We are in an era that promises a rational. treatment of cancer patients. Levy et al. Genome Research 22:2201, 2012 Vanderbilt university

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1 Enhancing Assessment of Leukemia with Next Generation Sequencing Michelle Afkhami, M.D. Medical Director, Clinical i l Molecular l Laboratory City of Hope National Medical Center How the Expert Treat Hematologic Malignancies

2 FINANCIAL DISCLOSURE NO CONFLICTS of INTEREST to DISCLOSE

3 We are in an era that promises a rational rather than empiric approaches for the treatment of cancer patients. Levy et al. Genome Research 22:2201, 2012 Vanderbilt university

4 The 20 Most Commonly Diagnosed Cancers Worldwide, 2012 Estimates

5 Estimated Number of new hematologic malignancies and deaths by sex, US, 2017 NAACCR*

6 Modern Pathologists Tissue pathology Molecular pathology MACRO HISTO/ CYTO IHC/ ICC IN SITU HYBRIDIZATIONS PCR Phenotype and Genotype are not mutually exclusive, but are complementary New molecular diagnostic approaches and New molecular diagnostic approaches and classifications are based on solid morphological interpretations

7 Genomic Landscapes of Common Forms of Human Cancer Average genes mutated 91% missense mutations 8% nonsense changes <2% splice site or UTR Bert Vogelstein et al. Science 2013;339:

8 Classification of leukemia was so easy before introduction of effective treatment Myeloid origin Acute Acute Myeloid Leukemia (AML) Chronic Chronic Myeloid Malignancies (CML, etc) Lymphoid origin Acute Lymphoblastic Leukemia (ALL) Chronic Lymphocytic Malignancies (CLL, etc)

9 Classification of leukemia was so easy before introduction of effective treatment Myeloid origin Acute Acute Myeloid Leukemia (AML) Chronic Chronic Myeloid Malignancies (CML, etc) Lymphoid origin Acute Lymphoblastic Leukemia (ALL) Chronic Lymphocytic Malignancies (CLL, etc)

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11 Karyotype 46,XX,t(9;22)(q34;q11.2) - Philadelphia chromosome

12 Philadelphia chromosome: A breakthrough In 1960 the Philadelphia chromosome was first discovered by Peter Nowell and David Hungerford from University of Pennsylvania. In 1973, Janet Rowley identified the mechanism by which the Ph chromosome arises as a translocation at University of Chicago.

13 BCR-ABL1 Tyrosine Kinase Inhibitor In the late 1990s, a TKI, STI-571 (imatinib), ib) was identified by Novartis company. In 2000 Jon Kuriyan determined the mechanism by which STI-571 inhibits the BCR/Abl kinase domain. In 2001 it was marketed by Novartis as imatinib mesylate (Gleevec in the US, Glivec in Europe)

14 Classification is easy when there is NO effective treatment Broken leg: shoot Book of Equine Medicine Infected eye: shoot Splayed hoof: shoot Runny nose: shoot Fever: shoot

15 FISH showing the BCR (green), ABL (orange), and BCR-ABL fusion g (g ), ( g ), signals (arrow): A=positive (contains a residual ABL signal), B=normal

16 Historic Evaluation FLT3 NPM1 CEBPA Current Evaluation Evaluation Morphologic F t DNMT3A TET2 IDH1/ IDH2 Evaluation Features Fl KIT MLL WT1 BCOR Molecular profiling Flow Cytometric analysis FLT3 BCOR GATA2 RUNX1 TP53 NGS Cytogenetics and FISH analysis NPM1 CEBPA ASXL1 JAK2 EZH2 PCR analysis Molecular studies EVI1 CBL RAS RB1 studies RB1 MYC P16 MPL

17 Molecular Classification of leukemia Myeloid origin Lymphoid origin Acute FLT3, NPM1, KIT, CEBPA, IDH1, IDH2, ASXL1, DNMT3A, NRAS, RUNX1, TET2, TP53, ABL1 class fusion, CRLF2r_JAK2, JAK-STAT- EPORr- JAK2r, Chronic BCR/ABL (190,210,230) ATM, BIRC3, NOTCH1, SF3B1, TP53,

18 Is there still a role for microscopic evaluation in the diagnosis? Even in AML morphologic exam might be the quickest way for establishing a diagnosis Example: Quick diagnosis i of acute promyelocytic leukemia to start ATRA Does give information about cellularity, maturation patterns, stromal changes Establish baseline for evaluation of treatment efficacy Exclude relevant differential diagnoses

19 What is the molecular WHO update in AML classifications? Its incidence is expected to increase as the population ages. 1.2% of cancer deaths in the US.

