Genetic complexity in MPN, MDS/MPN and MDS

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1 Genetic complexity in MPN, MDS/MPN and MDS Nick Cross Wessex Regional Genetics Laboratory, Salisbury Faculty of Medicine, University of Southampton

2 Genetic complexity in chronic myeloid neoplasms Classes of mutations Complexity, subclonality and prognosis Specificity of mutations

3 Myeloproliferative disorders and myelodysplastic syndromes ET PV PMF CML CEL CNL MPN-U MPN CMML JMML acml MDS/MPN-U RARS-T MDS/MPN RCUD RCMD RARS RAEB MDS-U MDS Proliferation Effective haemopoiesis Dysplasia Ineffective haemopoiesis

4 Mutation discovery: cytogenetics, DNA arrays, exome/genome sequencing der(13) der(8) der(8) normal 13 normal 13 normal 8 der(13) normal 8 der(8) 52-M15 der(13) 16-L19 Reiter et al., Blood 1998;92: ; Gelsi-Boyer et al., Br J Haematol. 2009;145: ; Ernst et al., Nat Genet. 2010;42:722-6

5 Classes of somatically acquired driver mutation in MPN, MDS/MPN and MDS SIGNALLING TK fusions JAK2 MPL CBL NF1 NRAS KRAS CSF3R RIT1 PTPN11 FLT3 KIT SETBP1?? CALR?? EPIGENETIC TET2 DNMT3A IDH1/2 EZH2 ASXL1 PHF6 CREBBP EP300 mrna SPLICING SF3B1 SRSF2 U2AF1 ZRSR2 LUC7L2 PPRF8 TRANSCRIPTION/ REPAIR RUNX1 TP53 ETV6 BCOR CUX1 COHESIN STAG2 SMC1A SMC3 RAD21 CYTO 5q- -7/7q i(17q) del(11q) del(12p) del(20q) inv(3q) t(3;3)

6 No abnormality defines specific disease entities except BCR-ABL1 SIGNALLING TK fusions JAK2 MPL CBL NF1 NRAS KRAS CSF3R RIT1 PTPN11 FLT3 KIT SETBP1 CALR EPIGENETIC TET2 DNMT3A IDH1/2 EZH2 ASXL1 PHF6 CREBBP EP300 mrna SPLICING SF3B1 SRSF2 U2AF1 ZRSR2 LUC7L2 PPRF8 TRANSCRIPTION/ REPAIR RUNX1 TP53 ETV6 BCOR CUX1 COHESIN STAG2 SMC1A SMC3 RAD21 CYTO 5q- -7/7q i(17q) del(11q) del(12p) del(20q) inv(3q) t(3;3)

7 Drug targetable mutations? SIGNALLING EPIGENETIC mrna SPLICING TRANSCRIPTION/ REPAIR COHESIN CYTO TK fusions JAK2 MPL CBL NF1 NRAS KRAS CSF3R RIT1 PTPN11 FLT3 KIT SETBP1 CALR TET2 DNMT3A IDH1/2 EZH2 ASXL1 PHF6 CREBBP EP300 SF3B1 SRSF2 U2AF1 ZRSR2 LUC7L2 PPRF8 RUNX1 P53 ETV6 BCOR CUX1 STAG2 SMC1A SMC3 RAD21 5q- -7/7q i(17q) del(11q) del(12p) del(20q) inv(3q) t(3;3)

8 Classes of somatically acquired driver mutation in MPN, MDS/MPN and MDS MPN MDS/MPN MDS Signalling Splicing, cohesins Epigenetic, transcription

9 Signalling abnormalities Activate growth factor signalling pathways Thought to be largely responsible for proliferative phenotypes Gain of function mutations in transducers of signalling JAK2 V617F, KIT D816V, RAS mutations Tyrosine kinase fusion genes e.g. BCR-ABL1, FIP1L1-PDGFRA Loss of function mutations of negative regulators of signalling, eg CBL, SH2B3 (LNK)

