1. 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.

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

3. MOLECULAR PATHOLOGY LABORATORY Overview Mutations

4. MOLECULAR PATHOLOGY LABORATORY WHO 2008 AML Classification

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

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

7. MOLECULAR PATHOLOGY LABORATORY Chips are biosensors with 1-11 million pH meters each Ion Torrent PGM Semiconductor Sequencing pH sensing semiconductor sequencing by synthesis 35–400bp 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 (<30bp) mutations

8. MOLECULAR PATHOLOGY LABORATORY T A C template dNTP flow cycle Ion Torrent PGM: pH Measurement

9. MOLECULAR PATHOLOGY LABORATORY Ion Torrent PGM Semiconductor based Sequencing 9 Library construction Template clonal amplification Sequencing by PGM Data analysis

10. MOLECULAR PATHOLOGY LABORATORY

11. MOLECULAR PATHOLOGY LABORATORY Functional Groupings

12. MOLECULAR PATHOLOGY LABORATORY UIHC AML/MDS 30 Gene Panel 12 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

13. MOLECULAR PATHOLOGY LABORATORY 1. NPM1

14. MOLECULAR PATHOLOGY LABORATORY NPM1 Mutation Associated with Improved Survival; Meta-analysis Ann.Hematol.93.1279.2014.Port

15. MOLECULAR PATHOLOGY LABORATORY 2. Myeloid Transcription Factors CEBPA RUNX1 GATA2

16. MOLECULAR PATHOLOGY LABORATORY CEBPalpha 7-15% of AMLs have CEBPA mutations (most are single mutations) Double mutant/biallelic cases predict a favorable prognosis – Low frequency of other mutations or other cytogenetic abnormalities J.Clin.Onc.29.2739.2010.Green

17. MOLECULAR PATHOLOGY LABORATORY RUNX1 Alpha subunit of core binding factor transcription factor 13% of AMLs have RUNX1 mutations – Associated with poor outcomes in contrast to RUNX1 gene fusions, t(8;21) Prior H/o MDS or radiation exposure frequently Will become a WHO defined AML for de novo only J.Clin.Onc.30.3109.2012.Mendler < 60 yrs old≥ 60 yrs old

18. MOLECULAR PATHOLOGY LABORATORY GATA2 Indispensable transcription factor for hematopoiesis – Maintains the proliferative progenitor-cell phenotype Mutations are associated with bi-allelic CEBPA mutations and mostly favorable outcomes in cytogenetically normal AML Found in families with predisposition to MDS and AML Leukemia (2013) 27, 482–485. Fasan A < 60 yrs old≥ 60 yrs old

19. MOLECULAR PATHOLOGY LABORATORY Familial AML and MDS Associations; Germline Mutations CEBPA – familial AML GATA2 – familial MDS/AML RUNX1 – familial platelet disorder with propensity to myeloid malignancy All are heterogeneous with regard to their clinical presentation and progression

20. MOLECULAR PATHOLOGY LABORATORY 3. Activated Signaling FLT3 KIT CBL NRAS and KRAS

21. MOLECULAR PATHOLOGY LABORATORY FLT3 ITD Association with Worse Survival; Meta-analysis ITD mutations in 37-46% of cytogenetically normal AMLs Tyrosine kinase domain codons 835 and 836 mutated in 10-15% Constitutive kinase activity  activation of multiple signaling pathways  promotes cell proliferation and resistance to apoptosis Clinical Trials: first gen inhibitors not successful, 2 nd and 3 rd gen inhibitors in trials now Ann.Hematol.93.1279.2014.Port

22. MOLECULAR PATHOLOGY LABORATORY KIT Mutation Mutated in core binding factor AML (t(8;21), inv or t(16)) – Increased incidence of relapse and inferior outcome Treatment: Imatinib with standard chemo therapy has some activity – Phase 1/2 studies are investigating combination dasatinib and standard chemotherapy in core binding factor leukemias Leuk.28.1132.2014.Manara

23. MOLECULAR PATHOLOGY LABORATORY CBL Ubiquitin ligase – Targets a variety of tyrosine kinases for degradation by ubiquitination – Important for the termination of signaling of receptor tyrosine kinases Mutations more common in MDS (~5% of cases) than AML

24. MOLECULAR PATHOLOGY LABORATORY NRAS and KRAS GTP binding proteins - act downstream of tyrosine kinase receptors (FLT3 or KIT) Codon 12, 13, 61, and 146 mutations – inactivate intrinsic GTPase activity resulting in constitutive activation – Seen in 5-15% of AML and some MDS Hyperactive signaling via pro-proliferative PI3K/AKT/mTOR and RAF/MEK/ERK pathways is centrally implicated in malignant transformation driven by RAS mutations Treatment: several clinical trials for downstream targets including AKT and MEK inhibitors RAS mutations lead to constitutively active RAS

25. MOLECULAR PATHOLOGY LABORATORY To Summarize Favorable Prognosis NPM1 duplication CEBPA bi-allelic mutations – GATA2 mutations Poor Prognosis RUNX1 mutations FLT3 ITD and TKD KIT exon 8 and 17 Association with MDS GATA2 CBL Treatment/Clinical Trials FLT3 KIT RAS (via MEK or AKT inhibition)

26. MOLECULAR PATHOLOGY LABORATORY To Summarize Favorable Prognosis NPM1 duplication CEBPA bi-allelic mutations – GATA2 mutations Poor Prognosis RUNX1 mutations FLT3 ITD and TKD KIT exon 8 and 17 Association with MDS GATA2 CBL Treatment/Clinical Trials FLT3 KIT RAS (via MEK or AKT inhibition) Associated with familial MDS/AML

27. MOLECULAR PATHOLOGY LABORATORY 4. Tumor Suppressors WT1

28. MOLECULAR PATHOLOGY LABORATORY WT1; Prognostic Importance is Controversial Zinc finger transcription factor Exon 7 and 9 Mutations – 5-10% in AML – ~3% in MDS Prognosis: CN patients with WT1 mutations had worse overall survival Treatment: clinical trials of immunogenic WT1 peptide (confirmed WT1 expression) Leuk.29.660.2015.Krauth Normal karyotype patients

29. MOLECULAR PATHOLOGY LABORATORY 5. Epigenetic Regulation; DNA methylation and chromatin modification DNMT3A, IDH1, IDH2, TET2, MLL, ASXL1, EZH2

30. MOLECULAR PATHOLOGY LABORATORY High Frequency of Mutations in Regulators of Epigenetics 2010 NEJM.366.1079.2012.Patel

31. MOLECULAR PATHOLOGY LABORATORY Epigenetic Regulation of a Leukemogenic Locus BMC Cancer.12.304.2012.Murati

32. 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) – 5–16% of older patients (those aged 60 years and older) with AML have ASXL1 mutations – Associated with poor outcome in all studies reported to date

33. MOLECULAR PATHOLOGY LABORATORY Survival Associations NEJM.366.1079.2012.Patel

34. 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

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

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

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

38. 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

39. 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

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

41. 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

42. 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)

43. 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

44. MOLECULAR PATHOLOGY LABORATORY Summary Mutations

45. MOLECULAR PATHOLOGY LABORATORY

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