20 AML FAB Classification: French-American-British Classification 1976 Eight major subtypes (M0 M7 ) Morphology and Cytochemistry t

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22 The updated WHO 2016 Classification of AML AML, not otherwise specified (NOS) AML with recurrent genetic abnormalities AML with myelodysplasia-related changes Therapy-related myeloid neoplasms Myeloid sarcoma Myeloid proliferations related to Down syn

23 FAB (1976) Classification Connection to AML, NOS WHO 2008 category M0 AML with minimal differentiation M1 AML without maturation M2 AML with maturation M3 Acute Promyelocytic Leukemia M4 Acute Meylomonocytic Leukemia M5 Acute Monocytic/Monoblastic L. M6 Acute Erythroid Leukemia M7 Acute Megakaryoblastic Leukemia

24 AML, NOS WHO 2016 AML with minimal differentiation AML without maturation AML with maturation Acute monoblastic/monocytic Leukemia Pure Erythroid Leukemia Acute Megakaryoblastic Leukemia Acute Basophilic Leukemia Acute Panmyelosis with Myelofibrosis

25 AML with Recurrent genetic abnormality t(8;21);runx1-runx1t1 RUNX1T1 some maturation of neutrophilic line; rare in older patients; AML1/ETO fusion protein; FAB M2; flow cytometry CD19 t(15;17); PML-RARA: APL (granular and microgranular variants); t(11;17), t(5;17) inv(16) or t(16;16);cbfb-myh11 abnormal eosinophilic component; AMML with eos(m4eo) t(9;11); MLLT3- KMT2A (MLL) - monocytic; children; t(6;9); DEK-NUP214 Basophilia and multilinage dyplasia Inv3 or t(3;3); GATA2,MECOM t(1;22);rbm15-mkl1mkl1

26 Gene mutations for past 10 years Most commonly tested: FLT3 NPM1 Affecting DNA methylation: DNMT, TET, IDH, ASXL Tumor suppressor genes: TP53, WT1, NF1 Activated signaling: FLT3, RAS, NPM1

27 Two-hit model of leukemogenesis Loss of function of transcription factors needed for differentiation eg. AML1-ETO CBF -SMMHC PML-RAR Gain of function mutations of tyrosine kinases eg. FLT3, c-kit, NRAS, and K-RAS mutations BCR-ABL TEL-PDGF R Differentiation block + Enhanced proliferation AML Differentiation Leukemia

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29 AML with mutated NPM1 Mutation of the exon 12 of the NPM1 gene. Aberrant cytoplasmic expression of nucleophosmin NPM1 mutations occur in 45 64% of adult AML with a normal karyotype Mutually exclusive with partial tandem duplications of MLL and CEBPA

30 NPM1 NPM1 + AML: Good response to induction therapy and has a favorable prognosis The coexistence of FLT3-ITD and NPM1 mutations ti is associated with a poorer prognosis, but still has an improved prognosis compared to AML that t is NPM1 - and FLT3-ITD +

31 Mutational landscape of AML with normal cytogenetics: Biological and clinical implications Maria Paola Martelli, Brunangelo Falini, et al. Institute te of Hematology, University of Perugia, Perugia, Italy Blood Reviews 27 (2013) FLT3-ITD loses its prognostic effect in CN- AML patients >60 years and especially those aged >70 years On the other hand, NPM1 mutations maintain their positive effect on clinical outcome in this age group

