AML for dummies

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Disclosure of speaker’s interests M.J. Wondergem

Diagnostic methods

• morphology – May-Grunwald Giemsa stain

• cytochemistry – myeloperoxydase or Sudan black stain – non-specific esterase stain

• histochemistry • immunofenotyping • cytogenetics

– metaphase analysis, FISH • molecular biology

– PCR

ELN guideline, Dohner, Blood 2017

AML , 1 disease??

ELN guideline, Dohner, Blood 2017

ELN risk stratification

AML survival and risk factors

Diagnostic methods

• morphology – May-Grunwald Giemsa stain

• cytochemistry – myeloperoxydase or Sudan black stain – non-specific esterase stain

• histochemistry • immunofenotyping • cytogenetics

– metaphase analysis, FISH • molecular biology

– PCR

May-Grunwald Giemsa

Why still morphology for diagnosis AML?

Is it old-fashioned?

• Judging sample quality

• Suspicion of or diagnosis of AML/MDS • Classification of AML

– AML with myelodysplasia related changes – AML, NOS

• (classification of MDS)

Sometimes quality is dismal..

You wonder what has happened

squashed…..

exploded……

Watery…

• Judging sample quality

• Suspicion of or diagnosis of AML/MDS • Classification of AML -Recognition acute leukemia that needs intervention NOW -AML with myelodysplasia related changes -AML, NOS • (classification of MDS)

t(15;17)

Blast percentage

• AML and ALL : > 20% (some exceptions)

• MDS RAEB: 5 -20% WHO 2016: MDS-EB 1 or 2 – RAEB I: 5 -10% – RAEB II: 10 -20%

• other MDS < 5 %

– Refractory cytopenias MDS-SLD/MLD – MDS with 5q- – MDS unclassifiable MDS-U – pediatric- MDS

blast morphology

Mufti G J et al. Haematologica 2008;93:1712-1717

©2008 by Ferrata Storti Foundation

Lymfoblast or myeloblast??

myelodysplasia

• For AML: >50% in 2 cell lines or history of MDS or MDS related cytogenetic changes

dysplasiea erytropoiesis

beenmerg

-megaloblastoid

-abnormal nuclei

-nuclear fragmentation

-vacuolisation

dysplasia granulopoiesis

-hypogranulation

-Pseudo Pelger

-bizar forms (hypersegm)

dysplasia megakaryopoiesis

-micro-megakaryocytes

-monolobular

-separate nuclei

Acute Myeloïd Leukemia: definition

• > 20% blasts in bone marrow exception: • AML with recurrent cytogenetic abnormalities t(15;17), t(8;21), inv(16) of

t(16;16) • AML/MDS therapy related

• myeloperoxidase or Sudan black > 3% positive exception:

• Auer Rod: no MPO/SBB needed

• SBB and MPO negatief but immuno-phenotyping points to: – AML with minimal differentiation – Acute undifferentiated leukemia (AUL) – monocytic/monoblastic leukemia – pure erythroïd leukemia – acute megakaryoblastic leukemia – acute basophilic leukemia – blastair plasmacytoïd dendritische cel neoplasma

Diagnostic methods

• morphology – May-Grunwald Giemsa stain

• cytochemistry – myeloperoxydase or Sudan black stain – non-specific esterase stain

• histochemistry • immunofenotyping • cytogenetics

– metaphase analysis, FISH • molecular biology

– PCR

Cytochemistry

myeloperoxydase reactie of Sudan black reaction is positive in myeloid cells (strong) and monocytes (weak)

non-specific esterase reaction is positive in monocytic cells

(diffuse in cytoplasm)

Diagnostic methods

• morphology – May-Grunwald Giemsa stain

• cytochemistry – myeloperoxydase or Sudan black stain – non-specific esterase stain

• histochemistry • immunofenotyping • cytogenetics

– metaphase analysis, FISH • molecular biology

– PCR

immunofenotyping

example

0 1000Forward Scatter

0 1000Forward Scatter

100 101 102 103 104CD45 PerCP

100 101 102 103 104CD45 PerCP

R2

100 101 102 103 104CD34 APC

100 101 102 103 104CD34 APC

100 101 102 103 104CD15 FITC

100 101 102 103 104CD15 FITC

100 101 102 103 104CD61 FITC

100 101 102 103 104CD61 FITC

100 101 102 103 104CD65 FITC

100 101 102 103 104CD65 FITC

100 101 102 103 104c-TdT FITC

100 101 102 103 104c-TdT FITC

Role immunofenotyping for diagnosis AML

• Classification: MPO-negative AML • Prognosis: - • MRD: LAP (leukaemia associated phenotype) • Therapy: anti-CD33, ..

