Acute Leukemia Cytogenetics

Topic Presentation
HEMATOLOGY
Sansar Babu Tiwari, MBBS, PGY II
Department of Pathology
TUTH
1st July 2020
1

Case
A 52-year-old male
Features of lethargy
Complete blood count (CBC)
showed pancytopenia
• WBC: 1,620/µl
– Neutrophils - 11%
– Lymphocytes - 61%
– Monocytes - 27%
– Eosinophils - <1%
– Basophils - <1%
– Leukemic cells - 8%
• Hemoglobin-8.8 g/dL
• Platelets-98,000/µL
Fibrinogen and d-dimer is
increased.
2

Case
AML M3 Suspected on clinical grounds. Bone Marrow biopsy
with cytogenetics and flow cytometry ordered.
BM findings:
Bone marrow biopsy revealed about 100% cellularity, and that
80% of the nucleated elements were leukemic cells.
The leukemic cells showed medium to large size, irregular shape,
finely chromatinized nuclei with distinct nucleoli, and moderate
amount of blue cytoplasm with azurophilic granules. The
leukemic cells showed multiple Auer rods and frequently showed
faggot cells
3

Case
5
Patient is started on Isotretinoin therapy.
Did not show any improvement. So anthracycline derivative
(daunorubicin) was added.
Flow cytometry showed CD13, CD33, CD117, HLA-DR, and
cytoplasmic-MPO, Negative CD34
ZBTB16-RARA translocation was detected on the leukemia gene
screening test by reverse transcription-nested polymerase chain
reaction.
Cytogenetic study revealed t(11;17)(q23;q21)

Case
Diagnosis reviewed: t(11;17); ZBTB16-RARA
ATRA is now stopped and patient is doing well on
Standard induction therapy.
7+3 therapy:
• Cytarabine for 7 days
• Daunorubicin on Day1, 2 and 3
• If FLT3 mutation: Add Midostaurin (Tyrosine kinase
inhibitor) to the regimen.
8

Case Summary
The present case emphasizes the importance of
combining morphologic, immuno-phenotypic,
cytogenetic, and molecular studies to
distinguish variant APL cases from classical APL
cases before initiating chemotherapy, regardless
of whether the morphological study reveals
findings consistent with those of classical APL.
9

Cause-Mechanism-Effect
10
Cytogenetics
t(15;17)
Molecular
abnormality
PML-RARA
Immuno-
phenotype
Promyelocyte
(CD13, CD33)

Basis of Immuno-phenotyping
11

Morphological difference between
AML and ALL
12

14
Genetics: Chromosome language

15
Genetics: Chromosome language

20
Genetics: Cytogenetics
VS Paracentric inversion

Acute Myeloid Leukemia
22
Blast count
<20% = AML

Acute Myeloid Leukemia
• The Cancer Genome Atlas Research Network
– 200 cases of AML
– 13 mutations per case
– Overall: at least 23 recurrent mutations
23

AML with Recurrent genetic
abnormalities.
24

• CD34+: Immature cells
– t(8;21): 8 looks like B, so B cell markers seen (CD19, PAX5,
CD79a)
• RUNX: Run away from Killers (NK: CD56), to be safe. When CD56
present, bad prognosis. Auer Rods.
– inv(16): CD2 + (e)
– inv(3 = Mega): CD41, CD61. Aberrant expression of CD7
– BCR-ABL1: Aberrant expression of CD7 and CD19
– CEBPA: Absent Monocytic markers (CD14, CD64)
– RUNX1: Present Monocytic markers
• CD34-: Differentiated cells
– t(15;17): Promyelocyte
– t(9;11): Monocyte
– t(6;9): Monocyte (CD15+)
– t(1;22): Megakaryocyte (1+2=3=Mega)
– NPM1 - Myelomonocytic 26
Immuno-phenotype in AML

