Lecture 10 (cont.)/11/12 (final)

cytochemical stains:
MPO, SBB, SE, NSE

myeloperoxidase

Sudan black B

specific esterase
Naphthol AS-D chloroacetate esterase
(ONLY granulocytes are positive)

non-specific esterase
(α-naphthyl acetate and α-naphthyl butyrate)

blasts of AML from those of ALL

in primary granules of most granulocytic cells

leukemic myeloblasts

>80% blasts show MPO activity

Auer rods

lymphoblasts in ALL and lymphoid cells

blasts of AML from those of ALL
(like MPO but more sensitive)

lipids

granulocytes from myeloblast to mature neutrophil
(reaction gets stronger as cells mature)

monocytes

lymphoid cells

acute leukemias that are granulocytic from leukemias that are primarily of monocytic origin

reaction is positive in granulocytic cells and negative to weak monocytic cells

in primary granules of neutrophils

leukemic myeloblasts
Auer rods

reaction is strongly positive (diffuse) in monocytes that can be inhibited by adding sodium fluoride (NaF)
but negative in lymphoid cells and granulocytes

reaction is strongly positive (diffuse) in monocytes that can be inhibited by adding sodium fluoride (NaF)
but negative in lymphoid cells and granulocytes
(less sensitive than α-naphthyl acetate but more specific)

all substrates positive bc myeloid and monocytic cells are present

at least 20% of cells must show monocytic differentiation that is NSE positive and is inhibited by NaF

80% or more of the blasts are NSE positive and SE negative

MPO -
SBB -
NASDA -
ANBE & ANAE -/+ (focal)

MPO
SBB
NASDA

ANBE
ANAE

MPO
SBB
NASDA
ANBE & ANAE (diffuse)
(ALL STAINS)

SBB (-/ +)
ANBE & ANAE (diffuse)

MPO
SBB (-/ +)
NASDA

ANAE (localized)

MPO
SBB
NASDA
ANBE

in T cell ALL &
Hairy Cell Leukemia

tartrate-resistant acid phosphatase

confirmation diagnosis of HCL ;
if hairy cells present, stain remains positive after addition of tartaric acid to the incubation mixture

stains glycogen in all hematopoietic cells
but typical reaction (block of positivity) in lymphocytes/ lymphoblasts

stain is redundant in ALL diagnosis
still useful in AML and MDS to ID abnormal erythroblasts and dysplastic megakaryocytes

acute and chronic

proliferation of stem cells leading to accumulation of BLASTS, predominantly in BM which will lead to BM failure

acute lymphoblastic leukemia (ALL)
acute myeloid leukemia (AML)

malignant clone is able to differentiate resulting in accumulation of more MATURE cells

chronic lymphoblastic leukemia (CLL)
chronic myeloid leukemia (CML)

25% of childhood cancer
75% of childhood leukemias
peak age 2-5 yrs, but better response to therapy than adults
IN adults, poor prognosis and outcome

B-cell ALL
T-cell ALL

Fatigue, fever, mucocutaneous bleeding
Lymphadenopathy, hepatosplenomegaly
Eventual infiltration into meninges, testes, or ovaries
Lymphoblasts found in CSF.

anemia

neutropenia and infection

thrombocytopenia

meninges, testes/ ovaries
lymphoblasts can also be found in cerebrospinal fluid

(similar to B-ALL)
anemia, thrombocytopenia, organomegaly, bone pain!!!
but degree of leukopenia is less severe!!!

large mass in mediastinum leading to compromise of regional anatomic structures

9

BCR-ABL1

t(9;22)(q34.1;q11.2)

BCR-ABL1 t(9;22)

Philadelphia-chromosome positive
More common in adults
Responds to imatinib (Gleevec) <- drug used in CML

t(v;11)q23.3

common in very young infants!
translocation may occur in utero!
very poor prognosis!

t(12;21)

rare in adults
EXCELLENT prognosis in children (90% cure rate)

hypErdiploidy: less common in adults, favorable prognosis in children (> 46 chromosomes)
hypOdiploidy: poor prognosis in both adults and children (< 46 chromosomes)

L1, L2, AND L3

small blasts with thiiiin rim of cytoplasm

irregular clefts of nucleus

Huge, basophilic, mature cells
VACUOLES!! ~ STARRY SKYYYY
associated with Burkitt's lymphoma

mature B-cell ALL (L3)
& vacuolations

CD34, CD19, cytoplasmic CD22, TdT

CD34, CD19, CD10, cytoplasmic CD22, TdT

CD19, cytoplasmic CD22, cytoplasmic µ, TdT (variable)

CD2, CD3, CD4, CD5, CD7, CD8, TdT

teenaged males with mediastinal mass, elevated peripheral blast counts, meningeal involvement, and infiltration of extra marrow sites

terminal deoxynucleotidyl transferase

thrombocytopenia + anemia
leukocytosis or leukopenia (seen in chemotherapy)
circulating blasts in PBS

ALL

Because there is bone expansion due to increase of B-cells.

