AML for dummies€¦ · AML for dummies • (Potential ... • molecular biology – PCR ....
Transcript of AML for dummies€¦ · AML for dummies • (Potential ... • molecular biology – PCR ....
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AML for dummies
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• (Potential) conflict of interest • Potentially relevant company
relationships in connection with event
Sponsorship or research
funding Fee or other (financial)
payment Shareholder Other relationship, i.e. …
• None • Company names
Disclosure of speaker’s interests M.J. Wondergem
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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
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ELN guideline, Dohner, Blood 2017
AML , 1 disease??
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ELN guideline, Dohner, Blood 2017
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ELN risk stratification
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AML survival and risk factors
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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
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May-Grunwald Giemsa
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Why still morphology for diagnosis AML?
Is it old-fashioned?
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• Judging sample quality
• Suspicion of or diagnosis of AML/MDS • Classification of AML
– AML with myelodysplasia related changes – AML, NOS
• (classification of MDS)
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Sometimes quality is dismal..
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squashed…..
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exploded……
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Watery…
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• 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)
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t(15;17)
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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
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blast morphology
Mufti G J et al. Haematologica 2008;93:1712-1717
©2008 by Ferrata Storti Foundation
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Lymfoblast or myeloblast??
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myelodysplasia
• For AML: >50% in 2 cell lines or history of MDS or MDS related cytogenetic changes
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dysplasiea erytropoiesis
beenmerg
-megaloblastoid
-abnormal nuclei
-nuclear fragmentation
-vacuolisation
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dysplasia granulopoiesis
-hypogranulation
-Pseudo Pelger
-bizar forms (hypersegm)
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dysplasia megakaryopoiesis
-micro-megakaryocytes
-monolobular
-separate nuclei
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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
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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
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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)
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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
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immunofenotyping
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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
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Role immunofenotyping for diagnosis AML
• Classification: MPO-negative AML • Prognosis: - • MRD: LAP (leukaemia associated phenotype) • Therapy: anti-CD33, ..
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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
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Histochemistry
• Bone marrow biopsy -at diagnosis: when diagnosis AML is not clear yet, for alternative diagnosis: ALL, lymhoma, other cancers -dry tap
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CD34
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CD117
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MPO
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CD34
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CD117, MPO and CD68
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Lysozym and CD45
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pittfalls
• Dry tap: bone marrow biopsy for determination of remission status
• CD34 negative blasts: maturation
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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
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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
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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
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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
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Classification acute leukemia
WHO 2008 WHO 2008 WHO 2016
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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
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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
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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
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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
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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