27
Chromosomal abnormalities in AML

abnormalities: Balanced translocations
or inversions.
28

AML: t(8;21);RUNX1-RUNX1T1
- 1-5% of all AML
- Most frequent abnormality in children
- D/D: FAB M2
29Dr. Kiril Lyapichev, MD Anderson

- Myeloblast
- Indented nuclei
- Basophilic cytoplasm with a prominent perinuclear hof that may
contain azurophilic granules.
- Some may show large granules (Pseudo Chediak Higashi granules)
- Promyelocyte, myelocyte and metamyelocyte
- Often large and their cytoplasm has a waxy orange appearance and
lacks a granular texture in Romanowsky stain
- Pseudo Pelger Huet anomaly
- Neutrophils: Homogenous pink cytoplasm (Salmon pink)
- Auer rods, with tapering ends and present in blasts and
maturing neutrophils.
30

- Bone marrow eosinophilia is common but not
dysplastic as in inv(16)
- Erythroblastic and megakaryocytic lineage show
normal morphology
- Myeloid sarcoma may be present at diagnosis, in which
case the blast percentage may be low.
- Sometimes the manifestations of concurrent systemic
mastocytosis is obscured by the acute leukemic
infiltration of the bone marrow.
- Good prognosis except in CD56 or CKIT.
- More than 70% cases will show additional cytogenetic
abnormality including loss of sex chromosome or
del(9q)
31

- Blasts:
- Usually have myeloid markers like strong CD13, CD34,
HLA-DR, MPO; and weak CD33.
- Neutrophils:
- CD15, CD65
- Maturation asynchrony:
- CD15+ CD34+
- Lymphoid markers:
- 8 = B: CD19, CD79a, PAX-5
- NK cell:
- CD56
32

34
Myeloblasts with abundant cytoplasm
Indented nuclei and perinuclear hof
Auer rods

AML: inv(16) or t(16;16); CBFB-MYH11
- 5-8% of AML cases
- Previously classified as AMML with abnormal
eosinophils (FAB M4E0)
- Younger patient
- Extramedullary myeloid sarcoma (CNS
predilection)
- WBC count is significantly higher than in
t(8;21)
- No peripheral blood eosinophilia.
36

37
Dr. Kiril Lyapichev, MD Anderson

- Myeloblasts
- Monocytic component (may be NSE negative)
- Abnormal immature eosinophils without
maturation arrest.
- Atypical large purple-violet granules in addition to
eosinophilic granules in their cytoplasm
- Immature eosinophilic granule in late
promyelocyte and myelocyte stages of
development
- Mature eosinophils often show nuclear
hyposegmentation.
38

- Occasional cases with this abnormality lack
eosinophils, show only granulocytic
maturation without a monocytic component,
or show only monocytic differentiation.
- The naphthol AS-D chloroacetate esterase
(CAE) reaction which is normally negative in
eosinophils can be faintly positive in abnormal
eosinophils. (Significant finding)
39

- Immature blasts
- CD34 and CD117 (KIT)
- Granulocyte lineage
- CD13, CD33, CD15, CD65
- Monocyte lineage
- CD4, CD14, CD64
- Aberrant expression of CD2
- Used for monitoring MRD
40

- 40% cases have secondary genetic abnormality:
- Gain of chromosome 22 and 8
- Trisomy 22 is fairly specific for inv(16)  good
outcome
- Gain of chromosome 8 can be seen with patients with
other primary aberrations
- Del(7q)
- Gain of chromosome 21
- KIT mutation has higher risk of relapse and worse
survival (less significant than with t 8;21)
41

42

43

AML: t(15;17); PML-RARA
- AML with similar morphology resembling APL
(FAB M3) may be seen.
- Patients are relatively young and present with
hemorrhagic manifestations.
- Clinically:
- Anemia and thrombocytopenia (due to platelet
consumption in DIC)
- Clinical or laboratory evidence of DIC and
fibrinolysis, which may worsen with initial
cytolytic response to chemotherapy.
45