entire marrow replaced by blasts

clonal malignant disease of hematopoietic tissues that is characterized by
1. accumulation of abnormal blast (mostly in marrow)
2. impaired production of normal RBC

sequence of somatic mutations in multipotential HSC or more differentiated progenitor cell

ADULTS

based on morphology M0-M7

based on molecular genetics and cytogenetics

pallor, fever, easy bruising, splenomegaly (50%),
risk of DIC, skin and gum infiltration, lymphadenopathy (rare)

blasts in PB
WBC between 5-30 x10^3/µL
anemia, thrombocytopenia, neutropenia
(WBC could be normal, but anemia and thrombocytopenia are always present!!!)
BM hypercellular with >20% blasts (requires specific genetic abnormalities)

hyperuricemia, hyperphosphatemia, hypocalcemia, risk of acute renal failure, tumor lysis syndrome (when WBC are high)

acute myeloid leukemia, minimally differentiated
undifferentiated 5%

AML without maturation
no maturation 20%

AML with maturation
maturation 30%

acute promyelocytic leukemia
7%

acute myelomonocytic leukemia
M4eo: with eosinophilia

acute monocytic leukemia
5a: poorly diff
5b: well diff

acute erythroleukemia

acute megakaryocytic leukemia

RUNX1-RUNX1T1

5%

predominantly in children and young adults

Myeloblasts with dysplastic granular cytoplasm
Auer rods
Some maturation (similar to AML - M2 ~ WITH maturation)
Pseudo-Pelger Huet Cells and hypogranulation
Esosinophila may be seen

Pseudo-Pegler Huet cells and hypogranulation (less granules)
eosinophilia <- may be seen

genetic abnormality, regardless of blast count

5-8%

inv(16)(p13.1q22) or
t(16;16)(p13.1;q22)

1. Myeloblasts, monoblasts, and promyelocytes in PB and BM
2. Eosinophilia with dysplastic changes in BM
3. CNS involvement (replase)

genetic aberration regardless of blast count

5-8%

fused genes

PML-RARA

5-10%

differentiation block at PRO stage

hypErgranular promyelocytes, some with Auer rods (sometimes faggot cells)

DIC (release of procoagulant from promyelocytes)

microgranular variant (30-40% of APL cases), may be confused with other AML types

detection of (15;17) translocation sufficient for diagnosis, regardless of blast count

all-trans-retinoic acid (ATRA) and arsenic trioxide
(ATRA induces diff of promyelocytes)

t(9;11)

1. Increase in monoblasts and immature monocytes
2. Blasts are large with abundant cytoplasm and fine nuclear chromatin
3. Blasts may have pseudopodia, granules, and vacuoles
(M5)

Children
skin/ gum involvement may occur

CD13+
CD33+
CD34+
CD117+

MPO and SBB negative (-)

infants or older patients

<5%

CD13+
CD33+
CD34+
CD117+

MPO and SBB positive (+) in 3% blasts

Blasts 90% of non-erythroid cells in BM
<10% of cells show maturation up to promyelocyte

CD13+
CD34+
CD15+
CD11b+
HLA-DR+

Blasts >20% in BM
>10% maturing neutrophils
<10% blasts of monocytic lineage

negative for monocytic markers
CD36, CD14, CD64

differentiation block at pro stage

t(15;17)

DIC!!!!!!

CD13+, CD33+, CD64+
CD34-, CD11b-, HLA-DR-

very high WBC count!
presence of monocytic cells (monoblast & promonocytes) and neutrophilic cells

CD13+
CD33+
CD64+
CD14
CD4+
CD11c+

>80% cells of monocytic origin

CD14+
CD4+
CD64+
CD11c+
CD11b+

>80% cells of erythroid origin
>30% are pronormoblast
Dysplastic changes in erythroid precursors (multinucleation, N/ C asynchrony, vacuolization)
>50% of nRBCs on PBS
ringed sideroblasts, HJ bodies

aggressive and rapid aka bad prognosis

>50% blasts of megakaryocytic origin

CD41, CD42b, CD61 and cytoplasmic vWF by immunostaining

clonal hematopoietic disorders caused by gene mutations in HSCs:
increase in proliferation of and accumulation of mature erythrocytes, granulocytes, and platelets
(all neoplasms one line predominates)

chronic myeloid leukemia (CML)
Polycythemia vera (PV)
Essential thrombocythemia (ET)
Primary myelofibrosis (PMF)

JAK2 mutation (more frequent in PV)

Ph t(9;22) chromosome

erythrocytes**, reticulocytes, lymphocytes*, platelets, megakaryocytes*

*normal/ increased
**normal/ decreased

Erythrocytes**, LAP, erythropoiesis
**normal/ decreased

10:1

hypErleukocytosis (WBC > 300x10^9), risk of leukostasis

a life-threatening medical emergency that occurs when malignant blast cells obstruct small blood vessels, leading to organ damage.

