Introduction:
Hematopoietic
Neoplasms
(Acute Leukemia)
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Introduction: Hematologic Neoplasm
• Dysregulated Proliferation
• Benign
• Differentiated cells, organized growth
• Does not spread
• Malignant
• Anaplastic, proliferating cells (clones of abnormal cells)
• Often metastasizes (characteristically)
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WHO Classification of Hematologic Neoplasms
• Select Diseases
• Myeloproliferative neoplasms (MPNs)
• Acute myeloid leukemia (AML) and related
neoplasms
• Precursor Lymphoid Neoplasms (B & T
lymphoblastic leukemia/lymphoma)
• Mature B cell neoplasms (including chronic
lymphocytic leukemia, CLL)
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Molecular Basis of Cancer
• Oncogenes
• Proto-oncogene
Oncogene
• Proto-oncogenes
• Growth factors
• Growth factor receptors
• Proteins involved in signal transduction
• Signal transducers
• Transcription factors
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Molecular Basis of Cancer
• Tumor suppressor genes
• Code for proteins that inhibit cell growth in normal cells
(p53 & RB)
• Mutations lead to loss of function
• loss of protein’s normal, growth inhibitory effect
• Example: Mutation of p53
• Loss of function of p53 allows damaged cells to
proceed through cell cycle
• Unregulated proliferation of the damaged cell
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Molecular Basis of Cancer
• Epigenetics
• Heritable changes in gene expression not caused by changes
in DNA sequence
• Epigenetic changes
• DNA methylation of CpG dinucleotides
• Transcriptional silencing of a gene as a result of
hypermethylation of CpG dinucleotides in the gene’s
promoter region
• Histone deacetylation
• Hypo-acetylated histones bind tightly to phosphate
backbone of DNA and chromatin remains in its
heterochromatin state – genes are silenced
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Leukemogenesis
• Genetic susceptibility -> Down’s Syndrome
• Somatic mutation
• Ionizing radiation
• Drugs
• Chemicals
• Viral infection
• Immunologic dysfunction
• Other factors
• Secondary to treatment for primary disease
• Other hematologic disease (e.g., PNH)
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WHO Classification of Hematologic Neoplasms
• Morphology
• Cytochemical stains
• Immunophenotyping
• Detection of genetic abnormalities
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Cytochemical Reactions
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Myeloperoxidase (MPO)
Benzidene dihydrochloride + H2O2
MPO
Oxidized benzidene + H2O
• Positive in myeloblasts
• Negative in lymphoblasts
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Immature myeloid cells demonstrating MPO positivity
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Auer Rods
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Sudan Black B (SBB)
• Diazo dye – fat soluble dye
• Soluble in cellular lipids
• Phospholipids, neutral fats, and sterols
• Parallels MPO
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Immature myeloid cells demonstrating SBB positivity
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Non-specific Esterase (NSE)
naphthyl acetate
NSE
naphthol + acetate
Diazonium salt
colored product
• Found in monocytes and macrophages
• Can inactivate the enzyme in monocytes by adding sodium
fluoride to incubation mixture
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Immature monocytic cells demonstrating NSE positivity
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Periodic Acid Schiff (PAS)
Stains glycogen and
polysaccharides
Positive in mature
neutrophils, monocytes,
platelets, megakaryocytes
Positive in
erythroleukemia
(malignant erythroblasts)
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Malignant erythroblasts demonstrating PAS positivity
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Acute Myeloid Leukemias
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FAB Classification
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Acute Myeloid Leukemia
• General Findings:
• More common in adults
• Clinical presentation: pallor, weak, tired, bone pain
• Laboratory presentation (general):
• thrombocytopenia, variable WBC count, anemia,
• Bone marrow – hypercellular
• >20% blasts
• Auer rods
• MPO & SBB positive blasts
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Figure 2. A high power view cofirms these cells as blasts
Maslak, P. ASH Image Bank 2004;2004:101032
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Copyright ©2004 American Society of Hematology. Copyright restrictions may apply.
Figure 3. Other marrow elements are sparse
Maslak, P. ASH Image Bank 2003;2003:100838
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Copyright ©2003 American Society of Hematology. Copyright restrictions may apply.
Figure 1. An Auer rod (arrow)is appreciated in this blast from a bone marrow aspirate of a
patient with AML
Maslak, P. ASH Image Bank 2002;2002:100454
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Copyright ©2002 American Society of Hematology. Copyright restrictions may apply.