- Morphology:
- Promyelocytes with coarse azurophilic granules and
multiple Auer rods.
- Characteristic folded, reniform or bilobed nucleus.
- Hypergranular/typical variant: More common: Leukopenia.
Auer rod is typically larger and ultrastructurally, they have
hexagonal arrangement. If present, few promyelocytes in
PBS.
- Microgranular variant: Granules not visible on light
microscopy. Leukocytosis. Promyelocytes can be seen on
PBS.
- Abnormal promyelocytes with deeply basophilic
cytoplasm in relapse patients treated with
isotretinoin.
46

- Morphology:
- MPO reaction is always strongly positive in all
leukemic promyelocytes. Entire cytoplasm and
nucleus. SBB and CAE also positive.
- NSE is weakly positive in aprrox. 25% cases.
- When in doubt between microgranular APL and
monocytic AML, MPO often helps to break the tie.
- Treatment:
- Tretinoin (Maturation and macrophages contain
Auer rods) and Arsenic trioxide (Increased
osteoblasts surrounding trabeculae)
47

- Low or absent expression of:
- HLA-DR, CD34
- Positive for:
- CD 13 (heterogenous) and CD 33 homogenous)
- Weak positive:
- CD117
- Negative:
- CD15, CD65
- Aberrant expression:
- CD56 (worst outcome)
- CD2 (17-15=2)
51

AML: t(V;17); v-RARA
- Variant RARA translocations:
- ZBTB16 { t(11;17); ZBTB16-RARA)
- Regular nuclei
- Many granules
- Absence of Auer rods
- Pelgeroid neutrophils
- Strong MPO activity
- NUMA1 t(11;17) (Respond to tretinoin)
- NPM1 t(5;17) (Respond to tretinoin)
- STAT5B t(17;17)
52

AML: t(9;11); KTM2A-MLLT3
- Common in children, but can occur at any age
- D/D: M5a
- Clinical features:
- DIC
- Extramedullary myeloid sarcoma and/or tissue
infiltration (gingiva, skin)
- Monoblasts ± promonocytes usually show strong
positive NSE.
- Strong expression of HLA-DR, CD4, CD33, CD65
- Few monocytic differentiation: CD14, CD64
- Few immature markers: CD34 (usually negative)
53

AML: t(9;11); KTM2A-MLLT3
54
Dr. Kiril Lyapichev, MD Anderson

AML: t(6;9); DEK-NUP214
- Occurs in both children and adults
- D/D: FAB M2 or M4
- Basophilia and multilineage dysplasia (b;d)
- Clinical features:
- Pancytopenia
- WBC usually lower than in other AML. (12000/MicroL)
- Morphologically similar to AML with maturation
and AMML.
- Bone marrow or PB basophils ≥2%.
- Erythrocytic and granulocytic dysplasia
- Ring sideroblasts (69) are also seen.
56

AML: t(6;9); DEK-NUP214
- MPO ± NSE
- Blasts: Non-specific myeloid immuno-
phenotype: HLA-DR, CD13, CD33
- Few immature markers:
- CD34, KIT
- tDT in 50%
- Monocyte marker: CD64
- Basophils: CD123, CD33 and CD38 but
negative HLA-DR.
- Poor prognosis
57

AML: inv(3) or t(3;3); GATA2, MECOM
- Common in adults
- D/D: Any FAB except M3
- Normal or elevated platelet count
- Increased dysplastic megakaryocyte with
unilobed or bilobed nuclei and tri(3)lineage
dysplasia in the bone marrow.
- PBS:
- Hypogranular pelgeroid neutrophils ± Blasts
- Bare megakaryocyte nuclei may be present
- BM:
- Hypocellular BM in some cases
- Morphologically similar to M1, M4, M7
59

- HLA-DR, KIT, CD13, CD33, CD34
- High CD34 expression with inv(3) than t(3;3)
- Megakaryocytic markers in few:
- CD41, CD61
- Aberrant expression of CD7 (lymphoid marker)
- Second abnormality:
- Monosomy 7 in half of the cases
- 5q deletions
- RAS mutations (NRAS in 27%, KRAS in 11%, NF1 in
9%)
60