10 Targeted therapy for myeloid disorders with activated tyrosine kinases Mastocytosis with KIT D816V MPN with JAK2 V617F midostaurin JAK2 inhibitors MPN with PDGFR Fusion genes imatinib BCR-ABL1 positive CML imatinib MPN with FGFR1 Fusion genes MPN with FLT3 Fusion genes MPN with JAK2 Fusion genes ponatinib dovitinib FLT3 inhibitors ruxolitinib Less aggressive More aggressive

11 Tyrosine kinase fusions in MLN-eo and related MPN GOLGB11 3q12 FIP1L1 4q12 KIF5B 10p11 CDK5RAP2 9q33 STRN 2p24 FOXP1 3p14 FLT3 13q12 NTRK3 15q25 LYN 8q12 SYK 9q22 PDGFRA 4q12 N=7 RET 10q11 SPTBN1 2p16 TPM3 1q21 PDE4DIP 1q22 WDR48 3p22 GOLGA4 3p22 PRKG2 4q21 CEP85L 6q22 HIP1 7q11 KANK1 9p24 CCDC6 10q21 GPIAP1 11p13 ERC1 12p13 BIN2 12q13 ETV6 12p13 PDGFRB 5q33 N=25 SART3 12q23 DTD1 20p11 RABEP1 17p13 GIT2 12q24 ABL 9q34 JAK2 9p34 MYO18A 17q11 SPECC1 17p11 NDE1 16p13 TP53BP1 15q22 NIN 14q24 TRIP11 14q32 CCDC88C 14q32 PCM1 8p21 TPR1 1q25 RPN1 3q21 N=13 BCR 22q11 FGFR1 8p11 LOC q13 ZNF198 13q12 FGFR1OP 6q27 CNTRL 9q33 LRRFIP1 2q37 RANBP2 2q13 CUX1 7q22 TRIM24 7q34 FGFR1OP2 12p11 CFS1 12q15 KIT 4q12 ALK 2p23 September 2014: 60 tyrosine kinase fusion genes

12 Identification of BCR-ABL1 negative, imatinib responsive patients All known imatinib-responsive fusions are associated with cytogenetically visible abnormalities except for FIP1L1- PDGFRA. Screen patients with Eos-MPN or persistent unexplained eosinophilia for FIP1L1-PDGFRA (and BCR-ABL1). Only screen for other fusions if indicated by karyotype: abnormalities of 4q11-12 (PDGFRA), 5q31-33 (PDGFRB), 9q34 (ABL) If cytogenetics fails consider split apart FISH Screen suspected mastocytosis for KIT D816V; only screen for FIP1L1- PDGFRA if eosinophilia present.

13 Identification of BCR-ABL1 negative, imatinib responsive patients Most BCR-ABL1 negative imatinib responders are male (>10:1 ratio for PDGFR fusions) with eosinophilia. BCR-ABL1 negative imatinib responders are very rare.

14 JAK2 fusions: clinical responses to ruxolitinib Schwaab et al., Ann Hematol. 2015;94(2):233-8

15 STAT5 phosphoflow to identify potential responders to TKI therapy? Roberts et al., N Engl J Med. 2014;371: Untreated vs +imatinib, dasatinib, ruxolitinib, ponatinib (1 hour) Overnight fix, permeabilisation 120 Sample 1 and Sample 2 response with dasatinib untreated dasatinib untreated dasatinib unt dasatinib

16 Signalling abnormalities in MDS/MPN Up to 50% of CMML cases have abnormalities that activate TK/RAS signalling Higher frequency in proliferative (WBC > /L) vs dysplastic CMML 10-60% of CNL/aCML cases have activating CSF3R mutations Maxson et al., NEJM 2013;368:11-20 Gene Mutated in CMML NRAS 11% CBL 10% JAK2 8% KRAS 8% NF1 4% FLT3 3% PTPN11 3% KIT <1% Gelsi-Boyer et al. Br J Haematol. 2010;151: Itzykson et al, J Clin Oncol, 2013;31: Haferlach et al. Leukemia. 2012;26:834-9 Plon S. Genet Med. 2011;13:203-4

