1. How to make sense of genetic studies in AML and MDS Elie Traer September 13, 2012

2. Outline Genetic tests and methodology – Cytogenetics, i.e. large chromosomal abnormalities – FISH, smaller chromosomal changes – Genetic mutations AML – Evolution of genetic tests in diagnosis and prognosis – Risk groups and treatment – Future MDS – Current prognostication in acute leukemias What to order How it affects treatment

3. Cytogenetics - Methodology Cytogenetics (karyotype) – Cells arrested in metaphase with mitotic inhibitors – Depends upon chromatin condensation during metaphase (metaphase spread) Most cells are in interphase – Staining with Geimsa stain produces distinctive G-banding patterns Metaphase Of course, not everything is neatly arranged in real life… Interphase

4. Normal cytogenetics – cleaned up 22 paired chromosomes + XY = 46 total (image from NHGRI)

5. Classic cytogenetics Advantages: – Whole chromosome analysis of individual cells – Bone marrow aspirate relatively easier Compared to solid tumors Disadvantages: – Time consuming – Need metaphase spreads – Not sensitive for small genetic deletions or changes

6. FISH – a “new” technique for cytogenetics FISH = fluorescence in situ hybridization Technique developed in part by Joe Gray Proc Natl Acad Sci U S A. 1986 May;83(9):2934-8 Proc Natl Acad Sci U S A. Fluorescently labeled probes targeted to known areas of chromosomes Advantages compared to cytogenetics – Don’t need metaphase – Sensitive to small changes – Can target any part of chromosome BUT, you have to know what you are looking for

7. FISH – CML Red probe to chromosome 22 Green probe to chromosome 9 Fusion creates red-green or yellow color InterphaseMetaphase Probes come together

8. Cytogenetics in leukemia Oldest method for looking at chromosomes – >300 cytogenetic abnormalities in acute leukemia Most famous translocation is t(9;22), or Philadelphia chromosome – fusion of BCR and ABL genes – led to development of imatinib Technique demonstrate clonality of leukemia – CML with specific secondary cytogenetic abnormalities Cytogenetic abnormalities associated with disease subtypes and prognosis

9. How important are genetics? FAB CLASSIFICATION oM1 myeloblastic undifferentiated oM2 myeloblastic with differentiaion oM3 promyelocytic oM4 myelomonocytic oM5 monoblastic oM6 erythroleukaemia oM7 megakaryoblastic WHO CLASSIFICATION 2008 oAML with recurrent genetic abnormalities oAML with multilineage dysplasia oAML therapy related oAML not otherwise categorised oAML of ambiguous lineage

10. WHO 2008 recurrent cytogenetics t(8;21)(q22;q22) Inv(16)(p13.1q22) or t(16;16)(p13.1;q22) t(15;17)(q22;q12) t(9;11)(p22;q23):MLLT3-MLL t(6;9)(p23;q34) t(1;22)(p13q13) *Considered acute leukemias regardless of blast count

11. Cytogenetic risk groups Favorable (~20%) – CBF: t(8;21)(q22;q22), Inv(16)(p13.1q22) or t(16;16)(p13.1;q22) – APL: t(15;17) Intermediate (~60%) – Normal cytogenetics (~50% of all AML cases) – t(9;11)(p22;q23):MLLT3-MLL – Any cytogenetic abnormality not classified as favorable or adverse (trisomy 8) Adverse (~20%) – Inv(3)(q21q26.2) or t(3;3)(q21;q26.2) – t(6;9)(p23;q34) – t(v;11)(v;q23): MLL rearranged – - 5 or del(5q) – -7 – Abnl 17p – Complex karyotype

12. Core Binding Factor Leukemias t(8;21) and inv16/t(16;16) atlasgeneticsoncology.org

13. t(8;21)(q22;q22) RUNX1-RUNX1T1 RUNX1 = AML1 = CBFa = 21 RUNX1T1 = ETO = 8 Approximately 8% of AMLs - predominately in younger patients Blasts have cytoplasmic hoffs, occasional Auer rods, occasional salmon- colored granules Dysplastic features in maturing neutrophils Favorable prognosis when presenting with white blood cell count less than 20 x 10 9 /L and NO KIT mutation Jaffe Hematopathology

14. Inv(16)(p13.1q22) or t(16;16)(p13.1;q22) CBFB = 16q22 MYH11 (smooth muscle myosin heavy chain) = 16p13 5-8% of AML All age groups, predominately in younger patients Blasts have myelomonocytic features Abnormal eosinophils with large granules in bone marrow (M4Eo) – no peripheral eosinophilia Good prognosis when NO KIT mutation is present May be missed on routine karyotyping, need FISH