32 CEBPA CCAAT/enhancer binding protein alpha Occur in 6 15% of de novo AML and in 15 18% of CN-AML AML with mutated CEBPA tends to have Higher hemoglobin levels Lower platelet counts Lower lactate dehydrogenase levels Higher PB blast cell counts

33 AML with biallelic mutations of CEBPA AML with a normal karyotype yp and CEBPA mutation is associated with a favorable prognosis, similar to that of AML with inv(16)(p13.1q22) or t(8;21)(q22;q22). q )

34 AML with BCR-ABL1 AML with a normal karyotype yp and CEBPA mutation is associated with a favorable prognosis, similar to that of AML with inv(16)(p13.1q22) or t(8;21)(q22;q22). q )

35 FLT 3 -activating mutation fms-like tyrosine kinase 3 (FLT3) gene One of the most common molecular abnormality of AML A member of the PDGFR family Encodes a tyrosine kinase receptor that is involved in hematopoietic stem cell differentiation and proliferation.

36 FLT3 Extracellular domain Transmembrane and Juxtamembrane domains Tyrosine kinase domain Dimerization cause activation and recruits proteins leading to activation of AKT, MAPK, and STAT, which interact with HSP90 which protects them from inactivation and chaperones the active mediators to the nucleus to mediate vital cellular functions

37 DNMT3A Prediction of response to therapy (decitabine) in AML Found in 17-19% of AML

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39 Top 20 genes in AML

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45 Case 64 year-old female with pancytopenia PBS P t i ith 14% i l ti PBS: Pancytopenia with 14% circulating blasts

46 BM Aspirate

47 Bone marrow core biopsy

48 Diagnosis Bone marrow biopsy (HP15-861): Acute myeloid Leukemia (35.5% BLASTS) Flow cytometry: 38% blasts with CD13, CD34, CD117, and HLA-DR expression. IDH1/2 was ordered.

49 Next generation sequencing Yielding rapid and relatively low-cost data sets Introducing new prognostic markers in AML such as DNMT3A and IDH1

50 TOP 20 genes in Acute myeloid leukemia

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53 Overview of CMDL at COH In operation since 1990s as a clinical reference laboratory CAP certified, CLIA licensed Clients: City of Hope Medical Center Last 3 years, 681 non-coh facilities from: United States, Canada, Mexico, Brazil, Belgium, Spain, Turkey, Iran, Israel, China, Egypt, Australia

54 Clinical Molecular Diagnostics Laboratory Services: Solid Tumors Hematological Malignancies Germline Testing Molecular Microbiology

55 Molecular Testing at CMDL, COH CLIA & CAP Certified Laboratory Application of molecular techniques & knowledge of the molecular mechanisms of disease in: Diagnosis Prognosis Treatment

56 Molecular Testing BRAF V600E BRAF V600E BRAF V600E

57 Targeted next generation sequencing of clinically significant gene mutations and translocations in leukemia Eric J Duncavage, Haley J Abel, Philippe Szankasi, Todd W Kelley, and John D Pfeifer Modern Pathology (2012) 25,

58 Leukemias are currently subclassified based on the presence of recurrent cytogenetic abnormalities and gene mutations. Basis for risk-adapted therapy

59 Targeted next generation sequencing Full spectrum of prognostically significant gene mutations including: Translocations, Single nucleotide variants (SNVs) Insertions/deletions (indels) Can be identified simultaneously in multiplexed sequence data. Yielding rapid and relatively low cost data Yielding rapid and relatively low-cost data sets

60 PCR and Sanger sequencing Commonly tested genes: FLT3 and NPM1 tend to have a limited spectrum of mutations, making them amenable to PCR The broader range of mutations such as CEPBA, DNMT3A, and KIT needs the use of direct sequencing Adding to the expense and turn-around time of testing.

