aml for dummies€¦ · aml for dummies • (potential ... • molecular biology – pcr ....
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AML for dummies
• (Potential) conflict of interest • Potentially relevant company
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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