Diagnostic methods

• morphology – May-Grunwald Giemsa stain

• cytochemistry – myeloperoxydase or Sudan black stain – non-specific esterase stain

• histochemistry • immunofenotyping • cytogenetics

– metaphase analysis, FISH • molecular biology

– PCR

Histochemistry

• Bone marrow biopsy -at diagnosis: when diagnosis AML is not clear yet, for alternative diagnosis: ALL, lymhoma, other cancers -dry tap

CD34

CD117

MPO

CD34

CD117, MPO and CD68

Lysozym and CD45

pittfalls

• Dry tap: bone marrow biopsy for determination of remission status

• CD34 negative blasts: maturation

Diagnostic methods

• morphology – May-Grunwald Giemsa stain

• cytochemistry – myeloperoxydase or Sudan black stain – non-specific esterase stain

• histochemistry • immunofenotyping • cytogenetics

– metaphase analysis, FISH • molecular biology

– PCR

Diagnostic methods

• morphology – May-Grunwald Giemsa stain

• cytochemistry – myeloperoxydase or Sudan black stain – non-specific esterase stain

• histochemistry • immunofenotyping • cytogenetics

– metaphase analysis, FISH • molecular biology

– PCR

Diagnostic methods

• morphology – May-Grunwald Giemsa stain

• cytochemistry – myeloperoxydase or Sudan black stain – non-specific esterase stain

• histochemistry • immunofenotyping • cytogenetics

– metaphase analysis, FISH • molecular biology

– PCR

Role cytogenetics/molecular diagnostics for diagnosis AML

• Classification: AML with recurrent cytogenetic abnormalities

• Prognosis: risk classification • MRD: bv NPM-1 • Therapie: t(15;17), FLT3ITD, IDH1or 2

Classification acute leukemia

WHO 2008 WHO 2008 WHO 2016

WHO 2008: Acute Myeloïd Leukemia

• 1. AML with recurrent cytogenetic abnormalities • 2. AML with myelodysplasia related changes • 3. Myeloïd neoplasm, therapy related • 4. AML not otherwise specified (NOS) • 5. Myeloïd sarcoma • 6. Myeloïd proliferations related to Downs syndrome • 7. Blastic plasmacytoïd dendritic cell neoplasm

WHO 2008: Acute Myeloïd Leukemia

• 1. AML with recurrent cytogenetic abnormalities • 2. AML with myelodysplasia related changes • 3. Myeloïd neoplasm, therapy related • 4. AML not otherwise specified (NOS) • 5. Myeloïd sarcoma • 6. Myeloïd proliferations related to Downs syndrome • 7. Blastic plasmacytoïd dendritic cell neoplasm

• Diagnosis mainly IF/IHC: CD4, CD123, often CD56, CD68 in 50%.

Skin abnormalities

BAL => MPAL (mixed phenotype acute leukaemia)

• Myeloïd: – MPO (cytochem or ab)

– Monocytic marker (> 2 van: NSE, CD11c, CD14, CD64, lysozym)

• B lymphoïd: – CD19++, with 1 of: CD79a, cCD22, CD10 – CD19 weak with strong expression of 2 of: CD79a,

cCD22, CD10 • T lymphoïd:

– cyt and/or sCD3

59

AUL

Acute ongedifferentiated leukemia • Negative for myeloïd, B en T cell markers • Often positive for blast marker CD34, tdt

Exclusion other tumors with extended panel: • Cave plasmacytoid dendritic cell tumor, megakaryo,

basophilic, NK-cell of solid tumor

45

Remission detection

• Morphology: <5% blasts • Immuno-phenotyping: MRD • Immuno-histochemistry:

– problem: % blasts (CD34/CD117): maturation arrest – Limited amount of cells, blasts can also be caused by

regeneration