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Professor Hélène Antoine-Poirel, MD, PhD

Center for Human Genetics

INTERUNIVERSITY CERTIFICATE IN HUMAN GENETICS

Université catholique de LouvainBrussels,19/02/2016

de DUVEINSTITUTE

GENETICS OFHEMATOLOGICAL MALIGNANCIES

Genetics of hematological malignancies

I - Introduction

II - Diagnostic

III- Prognosis

IV – Minimal residual disease

V - Targeted treatment

VI – Genetic predisposition

I - INTRODUCTION

Normal hematopoiesis

I - INTRODUCTION

Hematopoiesis : transcription factors

I - INTRODUCTION

Hematopoiesis : growth factors

I - INTRODUCTION

Neoplasms of hematopoietic precursors

Clonal proliferation of bone marrow hematopoietic p recursors

With a block of differentiation

= acute

Without a block of differentiation

= chronic

QuantitativeAnomaly

QualitativeAnomalyMyeloid Lymphoid

MyeloproliferativeNeoplasm

(MPN)

MyelodysplasticNeoplasm

(MDS)

AcuteMyeloblastic

Leukemia(AML)

AcuteLymphoblastic

Leukemia(ALL)

One or more myeloid lineage

Blast cells

Acute transformation

www.koreahealthlog.com

I - INTRODUCTION

Neoplasms of mature lymphoid cells

Clonal proliferation developed from a mature lympho id cell

Classification according to the cell of origin :- B-cell or T-cell lineage- differentiation and / or activation status (B & T- cell immune response)

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• All cells derive from the same ancestral cell where occurred the acquired initiating mutation

• Thrombocytosis, polycythemia, myelofibrosis : Myeloproliferative neoplasm or reactive proliferation ?

– JAK2 V617F mutation = clonal myeloproliferative neoplasmpolycythemia vera / Vaquez (95%)

essential thrombocytosis (50-60%)myelofibrosis (50-60%)

– CalR (ex9) = clonal myeloproliferative neoplasmessential thrombocytosis (25%)

myelofibrosis (35%)

II - DIAGNOSIS

Clonality = marker of autonomous proliferation

CalR

CalR

II - DIAGNOSIS

Myeloproliferative neoplasms

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•(AML minimally differentiated M0)

•AML without maturation M1

•AML with maturation M2

•Promyelocytic acute leukemia M3

•Myelomonocytic leukemia M4

•AML-M4 with abn eosinophils M4Eo

•Monoblastic/monocytic AL M5a/b

•Erythroid AL M6

•Megacaryoblastic AL M7

FAB Classification (1976)

Morphology (+ immunophenotyping 1991)

II - DIAGNOSIS

Classification of acute myeloid leukemias (AML)

• Uses all available information to define entities : – Clinics

– Morphology

– Immunophenotype

– Genetics

• No real « gold standard » criteria

• Aims : – To define « real disease » entities

– Reproducibility

– Easy to use in the daily practice

II - DIAGNOSIS

2008 WHO Classification : multidisciplinarity

AML with recurrent genetic abnormalities

AML with balanced translocations / inversionst(8;21)(q22;q22) RUNX1-RUNX1T1inv/t(16;16)(p13;q22) CBFB-MYH11 abnormal eosinophils (M4Eo)t(15;17)(q22;q12) PML-RARa promyelocytic (M3)t(9;11)(p22;q23) MLLT3-MLLt(6;9)(p23;q34) DEK-NUP214 basophiliainv/t(3;3)(q21;q26.2) RPN1-EVI1t(1;22)(p13;q13) RBM15-MKL1 megacaryoblastic (M7)

AML with gene mutationsNormal karyotype FLT3-ITD, FLT3-TKD

MLL-PTDNPM1 mutations (ex12)CEBPA mutations

t(8;21), inv/t(16;16) KIT mutations

II - DIAGNOSIS

Classification of acute myeloid leukemias (AML)

2008 WHO Classification

Genetics + Morphology + Immunophenotyping

AML with myelodysplasia-related changesfollowing a myelodysplastic syndrome (MDS) or myeloproliferative disorderwithout prior MDS