A differential report notes the presence of more than
20% blasts. This number supports the diagnosis of:
A. CML
B. AML
C. CLL
D. MDS
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An Auer rod is best described as:
A. Threads of chromosomal DNA
found within the cytoplasm
B. Inappropriately fused primary
granules
C. Excessive accumulations of
ribosomal RNA
D. Golgi apparatus that have not
been enzymatically degraded
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WHO Classification of Acute Myeloid
Leukemias
• AML with recurrent genetic abnormalities
• AML with myelodysplasia-related changes
• Therapy-related myeloid neoplasms
• AML not otherwise categorized
• Myeloid sarcoma
• Myeloid proliferations related to Down syndrome
https://ashpublications.org/blood/article/127/20/2391/35255/The2016-revision-to-the-World-Health-Organization
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Acute Myeloid Leukemia
• AML not otherwise categorized
• Acute myeloid leukemia without maturation
• One of most common in adults
• Bone marrow
• >90% of non-erythroid cells are blasts
• Maturation beyond blast stage is <10%
• MPO and SBB positivity in >3% blasts
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Figure 3. Multiple small nucleoli are apparent in these blasts
Maslak, P. ASH Image Bank 2004;2004:101032
PB: AML without maturation
Copyright ©2004 American Society of Hematology. Copyright restrictions may apply.
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Figure 1. Bone marrow aspirate shows increased blasts
Maslak, P. ASH Image Bank 2002;2002:100473
BM: AML without maturation
Copyright ©2002 American Society of Hematology. Copyright restrictions may apply.
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Acute Myeloid Leukemia
• AML with recurrent genetic abnormalities
• Acute myeloid leukemia with t(8;21)(q22;q22)
• Peripheral blood: observe pseudo Pelger-Huet cells
• Bone marrow: 30-90% blasts
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Acute Myeloid Leukemia
• AML with recurrent genetic abnormalities
• Acute myeloid leukemia with t(8;21)(q22;q22)
• Peripheral blood
Hyposegmentation
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Hypogranulation
Figure 1. Blasts are increased but other myeloid elements are present as well (MacNeal
Tetrachrome 400x)
Maslak, P. ASH Image Bank 2002;2002:100480
BM: AML with t(8:21)
Copyright ©2002 American Society of Hematology. Copyright restrictions may apply.
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Figure 7. Diagrammatic representation of balanced translocation which defines this distinct
biologic AML subtype
Maslak, P. ASH Image Bank 2001;2001:100215
Cytogenetic analysis: t(8;21)
Copyright ©2001 American Society of Hematology. Copyright restrictions may apply.
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Acute Myeloid Leukemia
• AML with recurrent genetic abnormalities
• Acute myeloid leukemia with t(8;21)(q22;q22)
• Genes involved
• Chr 8: RUNX1T1 (also called ETO)
• Chr 21: RUNX1 (formerly AML1)
• Hybrid gene: ETO/RUNX1
• Fusion protein = ETO/RUNX1
• acts as transcriptional repressor because fusion protein can’t
bind to DNA and promote transcription of genes/proteins
that promote cellular differentiation
• Net effect: cell differentiation is blocked and proliferation of
leukemic cells is increased
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Acute Myeloid Leukemia/ APL
• AML with recurrent genetic abnormalities
• Acute myeloid leukemia with PML-RAR
• Predominantly malignant promyelocytes present
• Possess multiple auer rods (faggot cells)
• Common complication: disseminated intravascular
coagulation (DIC)
• Two types:
• Hypergranular: folded or bilobed nucleus
• Microgranular: WBC markedly increased; multilobed
nucleus
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Figure 1. Peripheral smear from a 15 year old male shows blast-like cells with agranular
cytoplasm and convoluted nuclei
Lazarchick, J. ASH Image Bank 2004;2004:101007
Microgranular Variant
Copyright ©2004 American Society of Hematology. Copyright restrictions may apply.
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Figure 4. The "bi-lobed" nuclear morphology typical of hypogranular promyelocytes is seen
in this view
Lazarchick, J. ASH Image Bank 2004;2004:101007
PB: Microgranular Variant
Copyright ©2004 American Society of Hematology. Copyright restrictions may apply.
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Figure 5. A low power view (H&E 4x) of the marrow shows essentially 100% cellularity with an
apparent monomorphic infiltrate
Lazarchick, J. ASH Image Bank 2004;2004:101007
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Copyright ©2004 American Society of Hematology. Copyright restrictions may apply.
Figure 7. Another view of the same biopsy illustrating the nuclear abnormalities previously
described
Lazarchick, J. ASH Image Bank 2004;2004:101007
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Copyright ©2004 American Society of Hematology. Copyright restrictions may apply.
Figure 1. This cell found in the bone marrow of a patient with APL contains multiple Auer
rods in the cytoplasm
Maslak, P. ASH Image Bank 2002;2002:100587
Faggot cell
Copyright ©2002 American Society of Hematology. Copyright restrictions may apply.
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Figure 3. Promyelocytes are heavily granulated
Maslak, P. ASH Image Bank 2004;2004:101126
Hypergranular variant
Copyright ©2004 American Society of Hematology. Copyright restrictions may apply.
