF2, U2AF1) Mutations Can be gain of function through exon skipping or alternative splicing or loss of function by intron retention of target genes. More prevalent in secondary than in de novo AML Usually mutually exclusive - suggesting functional redundancy or combined lethal effect

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MOLECULAR PATHOLOGY LABORATORY Spliceosome Mutations 60 % of CMML cases harbor mutations 50% in SRSF2, 20% in ZRSF2, SF3B1, U2AF35, U2AF65 and SF3A1 SF3B1 mutations Significantly associated with appearance of ring sideroblasts and in 75% of RARS-T SRSF2 mutations cluster to hotspot residue Pro95 Co-occur with TET2, ASXL1, RUNX1, or IDH mutations Predict increased risk of progression from myeloproliferative neoplasms to secondary AML

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MOLECULAR PATHOLOGY LABORATORY SF3B1 in MDS Conveys Favorable Prognosis Blood.124.1513.2014.Malcovati

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MOLECULAR PATHOLOGY LABORATORY 7. SETBP3 Possibly involved in DNA replication 25% of aCML 10% advanced MDS 5% CMML Associated with poor overall survival and high risk of leukemic evolution Nat.Gen.45.18.2013.Piazza

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MOLECULAR PATHOLOGY LABORATORY Summary Favorable Prognosis NPM1 duplication/FLT3 wt CEBPA bi-allelic mutations GATA2 mutations DNMT3A when treated with high dose daunorubicin IDH1/IDH2 mutations SF3B1 Poor Prognosis RUNX1 mutations FLT3 ITD and TKD KIT exon 8 and 17 mutations DNMT3A R882 mutation ASXL1 mutations TET2 mutations SETBP1 mutations Association with MDS GATA2 CBL SF3B1, ZRSR2, SRSF2, U2AF1 SETBP1 Treatment/Clinical Trials FLT3 KIT RAS (via MEK or AKT inhibition)

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MOLECULAR PATHOLOGY LABORATORY UIHC AML/MDS 30 Gene Panel 43 Pathway / ClassificationGenes 1.NucleophosminNPM1 2.Myeloid Transcription FactorsRUNX1, CEBPA, GATA2 3.Activated SignalingFLT3, KIT, CBL, JAK2, KRAS, NRAS, HRAS, BRAF 4.Tumor SuppressorsTP53, PHF6, NF1, PTEN, PTPN11, WT1 5.Epigenetic Regulation/ Chromatin Modifiers DNMT3A, TET2, IDH1, IDH2, MLL, ASXL1, EZH2 6.SpliceosomeZRSR2, SF3B1, SRSF2, U2AF1 7.DNA ReplicationSETBP1

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MOLECULAR PATHOLOGY LABORATORY Summary Mutations

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MOLECULAR PATHOLOGY LABORATORY

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MOLECULAR PATHOLOGY LABORATORY Mutation Evolution in MDS Blood.122.3616.2013.Papaemmanuil