AML & myelodysplastic syndromes, therapy related Alkylants agentsIonizing radiation therapyTopoisomerase II inhibitorsOthers

AML not otherwise specified FABAML with minimally differentiation M0AML without maturation M1AML with maturation M2Acute myelomonocytic leukemia M4Acute monoblastic & monocytic leukemia M5a/bAcute erythroid leukemia M6Acute megacaryoblastic leukemia M7Acute basophilic leukemiaAcute panmyelosis with myelofibrosis

Myeloid sarcoma

II - DIAGNOSIS

Classification of acute myeloid leukemias (AML)

Chromosomal translocations targeting the Core Bing Factor : RUNX1/21q22 & CBFββββ////16q22

AML-M2 :t(8;21)(q22;q22)RUNX1-RUNX1T1

AML-M4Eo :t/inv(16)(p13q22)CBFββββ-MYH11

GM-CSF, IL-3, M-CSF-R, TCRαααα, TCRß p21/WAF1, MPO, NE, granzyme B, NP-3….

RUNX1

RUNX1

RUNX1

RUNX1T1

MYH11

II - DIAGNOSIS

Classification of acute leukemias

Dominant negative effect of the fusion protein ���� inihibition of the target genes involved in myeloid differentiation

RARαααα

R

R

R R R R

A

A

AA

AA

AA

PML-RARαααα

Acute promyelocytic leukemia : Chromosomal translocation targeting the Retinoic Acid Receptor- αααα

II - DIAGNOSIS

Classification of acute myeloid leukemias

t(15;17)(q22;q21)

Chromosomal translocations targeting MLL/11q23

II - DIAGNOSIS

Classification of acute myeloid leukemias

Roboz GJ ASH Education Book December 10, 2011 vol. 2011 no. 1 43-50

Molecular heterogeneity of AML with normal karyotyp e

II - DIAGNOSIS

Classification of acute myeloid leukemias

Murati A BMC Cancer 2012

II - DIAGNOSIS

NGS : 5 classes of cooperating mutations

Classification of acute myeloid leukemias

ALL of B-cell precursors

(Child / Ad%)t(9;22)(q34;q11.2) 2-4% 25% BCR-ABLt(v;11q23) 2-3% 2-3% MLL Rgtt(12;21)(p13;q22) ~25% 0% ETV6-RUNX1Hyperdiploidy >50 ~25% <5%Hypodiploidy 1%t(5;14)(q31;q32) IL3-IGHt(1;19)(q23;p13.3) 6% TCF3-PBX1

ALL of T-cell precursors1p32 25% SIL-TAL1del(9)(p21) 30% p16t(10;v)(q24;v) 7-31% TLX1t(5;14)(q35;q32) 13-20% TLX3t(10;11)(p13;q14) 9-30% CALM-AF10t(11;v)(q23;v) 8% MLL-v9q34 6% NUP214-ABLinv(7)(p15q35) 5% TCRββββ-HOXA

FAB (1976) ���� GEIL/EGIL (1994/95) ���� WHO (2001/08)

Morphology + Immunophenotype + Genetics

ALL-L1

ALL-L2

ALL-L3

Pro-B-ALL

Common ALL

Pre-B-ALL

Mature B-ALL

Pro-T-ALL

Pre-T-ALL

Cortical T-ALL

Medullary T-ALL

II - DIAGNOSIS

Classification of acute lymphoid leukemias (ALL)

II - DIAGNOSIS

Classification : B -cell lymphomas

Ash A. Alizadeh, Nature 2000

NFKB activation

REL amplificationBCL2 translocation

(c)

II - DIAGNOSIS

Classification: Diffuse Large B -Cell Lymphoma• Clinical heterogeneity of DLBCL

• Non reproducibility of morphology and immunohistochemistry

• Gene expression profiling : 2 main molecular subtypes of DLBCL :– derived from the lymph node germinal center (GCB)

– derived from an activated B-cell (ABC)

Impact of cytogenetics on survival

(Moorman AV Blood 2007)

III - Prognosis

Childhood ALL

Hyperdiploidy

Cytogenetics : 1366 (90%) of 1522 patients

Successful : 1003 cases (73%)

Abnormal clone : 796 cases (79%)

