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How to make sense of genetic studies in AML and MDS

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

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How to make sense of genetic studies in AML and MDS

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  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 NatlAcadSci U S A. 1986 May;83(9):2934-8 • 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 Interphase Metaphase 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 • M1 myeloblastic undifferentiated • M2 myeloblastic with differentiaion • M3 promyelocytic • M4 myelomonocytic • M5 monoblastic • M6 erythroleukaemia • M7 megakaryoblastic WHO CLASSIFICATION 2008 • AML with recurrent genetic abnormalities • AML with multilineage dysplasia • AML therapy related • AML not otherwise categorised • AML 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 cytoplasmichoffs, 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 109/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 Estimate (CI) At Risk Deaths at 5 Years 100 Favorable 121 53 55% (45-64%) Intermediate 278 168 38% (32-44%) Unfavorable 184 162 11% ( 7-16%) 80 60 Cumulative Percent 40 20 Heterogeneity of 3 Groups: p<.0001 0 0 2 4 6 8 Years After Entering Study Slovak et al. Blood, 2000

  17. Is cytogenetic analysis old news? • Presence of a monosomy (ie chromosome 7 deletion) with 2 additionalchromosomaldeletions or withcomplexcytogenetics • Associatedwith a poor CR rate and OS (4%) • Manyrecentstudies have confirmed • phrases such as dismaloutcomeand veryunfavorable 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 • FLT3 is receptor tyrosine kinase • Japanese groups originally found internal tandem duplication • Nakao et al. Leukemia1996 • ITD leads to activation of kinase • Can be detected by PCR ITD normal ASH Education Book January 1, 2001 vol. 2001 no. 1 541-552

  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 Kottaridiset al. Blood 2001

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

  27. NPM1 mutations • Most frequentmolecularabnormality in normal cytogenetics AML: 50-60% • Usually 4 nucleotide insertion • Can bedetectedwith Sanger sequencing Chen et al. Arch Pathol Lab Med. 2006

  28. NPM1 mutation • Nuclear transport protein • Mutation associatedwithabnormallocalization of protein in cytoplasm • C-terminal mutations detected in 85% • NLS domain • Frequentlyoccurswith 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 J ClinOncol. 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

  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-treatanalysis • 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 Favorable CG Intermediate 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) Poor CG

  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. CytogeneticsR-IPSS Blood. 2012;120(12):2454

  47. Scoring system R-IPSSCytogenetics 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

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