Cytogenetic and molecular abnormalities in AML. Dr Elizabeth Tegg Director of haematology Pathology West

Transcription

1 Cytogenetic and molecular abnormalities in AML Dr Elizabeth Tegg Director of haematology Pathology West

2 Outline Classification of AML Types of genetic changes Next generation sequencing in HM

3 Outline Classification of AML Types of genetic changes Next generation sequencing in HM

4 Genetics: Importance Diagnosis Prognosis Different treatment options

5 Classification of HM Current WHO classification 2001, 2008, 2016 Morphology Immunophenotype Cytogenetics Molecular genetic Clinical features

6 Current Classification: WHO of AML 1.AML with recurrent genetic abnormalities AML with t(8;21)(q22;q22) AML with inv(16)(p13q22) or t(16;16)(p13;q22) Acute promyelocytic leukamia AML with t(15;17)(q22;q12) AML with 11q23 (MLL) abnormalities AML with t(6;9)(p23;q34) AML with inv(3)(q21q26.2) AML with t(1;22)(p13;q13) AML with gene mutations FLT3, NPM1 2.AML with myelodysplasia related changes 3.Therapy related myeloid leuakemia Alkylating agent related Topoisomerase II inhibitor-related 4.AML not otherwise specified AML, minimally differentiated AML without maturation AML with maturation Acute myelomonocytic leukaemia Acute monoblastic and monocytic leukaemia Acute erythroid leukaemia Acute megakaryoblastic leukaemia Acute basophilic leukameia Acute panmyelosis with myelofibrosis 5. Myeloid sarcoma 6. Myeloid proliferations related to Downs syndrome 7. Blastic plasmacytoid dendritic cell neoplasm

7 Outline Classification of AML Types of genetic changes Next generation sequencing in HM

8 Types of genetic changes Chromosomal DNA Epigenetic Changes in the tumour compared to germline

9

10 Cytogenetics

11 Cytogenetics Classic abnormalities Cryptic abnormalities All the above have been reported as also being cryptic A translocation that is undetectable to the eye FISH

12 Conventional Cytogenetics Aim: to get the maximal number of the cell of interest dividing and then halt cell division so that chromosomes can be visualised at their clumpest stage of the cell division (Prometaphase/metaphase). Pros: good overview of the whole genome of the disease Cons: Can be difficult to get cells dividing, low resolution

13 chromosomes for Conventional cytogenetics Culture: LIVE CELLS Mitogen Spindle inhibitor: HALTS DIVISION Hypotonic solution Fixative Banding: GTL Analysis

14 Cell cycle

15 Mitosis

16

17 Clones Defined as a cell population derived from a single progenitor. It is common practice to infer a clonal origin when a number of cells have the same or closely related abnormal chromosome complements. The clone size is given in square brackets [ ] after the karyotype At diagnosis we look at only 20 metaphases Follow up 40 metaphases

18 Stemline, sideline and clonal evolution Cytogenetically related clones (subclones) are presented as far as possible in order of increasing complexity, irrespective of the size of the clone. Stemline (sl) is the basic clone of a tumour and listed first Sideline (sdl) all additional derived clones 46,XX,t(9;22)(q34;q11.2)[3]/47,XX,+8,t(9;22)(q34;q11.2)[17] ml: 47,XX,+8,t(9;22)(q34;q11.2)[17] sl: 46,XX,t(9;22)(q34;q11.2)[3] sdl: 47,XX,+8,t(9;22)(q34;q11.2)[17] 46,XX,t(9;22)(q34;q11.2)[17]/47,XX,+8,t(9;22)(q34;q11.2)[3] ml: 46,XX,t(9;22)(q34;q11.2)[17] sl: 46,XX,t(9;22)(q34;q11.2)[17] sdl: 47,XX,+8,t(9;22)(q34;q11.2)[3]

19 Karyotype Count chromosomes: Modal number Near-haploid (23+/-) <34 Hypohaploidy <23 Hyperhaploidy Near-diploid (46+/-) Hypodiploidy Hyperdiploidy Near-triploid (69+/-) Hypotriploidy Hypertriploidy Near-tetraploidy (92+/-) Hypohaploidy Hyperhaploidy