61 FISH Detection of recurrent, balanced chromosomal translocations is critical to leukemia prognosis FISH offers increased sensitivity over conventional cytogenetics But Unable to identify novel translocation partners, such as those involving the MLL locus, or variant breakpoints Requires the use of multiple probes again Requires the use of multiple probes, again increasing the cost and complexity of testing

62 2 d Generation NGS Platforms CMDL Targeted sequencing Life Technology Ion Torrent Up to Gb per run Fast: 4-24 hours Depth of coverage >1000x Simplified data analysis Illumina MiSeq

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64 Hematological Malignancies Hematological Malignancies NGS panel Myeloid neoplasms Lymphoid neoplasms

65 Hematological Malignancies: Mutation assay by NGS of 74 genes OncoHemeMutations ASXL1 CCND1 EP300 IDH1 KMT2D NRAS SF3B1 WHSC1L1 ATM CCND3 ETV6 IDH2 KRAS PAX5 SPI1 WT1 BCOR CD79A EZH2 IL7R MEF2B PDGFRA SRSF2 ZRSR2 BIRC3 CD79B FBXW7 JAK1 MIR142 PHF6 STAG2 BRAF CDKN2A FLT3 JAK2 MPL PIK3CA SUZ12 BTK CEBPA GATA1 JAK3 MYC PTEN TCF3 CALR CHEK2 GATA2 KDM6A MYD88 PTPN1 TET2 CARD11 CREBBP HRAS KIT NOTCH1 PTPN11 TP53 CBL CSF3R ID3 KMT2A NOTCH2 RUNX1 U2AF1 CBLB DNMT3A IKZF1 KMT2C NPM1 SETBP1 WHSC1

66 Mini OncoHem NGS Assay Mutation analysis by Next Generation Sequencing Peripheral blood, bone marrow, and formalin-fixed paraffin-embedded tissue (FFPE) NPM1 KIT IDH1 IDH2 JAK2 MPL BRAF ABL1

67 Mini OncoHeme e Assay CEBPA by Sanger Sequencing NPM1 by PCR FLT3 TKD and ITD with signal ratio by PCR

68 Hematological Malignancies: Fusion Assay by NGS of f23 genes OncoHemeFusions ABL1 ABL2 ALK BCR CBFB CRLF2 CSFIR FGFR1 JAK2 KMT2A MECOM MKL1 NOTCH1 NUP214 PDGFRA PDGFRB PICALM RARA RBM15 RUNX1 RUNX1T1 TAL1 TCF3

69 Other Hematological Malignancies Assays PCR assays Immunoglobulin heavy and light chain gene rearrangements with BIOMED-2 primers T-cell receptor gamma and beta chain gene rearrangements with BIOMED-2 primers Quantitative t(9:22); BCR/ABL1 Quantitative t(15;17); PML/RARA Inv 16 t(8;21) t(14;18) Calreticulin (CALR) mutations

70 NGS Germline Cancer Predisposition Panels Targeted to breast, colon, ovary, pancreas, & kidney APC ATM BAP1 BARD1 BLM BMPR1A BRCA1 BRCA2 BRIP1 CDH1 CDK4 CDKN2A CHEK2 CTNNA1 EPCAM FAM175A FANCC FH FLCN HOXB13 MET MLH1 MRE11 MSH2 MSH6 MUTYH NBN PALB2 PMS2 PTEN RAD50 RAD51C RAD51D SDHB SDHC SDHD SMAD4 STK11 TP53 TSC1 TSC2 VHL XRCC2 Hong et al, AMP 2015 National Meeting Telatar et al, AMP 2015 National Meeting

71 Next.?

72 Era of Precision Medicine Tissue-based diagnosis still has the central role in diagnosis.

73 Proposed testing algorithm Targeted Mutation Panels genes: Mutations, deletions, gene fusions First line of testing Low cost/better reimbursement Mutation/ fusions + Mutation - Targeted Therapy Whole exome and transcriptome analysis Search for novel and unexpected variants Rare fusion types, splicing variants Copy number changes

74 So many genes, so little time. Communication for Collaboration Genomic Tumor boards Disease Teams/Programs Phase 1 Therapeutics meeting Inservice training for mid-levels Biannual review of existing clinical tests Collaborations with BRI researchers through the Molecular Core

75 NGS data analysis requires integration of clinical,,pathological, and genetic interpretations: Multidisciplinary Genomic Tumor Board

76 Things you can count on Death Taxes A new lymphoma/ leukemia classification based on new molecular l findings!

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