17 Epigenetic mutations Epigenetics: heritable (through cell generations) changes in gene expression without changes in DNA sequence Modification of: DNA (methylation of CpG) DNMT3A, TET2, IDH1/2, WT1 histones (methylation, acetylation etc) EZH2, ASXL1 Zhou et al. Nat Rev Genet. 2011;12:7-18

18 Epigenetic mutations in MPN and MDS/MPN CH 2 OH CH 2 OH DNMT3a: ET PV PMF CMML 1-5% 5-10% 5-10% 2-10% CH 3 DNMT3a CH 3 Cross, Am Soc Hematol Educ Program. 2011: Jankowska et al., Blood. 2011;118: Grossmann et al. Leukemia. 2011;25:877-9 Itzykson R et al, J Clin Oncol. 2013;31:

19 Epigenetic mutations in MPN and MDS/MPN α-kg TET2 CH 2 OH CH 2 OH TET2: CH 3 CH 3 ET PV PMF CMML 1-5% 5-10% 5-10% 40-60% DNMT3a Cross, Am Soc Hematol Educ Program. 2011: Jankowska et al., Blood. 2011;118: Grossmann et al. Leukemia. 2011;25:877-9 Itzykson R et al, J Clin Oncol. 2013;31:

20 Epigenetic mutations in MPN and MDS/MPN IDH1/2 isocitrate α-kg TET2 CH 2 OH CH 2 OH TET2: CH 3 CH 3 ET PV PMF CMML 1-5% 5-10% 5-10% 40-60% DNMT3a Cross, Am Soc Hematol Educ Program. 2011: Jankowska et al., Blood. 2011;118: Grossmann et al. Leukemia. 2011;25:877-9 Itzykson R et al, J Clin Oncol. 2013;31:

21 Epigenetic mutations in MPN and MDS/MPN IDH1/2* isocitrate α-kg 2HG TET2 CH 2 OH CH 2 OH IDH1/2: ET PV PMF CMML 1-5% 5-10% 5-10% 1-5% CH 3 DNMT3a CH 3 Cross, Am Soc Hematol Educ Program. 2011: Jankowska et al., Blood. 2011;118: Grossmann et al. Leukemia. 2011;25:877-9 Itzykson R et al, J Clin Oncol. 2013;31:

22 Epigenetic mutations in MPN and MDS/MPN IDH1/2* isocitrate α-kg H3K27me3 2HG WT1 TET2 CH 2 OH CH 2 OH IDH1/2: ET PV PMF CMML 1-5% 5-10% 5-10% 1-5% CH 3 DNMT3a CH 3 Cross, Am Soc Hematol Educ Program. 2011: Jankowska et al., Blood. 2011;118: Grossmann et al. Leukemia. 2011;25:877-9 Itzykson R et al, J Clin Oncol. 2013;31:

23 Epigenetic mutations in MPN and MDS/MPN PRC2 EZH2 IDH1/2* isocitrate α-kg H3K27me3 2HG WT1 TET2 CH 2 OH CH 2 OH EZH2: CH 3 CH 3 ET PV PMF CMML 1-5% 5-10% 5-10% 5-15% DNMT3a Cross, Am Soc Hematol Educ Program. 2011: Jankowska et al., Blood. 2011;118: Grossmann et al. Leukemia. 2011;25:877-9 Itzykson R et al, J Clin Oncol. 2013;31:

