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1 Enhancing Assessment of Leukemia and Lymphoma with Next Generation Sequencing Michelle Afkhami, M.D. Medical Director, Clinical Molecular Laboratory Assistant Professor, Department of Pathology City of Hope National Medical Center How the Expert Treat Hematologic Malignancies 2017
2 Objectives and Financial Disclosure List molecular targets useful for diagnosis and monitoring of myeloid malignancies List molecular targets useful for diagnosis and monitoring of selected lymphoid disorders 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 Personalized Cancer Medicine: Delivery of the right drug to the right patient at the right time Every type of cancer has distinct genomic profile that define clinical behavior and response to treatment in each individual Comprehensive Tumor Genotyping Establish specific molecular signature Match with appropriate targeted therapy
5 The 20 Most Commonly Diagnosed Cancers Worldwide, 2012 Estimates
6 Estimated Number of new hematologic malignancies and deaths by sex, US, 2017 NAACCR*
7 Modern Hematopathology Tissue pathology Molecular pathology Microscopy Immunophenotyping Karyotyping & FISH PCR, Sanger, and NGS Phenotype and Genotype are not mutually exclusive, but are complementary
8 Is there still a role for microscopic evaluation in the Leukemia diagnosis? Morphologic exam might be the quickest way for establishing a diagnosis Example: Quick diagnosis of acute promyelocytic leukemia to start ATRA Evaluation of cellularity, l maturation ti patterns, stromal changes Establish baseline for evaluation of treatment efficacy Exclude relevant differential diagnoses
9 Classification of Leukemia and Lymphoma Long time ago! Myeloid origin Acute Acute Myeloid Leukemia (AML) AML Chronic Chronic Myeloid Malignancies (CML, etc) CML Lymphoid origin Acute Lymphoblastic Leukemia (ALL) ALL Chronic Lymphocytic Malignancies (CLL, etc) CLL
10 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
11 French-American-British (FAB) Classification 1976 Eight g major subtypes (M0 M7 ) based on Morphology and Cytochemistry
12 Ph. chromosome and The magic bullet 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. In 2000, Jon Kuriyan determined the mechanism by which STI-571 inhibits the BCR/Abl kinase domain. In 2001 the first BCR/Abl kinase was marketed as imatinib mesylate.
13 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:
14 Most common Mutations in AML AML MDS MPN Diagnosis NPM1, CEBPA, RUNX1 SF3B1 JAK2, CALR, MPL, CSF3R Prognosis FLT3-ITD, DNMT3A, IDH1, IDH2, KIT, TET2, TP53, WT1. TP53, EZH2, ETV6, RUNX1, ASXL1, ASXL1, EZH2, ETNK1, IDH2, SETBP1, SF3B1, SRSF2 TCGA. N Engl J Med. 2013; 68(22): Bejar R. N Engl J Med. 2011;364(26):
15 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, KIT, RAS mutations BCR-ABL TEL-PDGF R Differentiation block + Enhanced proliferation AML Differentiation Leukemia
16 Molecular Classification of leukemia and Lymphoma in 2017 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-JAK2, Chronic BCR/ABL (CML), CSF3R (CNL), SETBP1, ETNK1 (acml) JAK2, MPL, CLAR, (MPN) ATM, BIRC3, NOTCH1, SF3B1, TP53,
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18 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.
19 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
20 AML, NOS WHO 2016 AML with minimal differentiation AML without t maturation ti AML with maturation Acute monoblastic/monocytic Leukemia Pure Erythroid Leukemia Previous Acute Erythroid Leukemia now can be AML with MDS-related features > 50% erythroid precursors will not have the % of blasts based on nonerythroid precursors. Acute Megakaryoblastic Leukemia Acute Basophilic Leukemia Acute Panmyelosis with Myelofibrosis
21 The updated WHO 2016 Classification of AML AML, not otherwise specified (NOS) AML with recurrent ctyogenetic abnormalities AML with myelodysplasia-related changes Therapy-related myeloid neoplasm Myeloid sarcoma Myeloid proliferations related to Down syn. Myeloid neoplasm's with Germline predispositions
22 AML with recurrent cytogenetic Genetics abnormalities 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 multilineage dysplasia Inv3 or t(3;3); GATA2,MECOM; Thrombocytosis, Multilineage dysplasia with small mono or bilobed megakaryocytes May have a MDS phase but > = 20% blasts required for Dx t(1;22);rbm15-mkl1mkl1
23 AML with recurrent cytogenetic Genetics abnormalities New categories AML with RUNX 1 mutation older men, poor prognosis AML with NPM1 Mutually exclusive with partial tandem duplications of MLL and bicebpa; good prognosis AML with CEBPA DM only changes prognosis (good) if present as a double mutant t AML with BCR- ABL1 Arber DA, Orazi A, Hasserjian R, et al. Blood. 2016;127(20): Mutations in 3 genes- RUNX1, ASXL1, and TP53- have been added in the risk stratification of the 2017 European Leukemia Net recommendations for AML.