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Acute Myeloid Leukemia
• AML with recurrent genetic abnormalities
• Acute myeloid leukemia with PML-RAR
• First identified mutation: t(15;17)(q22;q21)
• Genes involved
• Chr 15: PML = promyelocytic leukemia gene
• Chr 17: RAR = retinoic receptor  gene
• Hybrid gene: PML/RAR
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A laboratory professional reviewed the CBC of a patient who
had a WBC count of 5 × 103/μL with 32% blasts present. Follow
up testing with cytogenetics revealed t(15;17)(q22;q12). Which
of the following is a likely diagnosis for the patient?
A. APL
B. MDS
C. MPN
D. CLL
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The leukemia that belongs to the WHO classification of
AML with recurrent genetic abnormalities is:
A. APL with PML-RARA
B. Megakaryoblastic leukemia
C. Pure erythroid leukemia
D. AML minimally differentiated
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Figure 8. Diagram of the t(15;17)(q22;q21) rearrangement that characterizes classic APL
Maslak, P. ASH Image Bank 2004;2004:101126
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Copyright ©2004 American Society of Hematology. Copyright restrictions may apply.
Acute Myeloid Leukemia
• AML with recurrent genetic abnormalities
• Acute myeloid leukemia with PML-RAR
• Hybrid gene: PML/RAR
• Fusion protein = PML/RAR
• Binds to RA-responsive elements of specific target genes
& forms complex with RXR protein
• Complex recruits corepressor proteins and deacetylates
histones; thus prevents transcription of specific target
genes and blocks granulocytic differentiation
• Unlike normal RAR protein, physiologic levels of RA
can’t induce conformational change in fusion protein and
release corepressor molecules and activate gene
transcription
• Net effect: granulocytic differentiation is blocked
• Specific therapeutic intervention
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Predict the typical platelet concentration of a patient
with AML.
A. Marked decreased
B. Marked increased
C. Normal
D. Cannot be determined
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A PAS stain is ordered on a leukemic patient. The test is
performed, and the technologist confirms that the PAS stain
is positive with coarse granularity. Based on these findings,
from what is the patient most likely suffering?
A. Acute myelomonocytic
leukemia
B. Acute erythroleukemia
C. Acute megakaryoblastic
leukemia
D. AML with minimal
differentiation
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Acute Lymphoblastic
Leukemias
PRECURSOR LYMPHOID
NEOPLASMS
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Acute Lymphoblastic Leukemia
• General Findings
• Found primarily in young children (2-5 yr)
• Clinical presentation: easy bruising, fatigue, weight
loss, fever, bone pain
• Laboratory Presentation
• Peripheral blood: >50% of cases have increased
WBC, thrombocytopenia, anemia
• Bone marrow: >20% lymphoblasts
• lymphoblasts: MPO & SBB negative
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Figure 1. Cluster of blasts from a patient with ALL have a regular appearance and are devoid
of granules in the cytoplasm (MacNeal Tetrachrome 1000x)
Maslak, P. ASH Image Bank 2002;2002:100384
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Copyright ©2002 American Society of Hematology. Copyright restrictions may apply.
Figure 3. Bone marrow aspirate reveals increased blasts and immature lymphocytes (400X
MacNeal tetrachrome)
Maslak, P. ASH Image Bank 2002;2002:100400
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Copyright ©2002 American Society of Hematology. Copyright restrictions may apply.
WHO Classification of Lymphoid Leukemias/Lymphomas
• Precursor Lymphoid Neoplasms
• B Lymphoblastic leukemia/lymphoma
• B Lymphoblastic leukemia/lymphoma with recurrent genetic
abnormalities
• B Lymphoblastic leukemia/lymphoma not otherwise categorized
• T Lymphoblastic leukemia/lymphoma
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Precursor Lymphoid Neoplasms
• B Lymphoblastic Leukemia with recurrent genetic
abnormalities
• B lymphoblastic leukemia with t(12;21)
• Bone Marrow
• hypercellular
• greater than 20% blasts
• Cytochemical stains
• MPO: blasts are negative
• SBB: blasts are negative
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B Lymphoblastic Leukemia with
recurrent genetic abnormalities
• B lymphoblastic leukemia with t(12;21)
• Bone Marrow
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B Lymphoblastic Leukemia with
recurrent genetic abnormalities
• B lymphoblastic leukemia with t(12;21)
• What would immunophenotyping by flow cytometry reveal?
CD 34
CD 19
+
Tdt
HLA-DR
CD 10
+
Cytoplasmic CD
3
CD 33
CD 20
=
CD 2
Cytoplasmic μ
+
CD 5
CD 13
+
+
CD 14
CD 22
CD 7
CD 61
CD 24
CD 4
CD 25
CD 8
+
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Which of the following laboratory results are most
commonly found in ALL?
A. Eosinophilia & basophilia
B. Neutropenia &
thrombocytopenia
C. Neutrophilia &
thrombocytopenia
D. Lymphocytosis &
thrombocytosis
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