- Patients with BCR-ABL1 positive CML may
acquire inv(3) or t(3;3) and such a findings
indicates accelerated or blast phase of the
disease.
- Patients having t(9;22) and inv(3) or t(3;3) are
considered an aggressive phase of CML rather
than AML.
- Poor prognosis. Even poorer if Monosomy 7 is
present.
61

AML: t(1;22); RBM15-MKL1
- Maturation in the megakaryocytic lineage
(1+2=3=Mega)
- Associated with FAB M7
- Most commonly occurs in infants without
Down syndrome, with a female
predominance.
- This is a de novo AML restricted to infants and
young children aged ≤3 years.
- HSM, anemia, thrombocytopenia and a
moderately elevated WBC.
63

- Morphology:
- Similar to M7
- The megakaryoblasts are usually medium-sized to
large blasts with a round, slightly irregular or indented
nucleus with fine reticular chromatin and 1-3 nucleoli.
- Distinct blebs or pseudopod formation
- Micromegas common, but dysgenesis in erythroid and
granulocytes are usually not seen.
- Dense marrow fibrosis, may mimic that of metastatic
tumor.
- SBB and MPO negative
64

- Megakaryoblasts:
- CD 41 (Gp IIb/IIIa)
- CD 61 (Gp IIIa)
- CD 42b (Gp Ib)
- Myeloid:
- CD 13
- CD 33
- Cytoplasmic staining of CD41 and CD61 is more
specific and sensitive than membrane staining.
- FLT3-internal tandem duplication: Poor prognosis
65

AML: BCR-ABL1
- < 1% of all AML
- < 1% of BCR-ABL1 positive acute and chronic
leukemias
- D/D: FAB M0-M2
- It occurs primarily in adults with a possible male
predominance
- Leukocytosis
- In contrast to myeloid blast transformation in
CML, patients with AML have less frequent
splenomegaly (but if present it is massive) and
lower peripheral blood basophilia (<2%).
67

AML: BCR-ABL1
- Morphology:
- Less cellular marrow (80%) versus 95-100% in blast
transformation of CML.
- Dwarf megakaryocytes are less common than in CML.
- MPO and SBB: Positive
- CD34+
- Myeloid antigens:
- CD13 and CD33
- Aberrant expression of CD19, CD7 and TdT
- Second abnormality
- Loss of chr 7
- Gain of chr 8
- Poor prognosis
68

abnormalities: Gene mutations.
70

AML: Mutated NPM1
- This AML type has myelomonocytic or monocytic
features.
- D/D: FAB M4 or M5
- 2-8% of childhood cases
- 27-35% of adult cases
- Female predominance
- Anemia and thrombocytopenia
- Higher WBC and platelet count
- Extramedullary involvement: Gingiva, lymph
nodes and skin.
71

AML: Mutated NPM1
- Immunostaining with anti-NPM1 antibodies
(nuclear + cytoplasmic) reveals involvement of
two or more bone marrow lineages (myeloid,
monocytic, erythroid, megakaryocytic) in the
vast majority of cases.
- CD33 and CD13 expression
- KIT, CD123, and CD110 positive
- HLA-DR and CD34 negative
- If CD34 +, worst prognosis
72

73
Cup-shaped or
fish-mouth nuclei

AML: Biallelic mutation of CEBPA
- Provisional entity in 2008, definitive entity in
2016
- Children and young adults
- Morphological resemblance to AML with or
without maturation (FAB M1 or M2)
- Higher hemoglobin levels
- Lower platelet counts
- Lower LDH levels
- Multilineage dysplasia in 26% of cases of
denovo AML with mutated CEBPA
74

- Myeloid markers:
- CD15, CD65, CD13, CD33
- Blasts:
- HLA-DR, CD34
- Monocytic markers like CD14 and CD64 are
usually absent.
- Aberrant CD7 expression.
- Favourable prognosis
75