24 AML mutations Knowledge Activated signaling: g FLT3, RAS, NPM1 Secondary events and occur later during leukemogenesis. Affecting DNA methylation: DNMT, TET, IDH, ASXL Commonly acquired early and present in the founding clone Tumor suppressor genes: TP53, WT1, NF1 Bullinger L, Döhner K, Döhner H. Genomics of Acute Myeloid Leukemia Diagnosis and Pathways. J Clin Oncol March ;35(9):
25 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
26 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
27 The Cancer of Genome Atlas. Genomic and epigenomic landscapes of adult de novo acute myeloid leukemia. N Engl J Med. 2013; 68(22):
28 Red: Connect mutually exclusive genes Blue: Connect Co- Occurring events Black: Mutations with define favorable Cytogenetics The Cancer of Genome Atlas. Genomic and epigenomic landscapes of adult de novo acute myeloid leukemia. N Engl J Med. 2013; 68(22):
29 Ten studies covering a total of 6219 subjects indicated bi CEBPA mutations ti were associated with favorable clinical outcome in patients with AML or in cytogenetically normal AML. No significant difference was found between monoallelic CEBPA mutation and wild type CEBPA in patients with AML or CN-AML (P > 0.05). Li HY, Deng DH, Huang Y, et al. Eur J Haematol. 2015;94(5): Green CL, Koo KK, Hills RK, et al. J Clin Oncol. 2010;28(16):
30 New Provisional Entity: AML with BCR-ABL1 Hard to differentiate from CML myeloid blast crisis, requires good history Deletion of Ag receptros (IGH, TCR), IKZF1 or CDKN2A may support diagnosis of de novo AML Konoplev S, et al, Leuk Lymphoma 54;138,2013 Soupir CP, et al. AM J Clin Pathol 127;642,2007
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33 IDH2: 9-13% IN AML and 3-6% MDS IDH1: 6-10% IN AML and 3% MDS Marcucci G, et al. J Clin Oncol. 2010;28(14):
34 Top 20 genes in AML
35 Genes encoding epigenetic modifiers: DNMT3A, ASXL1, TET2, IDH1, and IDH2 May persist after therapy, lead to clonal expansion during hematologic remission, and eventually lead to relapsed disease. Frequently found to be mutated in healthy elderly individuals along with clonal expansion of hematopoietisis (increased risk for the development of fhematologic cancers)
36 Can we define MDS with mutations profile? Analyzing of WES data of peripheral blood of 17,182 persons showed somatic mutations that drive clonal expansion of blood cells were a common finding in the elderly l and most frequently involved DNMT3A, TET2, or ASXL1. Jaiswal S, Fontanillas P, Flannick J, et al. Age-related clonal hematopoiesis associated with adverse outcomes. N Engl J Med. 2014;371(26): Arber DA, Orazi A, Hasserjian R, et al. Blood. 2016;127(20):
37 Reporting of the results Preliminary i report: morphology or and flow cytometry report in diagnosis of a new leukemia or relapse Opportunity to get more history, prior therapy, Initial report: Includes morphology and immunophenotyping Final report: Should be an integrated report Include all immunophenotyping, Cytogenetics and Molecular genetics results to approach the final diagnosis
38 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.