20 Order of abnormalities All abnormalities are listed in numerical order X, Y then 1-22 Numerical abnormalities before structural Structural abnormalities second in alphabetical order add, del, inv, t, 47,XX,+1,t(1;3)(p32;q21) 47,XX,t(1;3)(p32;q21),+21 If abnormalities occur to the same individual chromosome (ie is a derivative), abnormalities are listed according to breakpoint (pter to qter) and not separated by a comma 46,XX,der(1)t(1;3)(p32;q21)add(1)(q25)

21 Chromosomal abnormalities Numerical Monosomy Trisomies Structural Add Translocation Duplication Deletion Inversion Isochromosome Ring Marker Double minutes

22 What does this mean 46,XX[20] Normal female karyotype 70,XXX,+8[20] Near triploid cell line, with additional chromosome 8 (ie 3 copies of all chromosomes with 4 copies of 8) 46,XY,inv(16)(p13q22)[20] AML with inversion 16

23 Numerical Changes A very common mechanism Seen in both Myeloma, AML and ALL Hyperdiploidy is typical in ALL modal chromosome number is 54 Hyperdiploidy with ~47 chromosome is seen in AML/MDS Typical patterns of chromosomes involved

24 Cytogenetics: Numerical Monosomy 7 AML, MDS, MPN Trisomy 8 All haematological malignancies Trisomy 21 All haematological malignancies

25 Structural Abnormalities in Leukaemia Found in 65% of cases 124 different structural abnormalities have been described But a sub-group of 30 represent the majority of abnormalities seen Are translocations 65%, deletions 30% or inversions 5%.

26 Translocation: Reciprocal exchange of two chromosomal segments Deletion: Removal of a chromosomal segment Inversion: Inversion of a segment around the centromere

27 Chromosome Translocations Result in the juxtaposition of previously separate DNA sequences eg the t(9;22)(q34;q11.2) When this was first published (1973) the concept of a translocation of genetic material was new 9 der(9)elizabeth der(22) Tegg 22016

28 Deletions Deletions remove genes that Stop the cell proliferating out of control Whose normal function is to detoxify chemical agents That repair DNA damaged by several mechanisms

29 Deletions Most common deletions in leukaemia are Deletions of 5q/7q and 20q in MDS/AML Deletions of 6q, 9p and 12p in ALL

30 Inversions Are an uncommon mechanism The best known is the inv(16) which is diagnostic of AML M4 with eosinophilia Associated with the most favourable outcome for all classes of AML in adults

31 Inversion 16(p13q22) Vysis LSI CBFB Dual Color, Break Apart Rearrangement Probe CBFB 5 CBFB 3 CBFB The inv(16) interrupts the coding region of the CBFB and MYH11 genes and leads to a chimeric protein that competes with normal

32 Fluorescence in situ hybridization Fluorescently labelled probes Denature the probe and target DNA (Heat) Anneal stage: complimentary sequences pair Wash off unbound probe Counter stain Analyse under a fluoresent microscope

33 FISH probes Locus Specific Identifiers Break Apart Dual Colour/Dual Signal, TriColour/Dual Fusion Dual Colour/Single Fusion

34 FISH nomenclature All changed recently with the ISCN 2005 and in 2013 ish: metaphase nuc ish: interphase nuc ish(abl1 x3),(bcr x3),(abl1 con BCR x2)[400] nuc ish(abl1,bcr)x3(abl1 con BCR x2)[400] All 400 interphase cells showed two dual fusion signals with no evidence of a deletion of the derivative 9 ABL1 x 3 BCR x 3 ABL1 con BCR x 2

35 Break-apart probes Break-apart probes are made of 2 probes, the short form does not convey that the normal situation is presence of 2 fusion signals nuc ish(mll x 2)[400] nuc ish(5 MLL,3 MLL)x2(5 MLL con 3 MLL x2)[400] Interpret: 400 interphase cells show a normal signal pattern. No evidence of rearrangement of MLL

36 Break-apart probe: MLL DFBA red=5 MLL green=3 MLL nuc ish(mllx2)[400] nuc ish(mll x 2)(5 MLLsep3 MLL x 1)[400] nuc ish(5 MLL x 2,3 MLL x1)(5 MLL con 3 MLL x 1)[400]