76

AML: Mutated RUNX1
- Provisional entity in WHO 2016
- 4-16 % of cases of AML
- Common in older adults
- Prior radiation exposure or alkylating agents
- Lower hemoglobin and LDH
- Low WBC count
- Morphological resemblance to AML with monocytic or
myelo-monocytic features (FAB M4 or M5)
- HLA-DR, CD34, CD13 positive
- Variable expression of CD33, monocytic markers and
MPO
- Poor prognosis
77

Lymphoblastic leukemia: Recurrent
cytogenetic abnormalities.
79

Distinctive features and Considerations
in ALL
• Morphology is not helpful in differentiating
types of ALL as for AML.
• Immuno-phenotyping and cytogenetics are
important.
• t(5;14); IGH-IL3 has increased eosinophil as a
reactive population.
– Is the only morphological clue among ALL
80

81
Distinctive features and Considerations
in ALL

82
Role of lumbar puncture in ALL
Significance of PBS
during CSF analysis
for blasts.

83
Role of lumbar puncture in ALL

87
Genetic abnormalities in ALL

B-ALL: t(9;22) BCR-ABL1
- More common in adults
- Positive for CD10, CD19 and TdT
- Occasional positive for CD13, CD33
- CD25 is highly associated with B-ALL with BCR-
ABL1.
- Worst prognosis
88

B-ALL: t(9;22) BCR-ABL1
89
Black is a normal ABL
Green is p210
Red is p190

B-ALL: t(v;11) KTM2A-rearranged
- More common in infants < 1 year
- High WBC count
- High frequency of CNS involvement
- t(4;11): CD19+, CD10- proB phenotype and
also CD15+
- Others: t(19;11), t(9;11)
- t(4;11) has a poorer prognosis
90

B-ALL: t(12;21) ETV6-RUNX1
- Not in infants but common in children
- CD19+, CD10+, CD34+, CD20-
- Very good prognosis, with cure seen in >90%
- Even in with poor prognostic factors like high
WBC and age > 10 years, these patients do far
better
91

B-ALL: Hyperdiploidy
- > 50 chromosomes without translocations or
other structural aberrations
- Common in children (not in infants)
- CD19+, CD10+, CD34+
- Chromosome 21, X, 14 and 4 are usually
involved, 1,2 and 3 least common
- Trisomies 4 and 10 have good prognosis
- Overall 90% patients are cured
92

B-ALL: Hypodiploidy
- < 46 chromosomes
- Child and adults
- CD10+, CD19+
- Subtypes:
- Near-haploid ALL (23-29): Childhood: RAS or
receptor tyrosine kinase mutation
- Low hypodiploid (33-39): Loss of function
mutation in TP53 and/or RB1
- High hypodiploid (40-43)
- Poor prognosis: Near haploid the worst one.
94

B-ALL: t(5;14)- IGH/IL3
- Children and adults
- Eosinophilia may be the presenting feature
- This is a reactive population and not part of
the leukemic clone
- CD19+, CD10+ blasts in a presence of
eosinophilia suggest this diagnosis
- Unfavourable prognosis
97

B-ALL: t(1;19) – TCF3-PBX1
- Common in children
- CD19+, CD10+, Cytoplasmic mu heavy chain +
- Strong expression of CD9 and lack CD34
- Intermediate prognosis
99

B-ALL: BCR-ABL1 like
- Common in high risk ALL children, adolescents
and adults
- Down syndrome children have high frequency
of CRLF2 translocations
- High WBC count
- CD19+, CD10+
- Poor prognosis
100

B-ALL: iAMP21
- Amplification of a portion of chromosome 21
- Detected by FISH with a probe for RUNX1
- ≥5 copies of the gene
- Or ≥3 extra copies on a single abnormal
chromosome 21
- Common in older children
- Poor prognosis
101

Acute Leukemia Cytogenetics

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