LAP score is decreased in CML & basophils are present in CML & gout is present in urine

altered chromosome 9 and alt chr 22 (Philadelphia)
BCR/ABL fusion gene (ABL comes from 9)

hyperuricemia, uricosuria, risk of secondary gout, and uric acid stones

cytogenetic analysis for detection of t(9;22)!!!
detection of BCR-ABL1 transcript using qRT-PCR
detection of BCR-ABL1 fusion gene using fluorescence in situ hybridization (FISH)

decreased!

1. PBS incubated with substrate of ALP (naphthol-phosphatte substrate and diazo dye at an alkaline pH)
2. colored ppt formed on granules of neuts/ bands
3. 100 neuts and bands counted and scored 0-4+ depending on color intensity
4. score multiplied by number of cells and then added

15-170

poor response to therapy :(
more chromosomal changes, more anemia, more baso, more abnormal platelets (micromegakaryocytes, and megakaryocytic fragments)
decrease in number of mature neuts
Increase of circulating blast count from 10% to 19%
(Transformation into either acute lymphoid or acute myeloid leukemia)

transformation into either acute lymphoid or acute myeloid leukemia

panmyelosis (increase in all cell lines)

JAK2 mutation

related to signal transducers and activators of transcription (STAT) which are activated after binding of cytokines (EPO) leading to cell proliferation and differentiation

constitutive activation of JAK/STAT signaling pathway (independent of EPO)

3 major:
1. elevated Hb and HCT
2. BM hypercellularity with trilineage growth (panmyelosis)
3. ID of JAK2 mutation

minor: low serum EPO levels

erythrocyte morphology (normocytic), LAP score*

* normal or increased

splenomegaly
hepatomegaly

pseudo-Gaucher cells (rare)

80%

100% of BM volume may exhibit hematopoietic cellularity (no fat cells)

splenomegaly, hepatomegaly,
generalized vascular engorgement, circulatory disturbances, risk of hemorrhage, tissue infarction, thrombosis

LR: 2-4+
CML: 0-1+

LR: 2-4+
CML: 0-1+

LR: frequent
CML: rare

LR: 0
CML: 1-3+

LR: 0
CML: 1-3+

LR: High
CML: Low or 0

LR: Negative
CML: Positive >90%

PV: ALWAYS increased
SH: can be increased

PV: Decreased
SH: Increased

PV: normal
SH: decreased

PV: myelopoiesis
SH: only erythroid hyperplasia

PV has leukocytosis (>12) and increase plts (>400)
splenomegaly (>75%)

thrombosis, hemorrhage

Hb (>16.5) in M; Hb (>16) in F
BM panmyelosis
JAK2 mutation
other causes of erythrocytosis

primary (EPO decrease or Normal)
secondary (EPO increase)

polycythemia vera

hypoxia driven
hypoxia-independent

high altitudes
cardiopulmonary disease
defective O2 transport

renal cysts or tumors
extrarenal tumors

1. marked and persistent thrombocytosis (big increase)
2. WBCs elevated ( can see neutrophilia with slight left shift)
3. RBCs Normal or slightly decreased (rare nRBC)

600-2,000 x10^9/ L (>450)
large masses of platelet aggregates (large clusters)
abnormal plt morph (giant platelets)
megakaryocyte frags
Generally 'normal' looking but can vary in size, shape, granulation

neutrophilia with slight left shift

vascular occlusion or bleeding
headaches, dizziness, throbbing/ burning hands/ feet,
mucosal bleeding, seizures, myocardial infarction

function abnormal
aggregation abnormal
when plt reduced, normal plt function returns

hemorrhage (bleeding tendencies due to abnormal plt)
thrombosis (plts >2000 109/ L)

JAK2 64%
MPL 4%
CALR 15%

Hypercellularity with marked megakaryocytic hyperplasia

abnormal clone of megakaryocytes that produces cytokines which stimulate fibroblasts to produce collagen = fibrosis

fibroblastic growth factors released (PDGF-β) aka platelet drive growth factor

BM tissue gradually replaced by fibrous scar-like material (fibrosis),
dry tap BM during biopsy;
leads to progressive BM failure (overtime)
results in extramedullary hematopoiesis

JAK2 V617F (like PV and ET) &
found in 60% of PMF patients

immature granulocytes and nRBCs, teardrops, giant platelets, and other RBC shapes
increased platelets and micromegakaryocyte

result of narrowing and rigidity of splenic sinusoids

JAK2 60%
MPL 30%
CALR 5%

increase

normal or decrease

normal or increase

Ph t(9;22)
BCR-ABL1

right shift or increase

increase

right shift or increase

JAK2 V617F or other JAK2 mutation

right shift or
slightly decrease

right shift of slight decrease

increase

+-JAK2 or other mutation

right shift
increase or decrease

right shift or decrease

right shift
increase or decrease

JAK2 or other mutation

early WBC with myelocytes, metas, and few pros & blasts
increased eos and baso
N/N RBC
may have increased plt

extremely high RBC- very dense with no monolayer
increased WBC with some earlier forms but not pros or blasts
normal plt morph

extremely high plt with abnormal morph
may have slight left shift with increased WBC
N/ N RBC

tear drop poik in RBC
nRBCs
slight left shift with increased WBC
abnormal plt morph, MMK