Cytogenetic and molecular

abnormalities in AML

Dr Elizabeth Tegg

Director of haematology

Pathology West

Elizabeth Tegg 2016

Outline

• Classification of AML

• Types of genetic changes

• Next generation sequencing in HM

Elizabeth Tegg 2016

Outline

• Classification of AML

• Types of genetic changes

• Next generation sequencing in HM

Elizabeth Tegg 2016

Genetics: Importance

• Diagnosis

• Prognosis

• Different treatment options

Elizabeth Tegg 2016

Classification of HM

• Current WHO classification 2001, 2008,

2016

– Morphology

– Immunophenotype

– Cytogenetics

– Molecular genetic

– Clinical features

Elizabeth Tegg 2016

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

Elizabeth Tegg 2016

Outline

• Classification of AML

• Types of genetic changes

• Next generation sequencing in HM

Elizabeth Tegg 2016

Types of genetic changes

• Chromosomal

• DNA

• Epigenetic

• Changes in the tumour compared to

germline

Elizabeth Tegg 2016

Elizabeth Tegg 2016

Cytogenetics

Elizabeth Tegg 2016

Cytogenetics

• Classic abnormalities

• Cryptic abnormalities

– All the above have been reported as also

being cryptic

• A translocation that is undetectable to the eye

– FISH

Elizabeth Tegg 2016

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

Elizabeth Tegg 2016

chromosomes for Conventional

cytogenetics

• Culture: LIVE CELLS

• Mitogen

• Spindle inhibitor: HALTS DIVISION

• Hypotonic solution

• Fixative

• Banding: GTL

• Analysis

Elizabeth Tegg 2016

Cell cycle

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Mitosis

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Elizabeth Tegg 2016

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

Elizabeth Tegg 2016

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]

Elizabeth Tegg 2016

Karyotype

• Count chromosomes: Modal number– Near-haploid (23+/-) <34

• Hypohaploidy <23

• Hyperhaploidy 24-34

– Near-diploid (46+/-) 35-57• Hypodiploidy 35-45

• Hyperdiploidy 47-57

– Near-triploid (69+/-) 58-80• Hypotriploidy 58-68

• Hypertriploidy 70-80

– Near-tetraploidy (92+/-) 81-103• Hypohaploidy 81-91

• Hyperhaploidy 93-103

Elizabeth Tegg 2016

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)

Elizabeth Tegg 2016

Chromosomal abnormalities

• Numerical– Monosomy

– Trisomies

• Structural– Add

– Translocation

– Duplication

– Deletion

– Inversion

– Isochromosome

– Ring

– Marker

– Double minutes

Elizabeth Tegg 2016

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

Elizabeth Tegg 2016

Numerical Changes

• A very common mechanism

• Seen in both Myeloma, AML and ALL

• Hyperdiploidy is typical in ALLmodal chromosome number is 54

• Hyperdiploidy with ~47 chromosome is seen in AML/MDS

• Typical patterns of chromosomes involved

Elizabeth Tegg 2016

Cytogenetics: Numerical

• Monosomy 7

– AML, MDS, MPN

• Trisomy 8

– All haematological malignancies

• Trisomy 21

– All haematological malignancies

Elizabeth Tegg 2016

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%.

Elizabeth Tegg 2016

Translocation:

Reciprocal exchange of two

chromosomal segments

Deletion:

Removal of a chromosomal

segment

Inversion:

Inversion of a segment around

the centromere

Elizabeth Tegg 2016

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

der(22) 229 der(9)Elizabeth Tegg 2016

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

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Deletions

• Most common deletions in leukaemia are

– Deletions of 5q/7q and 20q in MDS/AML

– Deletions of 6q, 9p and 12p in ALL

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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

Elizabeth Tegg 2016

Inversion 16(p13q22)

Vysis® LSI® CBFB Dual Color, Break Apart Rearrangement Probe

5’CBFB

3’CBFB

CBFB

The inv(16) interrupts the coding region of the CBFB and MYH11 genes and

leads to a chimeric protein that competes with normal Elizabeth Tegg 2016

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

Elizabeth Tegg 2016

FISH probes

• Locus Specific Identifiers

– Break Apart

– Dual Colour/Dual Signal, TriColour/Dual

Fusion

– Dual Colour/Single Fusion

Elizabeth Tegg 2016

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 3ABL1 con BCR x 2

Elizabeth Tegg 2016

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

Elizabeth Tegg 2016

Break-apart probe: MLL DFBAred=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]Elizabeth Tegg 2016

Elizabeth Tegg 2016

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

Elizabeth Tegg 2016

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 Elizabeth Tegg 2016

Elizabeth Tegg 2016

Elizabeth Tegg 2016

Copy neutral-Loss Of

Heterozygousity• SNP arrays

– 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. 2010 March 19, 2010.Elizabeth Tegg 2016

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

Elizabeth Tegg 2016

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)

Elizabeth Tegg 2016

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

Elizabeth Tegg 2016

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

Elizabeth Tegg 2016

AML with inv(16)(p13q22) or

t(16;16)(p13;q22)

Vysis® LSI® CBFB Dual Color, Break Apart Rearrangement Probe

5’CBFB

3’CBFB

CBFB

The inv(16) interrupts the coding region of the CBFB and MYH11 genes and

leads to a chimeric protein that competes with normal Elizabeth Tegg 2016

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

Elizabeth Tegg 2016

Acute promyelocytic leukaemia AML with

t(15;17)(q22;q12)

Elizabeth Tegg 2016

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

Elizabeth Tegg 2016

Gene names

Elizabeth Tegg 2016

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: 183-190Elizabeth Tegg 2016

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

Elizabeth Tegg 2016

Elizabeth Tegg 2016

AML with inv(3)(q21q26.2)

• Maybe de novo or

arise from MDS

• Morphology:

increased atypical

megakaryocytes

Elizabeth Tegg 2016

AML with t(1;22)(p13;q13)

• Acute

megakaroblastic

leukaemia

• Rare

• Most commonly

occurs in Down

syndrome

Elizabeth Tegg 2016

AML with gene mutations

• Fms-related tyrosine kinase 3 (FLT3)

• Nucleophosmin (NPM1)

• CEBPA

• KIT

• WT1

• NRAS

• KRAS

Elizabeth Tegg 2016

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

Elizabeth Tegg 2016

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. 2010 January 21, 2010;115(3):453-74. Elizabeth Tegg 2016

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 prognosisElizabeth Tegg 2016

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?)Elizabeth Tegg 2016

KIT

• Located 4q11-12

• Member of type 3 tyrosine kinase family

• Generally test for KIT mutation in the core

binding factor AML

– Poor prognosis

Elizabeth Tegg 2016

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. 2006;94(2):179-83.Elizabeth Tegg 2016

Outline

• Classification of AML

• Types of genetic changes

• Next generation sequencing in HM

Elizabeth Tegg 2016

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. 2009 September 10, 2009;361(11):1058-66Elizabeth Tegg 2016

International Cancer Genome

Consortium

• http://www.icgc.org/

• 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.

Elizabeth Tegg 2016

Elizabeth Tegg 2016

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

– 300-400 coverage needed

• We know from cytogenetics that different clones are present

in tumours

– Passenger vs driver mutations

Elizabeth Tegg 2016

Perspectives for therapeutic targeting of gene mutations in acute myeloid

leukaemia with normal cytogenetics

British Journal of Haematology

Volume 170, Issue 3, pages 305–322, 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.

Elizabeth Tegg 2016

Elizabeth Tegg 2016

Elizabeth Tegg 2016