39 AML with myelodysplasia related changes Multilineag dysplasia (dyspoiesis): 50% of the cells in 2 non-blast cell lines Exclude AML with recurrent genetics abnormalities or prior MDS Absence of prior history of therapy
40 NPM1 and CEBPA mutation and dyspoiesis: Significant MLD found in the presence of NPM1 74/318 (23%) with no prognostic significant in MLD MLD found in 28/108 (26%) CEBPA mutated AMLs wit h no significant survival difference in cases with or without MLD Removed from de novo cases with MDS related cytogenetic abnormality if there is an NPM1 or CEBPA dm Falini et al. Blood Diaz-Beya M et al Blood 2010 Revise cytogenetic abnormalities Del9q is an MDS related entity only in the absence of NPM1 mutations NPM1 commonly associated with del9q and is likely not adverse in this setting
41 AML with NPM1 and CEBPA mutation and abnormal Karyotype: 14.7% of NPM1 mutated (+8,+4, -Y, del(9q) and +21) and 26% of bi-cebpa mutated t (del(9q), del(11q), -Y, and +10, and +21) AML cases have abnormal Karyotype Del9q currently is an MDS related entity only in the absence of NPM1 or CEBPA mutations due to no prognostic significance (Haferlach C et al Blood )
42 Classification of myeloid neoplasms with germ line predisposition p Arber DA, Orazi A, Hasserjian R, et al. Blood. 2016;127(20):
43 Clinical Molecular Diagnostics Laboratory Service Lines: Hematological Malignancies: M. Afkhami, MD and P. Aoun, MD MPH Solid Tumors: M. Afkhami, MD Germline Testing: M. Telatar, PhD FACMG Molecular Microbiology: P. Aoun, MD MPH Bioinformatics: R. Pillai, MD
44 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
45 Molecular Testing at CMDL, COH CLIA & CAP Certified Laboratory Application of molecular techniques & knowledge of the molecular mechanisms of disease in: Diagnosis Prognosis Treatment and predictive of response to therapy
46 Hairy cell Leukemia BRAF mutations Langerhans cell histiocytosis Juvenile xanthogranuloma (Erdheim Chester D.) Rosai-Dorfman disease unclassifiable splenic B-cell lymphoma/leukemia
47 MULLIGHAN et al.blood;2013;vol 122, 24
48 MULLIGHAN et al.blood;2013;vol 122, 24
49 Pasqualucci L, et al. Curr Opin Hematol. 2013;20;336-44
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51 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
52 CMDL: Hematological Malignancy assays Myeloid neoplasms Lymphoid neoplasms
53 CMDL Comprehensive Molecular Profiling Panels for Hematological Malignancies TAT ( days) OncoHeme Mutations Full gene sequencing of 73 genes by NGS ASXL1 CCND1 ETV6 IL7R MEF2B PDGFRA STAG2 WT1 ATM CCND3 EZH2 JAK1 MIR142 PHF6 SUZ12 ZRSR2 BCOR CD79A FBXW7 JAK2 MPL PIK3CA TCF3 FLT3 BIRC3 CD79B GATA1 JAK3 MYC PTEN TET2 BRAF CDKN2A GATA2 KDM6A MYD88 PTPN11 TNFAIP3 BTK CEBPA HRAS KIT NOTCH1 RUNX1 TP53 CALR CREBBP ID3 KMT2A (MLL) NOTCH2 SETBP1 U2AF1 CARD11 CSF3R IDH1 KMT2C (MLL3) NPM1 SF3B1 UBR5 CBL DNMT3A IDH2 KMT2D (MLL2) NRAS SPI1 WHSC1 CBLB EP300 IKZF1 KRAS PAX5 SRSF2 WHSC1L1 OncoHeme Complete OncoHeme Fusions Detection of fusions involving any partner of 23 key genes by NGS ABL1 ABL2 ALK BCR CBFB CRLF2 CSFIR FGFR1 JAK2 KMT2A (MLL) MECOM MKL1 NOTCH1 NUP214 PDGFRA PDGFRB PICALM RARA RBM15 RUNX1 RUNX1T1 TAL1 TCF3