(Moorman AV Blood 2007)

Impact of cytogenetics on survival

III - Prognosis

Adult ALL

Hyperdiploidy

Grimwade D et al. Blood 2010

III - Prognosis

Adult AMLImpact of cytogenetic entities on survival

III - Prognosis

Adult AMLPrognostic subgroups according to genetic results

(ex : Hovon)

IV– Minimal Residual Disease

Minimal Residual Disease

By

- Real-time PCR- Multi-color Flow cytometry- NGS (high coverage)

- Acute lymphoblastic leukemia- Chronic myelocytic leukemia- …

“faggot cells” = bundles of Auer rods

V– Targeted treatment

Promyelocytic acute leukemia (AML -M3/APL)

t(15;17)(q22;q21)

V– Targeted treatment

Promyelocytic acute leukemia (AML -M3/APL)

All Trans Retinoic Acid – ATRA (Vesanoid ®)– (∆ vitamin A)

t(15;17)(q22;q21) = fusion between

PML (15q22) and

RARA, retinoic acid receptor alpha (17q21)

LMA M3

Reduced sensitivity of the nuclear receptor to the ligand, retinoic acid

V– T<argeted treatment

Promyelocytic acute leukemia (AML -M3/APL)

1µM ATRA : differentiation induction with growth in hibition and apoptosis

V– Targeted treatment

Promyelocytic acute leukemia (AML -M3/APL)

RARαααα

R

R

R R R R

R R

R R

R R

R R

RR

RRRR

RR

A

A

A A

AA

AA

AA

AA

AA

AAAA

AA

PML-RARαααα

NPM-RARααααNuMA-RARαααα

ATRAsensitivity 45 mg/m2/jr

PLZF-RARαααα

STAT5b-RARαααα

Poor ATRAsensitivity

45 mg/m2/jr

Chronic myeloid leukemia (CML)

fusion between

BCR (22q11) and ABL1 (9q34)

Constitutively activated chimeric tyrosine kinase

CML

Tyrosine kinase inhibitor (STI571 / Imatinib / Glivec®)

mouseleukemia

t(9;22)(q34;q11)

V– Targeted treatment

Chronic Myelocytic leukemia (CML)

Inhibitors of tyrosine kinase activitycompetitive antagonists of the ATP-binding site of bcr-abl

Druker, B. J. Blood 2008;112:4808-4817

Chronic myeloid leukemia (CML)V– Targeted treatment

Chronic Myelocytic leukemia (CML)

006C1721.004

LSI BCR/ABL (Vysis)

006C1721.001

LSI BCR/ABL (Vysis)

Development of new tyrosine kinase inhibitors (Dasatinib, Nilotinib, …)

Glivec resistance

BCR-ABL amplificationABL tyrosine kinase domain mutations

and overexpression of drug-efflux proteins (ABCB1 a nd ABCG2)

V– Targeted treatment

Chronic Myelocytic leukemia (CML)

ABL KD MutationsSensitivity of BCR/ABL Kinase Domain Mutations to TKIs (values are given a s IC50

Redaelli, S. et al. J Clin Oncol; 27:469-471 2009

V– Targeted treatment

Chronic Myelocytic leukemia (CML)

• < 1% underestimated, esp. in adults↑ with NGS

• Mainly myeloid disorders : – Bone marrow failure

– Myelodysplasia / AML

• Genes involved in :– DNA repair : Fanconi complementation group

– Telomere maintenance : TERC, TERT

– Ribosome : RPS19, SDBS

– Hematopoïesis : CEPBA, RUNX1, GATA2, PAX5, ETV6

• Other disorders : low penetrance susceptibility

VI – GENETIC PREDISPOSITION

Hematological malignancies

• Crucial to identify for MDS/AML :– Selection of an unaffected familial donor in case of hematopoïetic

stem cell transplantation– Fanconi Anemia, Telomeropathies are associated with an

increased sensitivity to chemotherapy/radiotherapy� dose adaptation to limit toxicity

• Genetic test :– on a non invaded sample (hair follicle, skin, buccal swap) – before allo-graft

VI – GENETIC PREDISPOSITION

Hematological malignancies