37

38 FISH vs Conventional Pros On both metaphase or interphase cells Target genetic aberrations Cons Inability to provide a genomewide assessment Necessity of clinical information to drive what probes to use Requirement of a high quality fluorescence microscope with multiple filters

39 Molecular karyotype DNA based technology Array comparative genomic hybridization Bac (Bacterial artificial chromsomes) SNP arrays High probe density in cancer relevant regions of the genome Higher sensitivity Don t need dividing cells Detect CNV and LOH Not good for balanced translocations or inversions

40

41

42 SNP arrays Copy neutral-loss Of Heterozygousity Identification of PAX5 as a key target of genetic inactivation in B-ALL Identification of TET2 as a major tumour suppressor in MDS Need germline DNA for comparison Mechanism where heterozygous mutations become homozygous Heinrichs S, Li C, Look AT. SNP array analysis in hematological malignancies: avoiding false discoveries. Blood March 19, 2010.

43 What have we learnt from cytogenetics A lot of subtypes are defined by simple translocations Many of the numerical gains have patterns to them and it is consistently the same chromosome lost or gained

44 1. AML with recurrent cytogenetic abnormalities AML with recurrent genetic abnormalities AML with t(8;21)(q22;q22) AML with inv(16)(p13q22) or t(16;16)(p13;q22) Acute promyelocytic leukamia AML with t(15;17)(q22;q12) AML with 11q23 (MLL) abnormalities AML with t(6;9)(p23;q34) AML with inv(3)(q21q26.2) AML with t(1;22)(p13;q13)

45 AML with t(8;21)(q22;q22) 5-12% of AML, mainly younger patients genes involved RUNX1/ RUNX1T1 Can diagnose AML with <20% blasts with this abnormality Good prognosis

46 AML with inv(16)(p13q22) or t(16;16)(p13;q22) 10-12% of AML, predominantly younger pt genes involved CBF beta to MYH11 (smooth muscle mycin gene) Can diagnose AML with <20% with this abnormality Good prognosis

47 AML with inv(16)(p13q22) or t(16;16)(p13;q22) Vysis LSI CBFB Dual Color, Break Apart Rearrangement Probe CBFB 5 CBFB 3 CBFB The inv(16) interrupts the coding region of the CBFB and MYH11 genes and leads to a chimeric protein that competes with normal

48 Acute promyelocytic leukaemia with t(15;17)(q22;q12) 5-8% of AML, usually adults in mid life genes involved PML/RAR alpha Best prognosis if you survive the first week Variant translocations t(11;17)(q23;q21) t(5;17)(q23;q12) t(11;17)(q13;q21) t(11;17)(q23;q21) is resistant to ATRA, and is morphologically the same Independent prognostic factor is WCC at diagnosis <2 is good Numerous Auer rods

49 Acute promyelocytic leukaemia AML with t(15;17)(q22;q12)

50 AML with 11q23 (MLL) abnormalities Usually associated with monocytic features 5-6% of AML, mainly children Two clinical subgroups have a higher frequency 1. Aml in infants 2. Therapy related, usually after DNA topoisomerase II inhibitors gene involved MLL gene ( Drosophila trithorax gene) Is a developmental regulator which is structurally altered

51 Gene names

52 AML with 11q23 (MLL) abnormalities Mixed Lineage Leukaemia Over 90 reported translocation MLL translocations predicts early relapse and poor prognosis MLL Consists of at least 36 exons, encoding a 3969 amino-acid nuclear protein with molecular weight of nearly 430kDa It is thought to function as a positive regulator of gene expression in early embryonic development and haematopoiesis MLL translocation breakpoints cluster within an 8.3kb region spanning exons 5-11 The mechanisms by which these rearrangements results in leukaemia remain largely unknown Li et al Leukaemia 2005; 19:

53 AML with t(6;9)(p23;q34) Morphology Associated with M2 or M4 and basophilia DEK-CAN DEK-NUP214 Poor prognosis High association with FLT3-ITD