54 CMDL: Mutation assay by NGS of 73 genes OncoHemeMutations ASXL1 CCND1 EP300 IDH1 KMT2D NRAS SF3B1 WHSC1 ATM CCND3 ETV6 IDH2 KRAS PAX5 SPI1 WHSC1L1 BCOR CD79A EZH2 IL7R MEF2B PDGFRA SRSF2 WT1 BIRC3 CD79B FBXW7 JAK1 MIR142 PHF6 STAG2 ZRSR2 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 TNFAIP3 CBL CSF3R ID3 KMT2A NOTCH2 RUNX1 TP53 CBLB DNMT3A IKZF1 KMT2C NPM1 SETBP1 U2AF1 DLBCL: CD79A, CD79B, CARD11,MYD88, MYC, EZH2, BTK, PTEN, BRAF, A20 FL: MLL2, BCL2, EZH2, CREBBP, EP300, TNFAIP3, CARD11,LMO2, CLL: ATM, BIRC3, NOTCH1, SF3B1, TP53 Burkitt lymphoma : MYC, ID3, TCF3
55 Single gene Assays for hematological malignancies and related disorders (TAT 3-5 days) Mutation analysis by Next Generation Sequencing, Sanger and PCR Peripheral blood, bone marrow, and formalin-fixed paraffin-embedded tissue (FFPE) NPM1 CEBPA IDH1 IDH2 FLT3 KIT BRAF ABL1 JAK2 CALR MPL TP53 PCR: NPM1 and FLT3 TKD and ITD with signal ratio
56 Hematological Malignancies: Fusion Assay by NGS of 23 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
57 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
58 Blood Evaluation Morphologic Features Flow Cytometric analysis Cytogenetics and FISH analysis Molecular studies DNA and RNA will be extracted and hold indefinitely Order Molecular as needed Up-front NGS panels FLT3 NPM1 CEBPA DNMT3A TET2 IDH1/ IDH2 KIT MLL WT1 BCOR GATA2 RUNX1 TP53 ASXL1 JAK2 EZH2 EVI1 CBL RAS RB1 MYC P16 MPL,
59 Case 71 year-old male with history of MDS since 2014 and deletion chromosome 1 at OSH Transferred to COH form an OSH where her received hydrea and steroids for autoimmune hemolytic anemia PBS: WBC: K/ul; Hb:9 g/dl; Platelets:12 PBS: WBC:50,000 K/ul; Hb:9 g/dl; Platelets:12 K/uL
60 Peripheral blood and Bone marrow was sent for evaluation PBS: WBC: K/ul; Hb:7 8 g/dl PBS: WBC:38,100 K/ul; Hb:7.8 g/dl, MCV:103.8; Platelets: 24 K/uL Diff: 66% blasts
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62 Diagnosis Bone marrow biopsy: Acute myeloid Leukemia (77% BLASTS) extensively involving a markedly hypercellular bone marrow (95%) Mini Hem panel (quick TAT) and OncoHeme Mutation NGS panel was ordered.
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66 AML Data Spread sheet
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68 DNMT3A c.2645g>a; p.r882h
69 RUNX1 c.946dup; p.e316gfs*284
70 FLT3 ITD
71 Should we call this AML with RUNX1 mutation? First we have to clarify previous label l Arber DA, Orazi A, Hasserjian R, et al. Blood. 2016;127(20):
72 Recommendation for recent AML genetics classifications Karyotype analysis should be performed on all diagnostic samples Molecular genetic studies should be ordered based on assessment of morphology, immunophenotype, and Karyotype analysis or Up-front
73 Proposed testing algorithm Targeted Mutation Panels About 100 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 Era of Precision Medicine NGS data analysis requires integration of clinical, pathological, and genetic interpretations: Multidisciplinary Genomic Tumor Board
75 Things you can count on Taxes Death A new lymphoma/ leukemia classification based on new molecular l findings! At the moment there is still a role for morphology!!!
76 Acknowledgment Drs. Steve Forman and Steve Rosen Dr. Dennis Weisenburger Dr. Patricia i Aoun Dr. Milhan Telatar Dr. Raju Pillai Dr. Carrie Louie Dr. Jenny Kuo Maria Cuellar, MS Dongging Gu, MS Huma Qurishi, MS Barbara Choi, MS Vanina Tomasian, MS Contact: mafkhami@coh.org And our entire team of dedicated Curators, LTAs, and clinical laboratory scientists!
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