15. Acute promyelocytic anemia (APL) with t(15;17)(q22;q12) PML-RARA Proliferation of leukemic blasts blocked at the promyelocyte stage of differentiatoin – 5-8% of AML – Abundant cytoplasmic granules and Auer Rods – Weak or absent HLA-DR and absent CD34 expression – Low white count – Disseminated intravascular coagulation (DIC) – Medical Emergency! – Treat with all-trans-retinoic acid (ATRA)

16. Survival associated with cytogenetics Slovak et al. Blood, 2000 Years After Entering Study 0 20 40 60 80 100 02 Cumulative Percent 846 Favorable121 5355% (45-64%) Intermediate27816838% (32-44%) Unfavorable18416211% ( 7-16%) Estimate (CI) At Risk Deaths at 5 Years Heterogeneity of 3 Groups: p<.0001

17. Is cytogenetic analysis old news? Presence of a monosomy (ie chromosome 7 deletion) with 2 additional chromosomal deletions or with complex cytogenetics Associated with a poor CR rate and OS (4%) Many recent studies have confirmed – phrases such as dismal outcome and very unfavorable Breems J Clin Oncol 2008

18. Effect on overall survival Breems J Clin Oncol 2008

19. Cytogenetics summary Cytogenetics remains an important risk classification for acute leukemia, particularly AML Cytogenetics/FISH can only detect large genetic changes However, 50-60% of AML with “normal” cytogenetics – Really normal?

20. Mutations in leukemia (molecular markers) Smaller changes to DNA are not detected with cytogenetics/FISH – Mutations – Smaller duplications – Deletions

21. Quick genetic review DNA holds all instructions – 3 trillion base pairs mRNA translated from DNA – introns spliced out – hundreds to thousands of base pairs mRNA translated into protein

22. Mutations – methodology PCR – Method for amplifying known sections of DNA or mRNA – Can be quantitative (QPCR) BCR-ABL, PML-RARA – Can detect small variations in size (deletions or amplifications) e.g. FLT3 ITD Sanger sequencing – Direct sequencing of DNA Sequenom – Multiplexed PCR and MALDI-TOF Next generation sequencing – Whole exome/genome – Deep sequencing

23. PCR Primers surrounding area of interest Selective area amplified Can be sequenced or analyzed by gel electrophoresis

24. FLT3 internal tandem duplication ASH Education Book January 1, 2001 vol. 2001 no. 1 541-552 FLT3 is receptor tyrosine kinase Japanese groups originally found internal tandem duplication Nakao et al. Leukemia 1996 ITD leads to activation of kinase Can be detected by PCR ITD normal

25. FLT3 ITD is unfavorable risk marker FLT3 ITD detected in ~30% of normal cytogenetics AML Point mutations, i.e. D835 mutations found in ~10% Not prognostic Associated with higher relapse and worse overall survival Kottaridis et al. Blood 2001

26. Point mutations: CEBP  Transcription factor involved in neutrophil differentiation Mutations in multiple sites but most lead to early truncation of protein 10% mutations in normal cytogenetics AML Preudhomme et al. Blood 2002

27. NPM1 mutations Most frequent molecular abnormality in normal cytogenetics AML: 50-60% Usually 4 nucleotide insertion Can be detected with Sanger sequencing Chen et al. Arch Pathol Lab Med. 2006

28. NPM1 mutation Nuclear transport protein Mutation associated with abnormal localization of protein in cytoplasm C-terminal mutations detected in 85% – NLS domain Frequently occurs with FLT3 ITD Fallini N Engl J Med 2005; Döhner Blood 2005; Schnittger Blood 2005; Verhaak Blood 2005

29. NPM1 and CEBPa are favorable prognostic markers (without FLT3 ITD) Schlenk N Engl J Med 2008

30. c-Kit Receptor tyrosine kinase Activating mutations in c-Kit have been described in AML Most common mutation in exon 17 (D816V) Only has prognosis in core binding factor (CBF) AML, inv(16) and t(8;21) Paschka et al. J Clin Oncol, 24 2006 However, recent report suggests that this may be limited to t(8;21) Park et al. Leuk Res, 2011

31. Genetic (cytogenetic and molecular) abnormalities and prognosis in AML

32. Sequencing more genes and more samples is increasing known mutations Largely driven by technology – Dramatic cost reductions – ~$100,000 to sequence genome in 2008 Ley et al. Nature 2008 – Now about $2000 Prognosis still not clear for many of these genes

33. Mutations in AML NPM145-64% CN-AMLGood without other mutations present CEBPA10-18% CN-AMLGood with both alleles are mutated FLT3 ITD28-34% CN-AMLWorse KIT25-30% CBF AMLInferior* in CBF-AML FLT3 TKD5-10% all AML/11-14% CN-AML? IDH1/210-15% CN-AMLInferior – controversial WT110-13% CN-AML? RUNX15-13% all AMLfew studies – worse MLL-PTD5-11% CN-AMLWorse, but not an independent prognostic factor NRAS9-14% CN-AML, 40% CBF AML, 25-30% AML with inv(3) None KRAS5-17% CBF-AML TP53Complex/monosomal karyotpype and tx-related AML Inferior TET223% of CN-AML? ASXL1~15%, Exon 12Few studies, worse DNMT3A20%inferior BCOR6%? Others: CBL, JAK2, EZH2 J Clin Oncol. 2011 Feb 10;29(5):475-86