24 Epigenetic mutations in MPN and MDS/MPN PRC2 EZH2 ASXL1 IDH1/2* isocitrate α-kg H3K27me3 2HG WT1 TET2 CH 2 OH CH 2 OH ASXL1: ET PV PMF CMML 1-5% 5-10% 5-10% 40-50% CH 3 DNMT3a CH 3 Cross, Am Soc Hematol Educ Program. 2011: Jankowska et al., Blood. 2011;118: Grossmann et al. Leukemia. 2011;25:877-9 Itzykson R et al, J Clin Oncol. 2013;31:

25 No clear association between mutations in epigenetic regulators EZH2 TET2 ASXL1 Ernst et al., Nature Genetics. 2010; Grossmann et al., Leukemia 2011

26 TET2 mutations may be acquired before or after JAK2 V617F in MPN Delhommeau et al. N Engl J Med. 2009;360(22): Schaub et al. Blood. 2010;115(10):

27 Several somatically mutated genes in myeloid disorders are constitutionally mutated in rare developmental disorders EZH2 SETBP1 ASXL1 DNMT3A BCOR cohesins EP300 Weaver Syndrome Schinzel-Giedion Bohring-Opitz Syndrome Overgrowth syndrome OFCD syndrome Cornelia de Lange Rubinstein-Taybi syndrome MPN/MDS/T-ALL acml Myeloid malignancies Myeloid malignancies AML, MDS MDS/MPN, MDS Lymphoma, T-ALL, myeloid

28 Common consequences of EZH2, ASXL1 and other mutations? SETBP1 Blood 2012;119: MLL SET Deregulation of HOXA gene expression EZH2 May provide a stem cell advantage that promotes clonal expansion (landscaping mutations) EED SUZ12 ASXL1 Cancer Cell 2012;22:180-93

29 Genetic complexity in chronic myeloid neoplasms Classes of mutations Complexity, subclonality and prognosis Specificity of mutations

30 MDS: prevalence of mutations >10% 13 genes 30 genes >5% >3% 34% of total oncogenic mutations Papaemmanuil et al. Blood. 2013;122(22):3616-2

31 Prognostic significance of mutations in specific genes EZH2, RUNX1, TP53, ASXL1 mutations generally associated with poor prognosis in multiple studies (MPN, MDS/MPN, MDS) Is the level of mutation important, ie clonal vs subclonal? Papaemmanuil et al. Blood. 2013;122(22):3616-2

32 TP53 Mutation Status Divides MDS Patients with Complex Karyotypes into Distinct Prognostic Risk Groups Bejar et al., ASH 2014

33 Prognosis in MDS associated with somatic genetic complexity (111 candidate genes) Papaemmanuil et al. Blood. 2013;122(22):3616-2

34 Prognosis in CMML associated with somatic genetic complexity (19 genes; 312 cases) Itzykson et al, J Clin Oncol, 2013;31:

35 Prognostic significance of somatic complexity in myelofibrosis: ASXL1, EZH2, SRSF2, IDH1/2 Lasho, Guglielmelli et al. Blood 2013;122:104 ASH 2013

36 Which genes should be screened for in diagnostic labs. Who is going to pay? Is it worth paying for?

37 Is up front mutation profiling the best prognostic indicator? Collaboration between Jackie Boultwood, Andrea Pellagatti (Oxford) and Moritz Gerstung, Elli Papaemmanuil, Peter Campbell (Sanger Institute, Cambridge) 124 MDS cases: NGS mutation screen + GEP microarray data (Affymetrix GeneChip Human Plus 2.0 arrays) on bone marrow CD34+ cells from 124 MDS patients Built statistical models to disentangle the effect of 12 mutated genes and 4 cytogenetic alterations on gene expression, diagnostic clinical variables and outcome in patients with MDS Gerstung et al., Nat Commun Jan 9;6:5901

38 prognostic power Prognostic power of gene expression, mutations and clinical parameters Genetics, gene expression, blood and bone marrow counts all contain information for predicting survival Prognostic power of expression data was greater than that of genetics, cytogenetics or IPSS score Prognostic information present in genetics and cytogenetics is mostly contained in expression and blood/bone marrow count data and does not add independent prognostic information Gene expression data provide greatest prognostic information Gerstung et al., Nat Commun Jan 9;6:5901

