AML Genomics 11/27/17. Normal neutrophil maturation. Acute Myeloid Leukemia (AML) = block in differentiation. Myelomonocy9c FAB M5

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1 AML Genomics 1 Normal neutrophil maturation Acute Myeloid Leukemia (AML) = block in differentiation AML with minimal differen9a9on FAB M1 Promyelocy9c leukemia FAB M3 Myelomonocy9c FAB M5 2 1

2 Principle 1: AML incidence increases with age Source: SEER data Principle 2: AML emerges through branching evolu9on Nowell, PC Science

3 5 Predic9ons Where do muta9ons come from? Genomic instability? How many muta9ons? Distribu9on of muta9ons? Pre-leukemic state? Leukemic evolu9on? Mechanism of relapse? 6 3

4 Li Ding 7 8 4

5 % of the Human Genome in Each Tier 48.7% 1.3% 8.6% 41.4% Tier 1: Genes Tier 2: Conserved Tier 3: Non-repe>>ve Tier 4: rest 9 Lots of muta9ons; are they all required? Passengers Drivers 10 5

6 The number of AML muta9ons correlates with the age of pa9ent 11 Predic9ons? Pre-leukemic state 12 6

7 What is the genomic structure of normal aging hematopoie9c stem cells? Healthy Volunteers Sort single Lin - CD34 + CD38 - Culture 2-3 weeks Pick 3 colonies Total WBCs vs Exome Sequencing 13 Soma9c variants in healthy-donor HSPCs 14 7

8 The number of AML and HSC muta9ons correlates with the age of pa9ent 15 HSC muta9on rate x 10-9 variants/nt/yr Intergenera9onal muta9on rate: ~4 x variants/nt/yr 16 8

9 Muta9on spectrum: 7 healthy-donors HSCs vs 24 AML cases 17 C > T vs G > A muta9ons CG GC CG GC TG AC CA GT The Oncologist June 1, 2004 vol. 9 no

10 Clonal Expansion and Allelic Frac>ons. Genovese G et al. N Engl J Med 2014;371: Prevalence of Mosaic Hematopoiesis increases with age DNMT3A TET2 RB1 Laurie et al Nature Gene+cs 44:

11 21 Jaiswal et al

12 Xie et al. Nature Medicine 23 Leukemic Evolu9on Predic9ons? 24 12

13 Subclonal architecture 1170 reads at TET2 locus 490 reads with muta9on 42% of alleles carry muta9on Therefore, 84% of the bone marrow cells carry the muta9on. Each point represents a unique muta9on 25 Subclonal architecture 1170 reads at TET2 locus 490 reads with muta9on 42% of alleles carry muta9on Therefore, 84% of the bone marrow cells carry the muta9on. Monoclonal Leukemia Each point represents a unique muta9on 26 13

14 Subclonal architecture Monoclonal Leukemia Subclone Founding clone Each point represents a unique muta9on 27 Sub-clonal architecture 28 14

15 Model of AML muta9on acquisi9on 29 Predic9ons? Relapse 30 15

16 Clonal selec9on at relapse Relapse from this subclone Founding Subclones lost at relapse Klco et al. Cancer Cell Muta9ons Predic9ons? 32 16

17 Heterogeneity 33 Significantly Mutated Genes 200 cases 34 17

18 How many muta9ons does it take? TCGA NEJM Order of muta9on acquisi9on? Drivers vs passengers Ini9a9on vs progression Subclonal architecture by Next-gen Temporal changes in muta9ons 36 18

19 Cytogene9cs: single cell analysis t(15;17) and +8 XY, t(15;17), +8[16]/XY, t(15;17)[3], XY[1] 36 cases published with t(15;17) and of these report t(15;17) cells without +8 None have +8 without t(15;17) t(15;17), -7 t(8;21), -X, -Y Inv(16), +13, NRAS and FLT3 muta9ons in subclones NRAS IDH1 NPM1 FLT3 NPM1 SMC1A 38 19

20 Response of subclone to chemotherapy Subclone muta9on confers resistance Schnipger ASH 2012: TET2 gained in 1/423 cases. 39 Response of subclone to chemotherapy Subclone muta9on confers sensi9vity Schnipger ASH 2012: TET2 lost in 0/17 cases 40 20

21 Kroenke et al Blood Summary of muta9ons Founding clone/ini9a9on muta9ons: t(15;17), CBF, MLL, DNMT3A, TET2 Commonly subclonal muta9ons: +8, +22, -X, -Y, FLT3, NRAS/KRAS, WT1, KIT, CEBPA Uncertainty: NPM1, IDH1/

22 Chromosome X analysis 43 Wong et al. Nature 518: 552,

23 Residual disease detec9on: NPM1 Ivey et al. NEJM 374: 422, Klco et al. JAMA 314:

24 Genomic Features of AML Cell specific muta9ons are acquired in each HSPC over its lifespan. 3.2 x 10-9 variants/nt/yr 1 cell division/month Most muta9ons are randomly distributed across the genome. Muta9ons favor C>T transi9ons. Founding clones and subclones that evolve during leukemogenesis and leukemic relapse via branching evolu9on. DNMT3A and TET2 may act as ini9a9ng events. FLT3, RAS, KIT, CEBPA, WT1 are commonly progression events NPM1 and IDH1/2 early, but not always ini9a9ng. AML is not a disease of genomic instability. 47 Acknowledgements Genomics of AML PPG Tim Ley Peter Westervelt Sharon Heath Tami Lamprecht Washington University Genome Insitute Rick Wilson Elaine Mardis Li Ding Chris Miller Dave Larsen Bob Fulton Daniel Link Timothy Graubert John Dipersio Geoffery Uy Amanda Cashen Ravi Vij Our Patients

25 Random Distribu9on of Muta9ons 49 Random Distribu9on of Muta9ons Genes 1.5% Conserved 8% Non-redundant 45% 50 25

26 Contamina9on of leukemia variants in skin sample 51 Clonal selec9on during xenogras Founding PB AML transplant 12 weeks Analyze A single clone engrass per mouse Variability in the engrasing clone 52 26