54

55 AML with inv(3)(q21q26.2) Maybe de novo or arise from MDS Morphology: increased atypical megakaryocytes

56 AML with t(1;22)(p13;q13) Acute megakaroblastic leukaemia Rare Most commonly occurs in Down syndrome

57 AML with gene mutations Fms-related tyrosine kinase 3 (FLT3) Nucleophosmin (NPM1) CEBPA KIT WT1 NRAS KRAS

58 DNA sequence changes Point mutation: simple change in one base Missence: amino acid change Nonsence: changes to a stop codon Frame-shift mutation: one or more bases is inserted or deleted

59 Molecular Subgroups of Cytogenetically normal AML Dohner H et al. Diagnosis and management of acute myeloid leukemia in adults: recommendations from an international expert panel, on behalf of the European LeukemiaNet. Blood January 21, 2010;115(3):

60 AML with mutated NPM1 Mutations usually in exon 12 Aberrant cytoplasmic expression of nucleophosmin is a surrogate marker of this gene mutation Morphology: monocytic CC: normal Mutated in 45-64% of adult normal CC AML Good prognosis

61 FLT3 Located 13q12 Encodes a tyrosine kinase receptor that is involved in HSC differentiation and proliferation 2 primary types of mutations Internal tandem duplications (FLT3-ITD) (adverse outcome) Mutations affecting codon 835 or 836 of the second tyrosine kinase domain (TKD) (outcome?)

62 KIT Located 4q11-12 Member of type 3 tyrosine kinase family Generally test for KIT mutation in the core binding factor AML Poor prognosis

63 Epigenetic changes Disruption of DNA methylation Histone modification Chromatin compartments Esteller M. Epigenetics provides a new generation of oncogenes and tumour-suppressor genes. Br J Cancer. Elizabeth 2006;94(2): Tegg 2016

64 Outline Classification of AML Types of genetic changes Next generation sequencing in HM

65 Next generation sequencing in HM Sequencing of cytogenetically normal AML 50% of AMLs will be cytogenetically normal 12 acquired mutations in coding regions 2 in known genes (NPM1 and NRAS) 10 in genes not previously reported to be mutated in AML Mardis ER, Ding L, Dooling DJ, Larson DE, McLellan MD, Chen K, et al. Recurring Mutations Found by Sequencing an Acute Myeloid Leukemia Genome. New England Journal of Medicine September 10, 2009;361(11):

66 International Cancer Genome Consortium ICGC Goal: To obtain a comprehensive description of genomic, transcriptomic and epigenomic changes in 50 different tumor types and/or subtypes which are of clinical and societal importance across the globe.

67

68 Clinical utility WGS Ideally every HM to be sequenced Problems from a haematology point of view Normal DNA What is normal Every time a cell undergoes mitosis DNA changes occur Tumour DNA Heterogeneity with in the tumour cells coverage needed We know from cytogenetics that different clones are present in tumours Passenger vs driver mutations

69 Perspectives for therapeutic targeting of gene mutations in acute myeloid leukaemia with normal cytogenetics British Journal of Haematology Volume 170, Issue 3, pages , August 2015 The acute myeloid leukaemia (AML) genome contains more than 20 driver recurrent mutations. Here, we review the potential for therapeutic targeting of the most common mutations associated with normal cytogenetics AML, focusing on those affecting the FLT3, NPM1 and epigenetic modifier genes (DNMT3A, IDH1/2, TET2). As compared to early compounds, second generation FLT3 inhibitors are more specific and have better pharmacokinetics. They also show higher anti-leukaemic activity, leading to about 50% of composite complete remissions in refractory/relapsed FLT3-internal tandem duplication-mutated AML. However, rapid relapses invariably occur due to various mechanisms of resistance to FLT3 inhibitors. This issue and the best way for using FLT3 inhibitors in combination with other therapeutic modalities are discussed. Potential approaches for therapeutic targeting of NPM1-mutated AML include: (i) reverting the aberrant nuclear export of NPM1 mutant using exportin-1 inhibitors; (ii) disruption of the nucleolus with drugs blocking the oligomerization of wild-type nucleophosmin or inducing nucleolar stress; and (iii) immunotherapeutic targeting of highly expressed CD33 and IL3RA (CD123) antigens. Finally, we discuss the role of demethylating agents (decitabine and azacitidine) and IDH1/2 inhibitors in the treatment of AML patients carrying mutations of genes (DNMT3A, IDH1/2 and TET2) involved in the epigenetic regulation of transcription.

70

71