34. AML and MDS panel at OHSU Ion Torrent – next generation sequencing

35. A “torrent” of data ~25 base pairs of data Massive computing to align sequences Deep sequencing – average coverage of one nucleotide

36. Ion Torrent panel - GeneTrails R-tyrosine kinaseFLT3KITCSF3R C-tyrosine kinaseJAK2ABL1 Signaling molecule CBLCBLBNRASKRASHRAS Serine/threonine kinase BRAF Cytokine receptorMPL PhosphatasePTPN11 EpigeneticIDH1IDH2DNMT3ATET2MLL EZH2 UTX Splicing machinery SF3B1PRPF40B SF1SF3A1 ZRSR2 SRSF2 U2AF1/ U2AF2 Transcriptional factor NPM1GATA1CEBPA ETV6RUNX1WT-1 OtherASXL1SH2B3 (LNK) TP53

37. Why do extra testing? Clinical – Help with prognosis and/or treatment e.g. AML with multiple MDS-type mutations Prepare for the future – Ion Torrent can sequence multiple genes at once Replace multiple genetic tests More sensitive Prepare for future prognostic studies Cost is going down

38. Reality check – How does this influence therapy?

39. EORTC AML-10 trial After induction, all patients < 46y allocated to - alloSCT if they have a donor - ASCT otherwise Intent-to-treat analysis N= overall 1198 pts After induction, n=293 with a donor, n=441 without a donor Suciu et al, Blood 2003

40. EORTC AML-10: results in cytogenetics groups Intermediate CG Favorable CG Poor CG Favorable risk gets no improvement in OS with allo Intermediate group as well but good portion were certainly favorable risk by molecular studies (just not known at time)

41. RFS in a donor vs no donor basis: NPM1+ FLT3-ITD- Schlenk N Engl J Med 2008

42. And that’s why we do this 3+7 followed by HiDAC 3+7 followed by allo SCT if good match available, consider auto or just chemo 3+7 followed by allo SCT

43. Myelodysplastic syndrome MDS – classically defined – Cytopenias – Abnormal cell maturation (dysplasia) – Can transform to AML Arbitrary line = 20% blasts in marrow is AML Shared genetic abnormalities Heterogeneous disease (like AML) – Transplant is only cure, but timing is important – Genetic studies becoming more important Diagnosis/prognosis Defining treatment

44. Old classification - Morphology Up To Date

45. WHO 2008 classification Genetic abnormalities becoming more important – Added MDS with isolated 5q- More importantly, risk assessment and genetics becoming more refined – Revised-IPSS

46. Cytogenetics R-IPSS Blood. 2012;120(12):2454

47. Scoring system R-IPSS Cytogenetics influences prognosis Blood. 2012;120(12):2454

48. Like AML, mutations are becoming more important in MDS N Engl J Med. 2011 364(26):2496-506

49. Many concurrent mutations N Engl J Med. 2011 364(26):2496-506

50. Mutations can be used to assess risk N Engl J Med. 2011 364(26):2496-506

51. Some mutations associate with low risk MDS Blood 2011 118(24):6239-46 RARS – refractory anemia with ringed sideroblasts Lower risk disease by morphology

52. SF3B1 mutations - OS Blood 2011 118(24):6239-46 * Not independent of morphology

53. Ion Torrent panel - GeneTrails R-tyrosine kinaseFLT3KITCSF3R C-tyrosine kinaseJAK2ABL1 Signaling molecule CBLCBLBNRASKRASHRAS Serine/threonine kinase BRAF Cytokine receptorMPL PhosphatasePTPN11 EpigeneticIDH1IDH2DNMT3ATET2MLL EZH2 UTX Splicing machinery SF3B1PRPF40B SF1SF3A1 ZRSR2 SRSF2 U2AF1/ U2AF2 Transcriptional factor NPM1GATA1CEBPA ETV6RUNX1WT-1 OtherASXL1SH2B3 (LNK) TP53

54. Genetics and treatment Isolated 5q – Lenalidomide Epigenetic mutations (TET2, DNMT3A, ASXL1) – May predict better response to hypomethylating agents Leukemia 2011 25(7):1147-1153 High risk – Allogeneic transplant

55. Summary Cytogenetics and genetic tests are important for risk stratification and treatment Important to collect information at diagnosis – Especially AML Cytogenetics and FISH still important Mutations also prognostic More mutations being discovered – GeneTrails/Ion Torrent