39 Molecular MRD status provides the most powerful prognostic factor in many scenarios e.g. NPM1 mutant AML

40 Genetic complexity in chronic myeloid neoplasms Classes of mutations Complexity, subclonality and prognosis Specificity of mutations

41 Somatic myeloid mutations in the general population n=17,182; 160 genes Mutations rare <40 years old but seen in 10% >70 years old DNMT3A, ASXL1, TET2 Increased risk of hematologic malignancy (HR = 11.1) n=12,380; WES Mutations 1% <50 yrs; 10% >65 yrs DNMT3A, ASXL1, TET2 Increased risk of hematologic malignancy (HR = 12.9) 42% of hematologic malignancies arose in persons who had clonality Jaiswal et al., N Engl J Med ;371(26): Genovese et al., N Engl J Med. 2014;371(26):

42 Somatically acquired uniparental disomy (copy number neutral LOH) in healthy individuals Study of 108 elderly men using the Illumina 1M-Duo beadchip analysis plus 78 elderly monozygotic twins Somatic abnormalities seen in 3.4% of cases >60 years old Included abnormalities associated with malignancy del(5q), del(20q), 4q aupd Blood counts normal Forsberg et al., Am J Hum Genet ;90:217-28

43 Is the aupd at 4q in ULSAM-697 associated with an acquired TET2 mutation? Sanger sequencing entire TET2 coding sequence Found 21bp deletion that disrupts exon 4 Mutation found at ages 71, 82, 88 and 90 but absent in fibroblasts

44 Why do some JAK2 V617F positive patients develop PV and others develop ET? Strength of JAK2 signalling Higher JAK2 V617F allele burden (%V617F) in PMF and PV compared to ET Frequent homozygous JAK2 V617F clones in PMF and PV; rare in ET JAK2 exon 12 mutations associated with erythroid phenotype and show stronger signalling in model systems Constitutional genetic differences? MPN phenotype in retroviral transplant models dependent on mouse strain Zaleskas et al., PLoS One. 2006;1:e18 Godfrey et al., Blood Sep 27;120(13): Scott et al., N Engl J Med. 2007;356:

45 Constitutional genetic variation at HBSL1-MYB influences whether JAK2 V617F positive cases develop ET or PV n=1112 (556 ET, 556 PV) from the UK Top hit (excluding 9p) = rs in HBSL1-MYB polymorphic intergenic region Tapper et al. Nat Commun 2015 in press

46 Summary Complex interplay between somatic and inherited variants in myeloid disorders >40 recurrent somatically mutated genes identified that can be grouped into 5 principal functional classes Small subset of genes and overall somatic complexity prognostically significant [but GEP and MRD analysis may be more powerful] Driver mutations may be present at high level in healthy elderly individuals

47 Acknowledgements Salisbury Andy Chase Will Tapper Amy Jones Jo Score Catherine Bryant Thomas Ernst Will Leung Sanger/ICGC Mannheim Elli Papaemmanuil Peter Campbell Jyoti Nangalia Carlo Gambacorti-Passerini Tony Green Mario Cazzola Eva Hellstrom-Lindberg David Bowen Jackie Boultwood Andrea Pellagatti Andreas Reiter Juliana Schwaab Uppsala Jan Dumanski Lars Forsberg GWAS Alessandro Vannucchi Paola Guglielmelli Mario Cazzola Giovanni Barosi Robert Kralovics Heinz Gisslinger Konny Döhner Frank Stegelmann Andreas Hochhaus Katerina Zoi Heike Pahl Susanne Schnittger David Oscier Andrew Duncombe Tony Green Anna Godfrey Claire Harrison Rosemary Gale Adam Mead Anna Schuh Andrew Jack Paul Evans Jo Ewing Mike Griffiths