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
Principle 1: AML incidence increases with age Source: SEER data 2005-2009. 3 Principle 2: AML emerges through branching evolu9on Nowell, PC Science 1976. 4 2
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
Li Ding 7 8 4
% 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
The number of AML muta9ons correlates with the age of pa9ent 11 Predic9ons? Pre-leukemic state 12 6
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 1 2 3 Total WBCs vs Exome Sequencing 13 Soma9c variants in healthy-donor HSPCs 14 7
The number of AML and HSC muta9ons correlates with the age of pa9ent 15 HSC muta9on rate 2.4 4.0 x 10-9 variants/nt/yr Intergenera9onal muta9on rate: ~4 x 10-10 variants/nt/yr 16 8
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. 3 353-354 18 9
Clonal Expansion and Allelic Frac>ons. Genovese G et al. N Engl J Med 2014;371:2477-2487. 19 Prevalence of Mosaic Hematopoiesis increases with age DNMT3A TET2 RB1 Laurie et al Nature Gene+cs 44:642. 2012 20 10
21 Jaiswal et al. 22 11
Xie et al. Nature Medicine 23 Leukemic Evolu9on Predic9ons? 24 12
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
Subclonal architecture Monoclonal Leukemia Subclone Founding clone Each point represents a unique muta9on 27 Sub-clonal architecture 28 14
Model of AML muta9on acquisi9on 29 Predic9ons? Relapse 30 15
Clonal selec9on at relapse Relapse from this subclone Founding Subclones lost at relapse Klco et al. Cancer Cell 2014 31 Muta9ons Predic9ons? 32 16
Heterogeneity 33 Significantly Mutated Genes 200 cases 34 17
How many muta9ons does it take? TCGA NEJM 2013 35 Order of muta9on acquisi9on? Drivers vs passengers Ini9a9on vs progression Subclonal architecture by Next-gen Temporal changes in muta9ons 36 18
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 +8 16 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, +22 37 NRAS and FLT3 muta9ons in subclones NRAS IDH1 NPM1 FLT3 NPM1 SMC1A 38 19
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
Kroenke et al Blood 2013 41 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, IDH1/2 42 21
Chromosome X analysis 43 Wong et al. Nature 518: 552, 2015 44 22
Residual disease detec9on: NPM1 Ivey et al. NEJM 374: 422, 2016 45 Klco et al. JAMA 314: 811 2015 46 23
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. 48 24
Random Distribu9on of Muta9ons 49 Random Distribu9on of Muta9ons Genes 1.5% Conserved 8% Non-redundant 45% 50 25
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