Acute Promyelocytic

Leukemia
MLSCI 430
Rabia Yousofi
Tino Villatoro
Our Case Study
 A 29-year-old male was admitted to ER
looking pale with a rash on his extremities.
 He has had a persistent nose bleed for the
last 2 hours.
 Lab Values:
◦ Hemoglobin 87 g/L
◦ Platelets 15 x 109/L
◦ WBC 38 x 109/L
◦ PT (INR) 5.0
◦ PTT 62 seconds
◦ Fibrinogen 0.3 g/L
Further Lab Testing
 A review of the peripheral blood smear
revealed many abnormal cells.
 A bone marrow aspiration and biopsy was
later performed.
 Samples were sent for flow cytometry,
cytogenetics, and the molecular oncology
lab.
 A diagnosis of Acute Promyelocytic
Leukemia (APL) was made on the basis of
the tests performed.
What is Acute Promyelocytic
Leukemia?
 APL is characterized by the accumulation of
blasts that are blocked at the promyelocytic
stage of differentiation.

http://www.pnas.org/content/102/20/7174/F6.large.jpg
WHO vs. FAB
 Acute promyelocytic leukemia falls under
the old FAB classification as AML FAB M3
and M3v (for the microgranular variant).
 Under the new WHO classification, APL
falls under the category AML with recurrent
genetic abnormalities.
 This new classification recognizes the
molecular/genetic feature of APL, namely
the balanced translocation of chromosome
15 and 17.
PML-RARA: t(15;17)
 The t(15;17) is characteristic and virtually
diagnostic of APL.
 This translocation results in the fusion of the
PML gene on chromosome 15q22 and the
RARA (Retinoic Acid Receptor A) on
chromosome 17q21.
 Expression of the PML-RARA protein
results in a block in differentiation at the
promyelocyte stage by suppressing RARA
target genes.
Variant t(15;17)
 There is a common breakpoint within intron
2 of the RARA gene and three breakpoints
within the PML gene which results in the
formation of three variants.
 These breakpoints are:
◦ Intron 6 (bcr1; 55% of cases)
◦ Exon 6 (bcr2; 5% of cases)
◦ Intron 3 (bcr3; 40% of cases)
 Bcr3 is associated with M3v microgranular
form. It has a higher incidence of DIC and a
higher leukocyte count.
Variant APL Translocations
 Other variant translocations may occur
involving chromosome 17 that lead to APL:
◦ t(11;17)(q23;q21)
◦ t(5;17)(q35;q21)
◦ t(11;17)(q13;q21)
◦ der(17) (17q21.3-q23)

http://www.pathguy.com/lectures/m3.jpg
Variant Translocations
 t(11;17) (q23;q21)
◦ Most common and intensively studied variant
◦ Fuses the PLZF gene (promyelocytic leukemia
zinc finger) with RARA resulting in the expression
of a PLZF-RARA protein.
◦ Falls into an unusual morphologic spectrum of
APL, with features intermediate between M2
(AML with some maturation) and M3 (APL).
◦ Important to recognize, as this translocation is
insensitive to ATRA
Variant Translocations
 t(5;17) (q35;q21)
◦ Second-most common variant
◦ This variant translocates the nucleophosmin
gene on 5q35 into the RARa locus on 17q21
◦ Nucleophosmin is a nucleolar phosphoprotein
that plays a role in ribosomal RNA assembly; it
also has chaperoning activities, as well as
nuclease activity.
◦ The phenotype is identical to APL M3
◦ In-vitro studies have shown that promyelocytes
of t(5;17) are still sensitive to ATRA, and this has
been shown in one case study as well.
Variant Translocations
 t(11;17)(q13;q21)
◦ This is a rare APL variant
◦ Blood smear and bone marrow specimens show
a predominance of promyelocytes and dysplastic
maturing neutrophils.
◦ This variant is still sensitive to ATRA
 der(17) (17q21.3-q23)
◦ Morphologically similar to AML M1 (AML with
minimal differentiation) with a minority of marrow
blasts showing morphologic evidence for the M3v
microgranular variant of APL.
◦ Shows no response to ATRA
Cytogenetic Diagnosis
 Cytogenetic studies can reveal the
abnormal karyotype in APL.
 Several banding techniques are available,
including giemsa-trypsin banding, r-
banding, and c-banding.
 Metaphase chromosomes are treated with
trypsin and stained with Giemsa. This
creates a unique banding pattern for each
chromosome.
http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=cmed&part=A1548&rendertype=figure&id=A1551

http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=cmed&part=A1548&rendertype=figure&id=A1557
Fluorescent In-Situ Hybridization

http://en.wikipedia.org/wiki/File:FISH_%28Fluorescent_In_Situ_Hybridization%29.jpg
Minimal Residual Disease
 Quantitative reverse transcriptase PCR can
be used to detect the PML-RARA transcript.
 Patients with a higher transcript level tend
to have a worse prognosis.
 Treatment and remission can also be
monitored using real-time quantitative RT-
PCR.
Typical Features of Blast Cells
 Myeloblast

 Monoblast

 Proerythroblast

 Megakaryoblast

 Lymphoblast
Myeloblast
 < 1% of the normal bone marrow, not observed
in normal blood

 Vary in size, but are usually large

 Nucleus is delicate, large, round or Sl oval, with

prominent nucleoli. Stain purplish red with
Wright stain. Chromatin stains evenly

 Small to moderate amount of bluish

nongranular cytoplasm

 Three major types: Type I, II, and III

Type I
 Fine nuclear chromatin
 2 to 4 distinct nucleoli
 Moderate rim of pale to basophilic
cytoplasm
 Without azurophilic granules

Konoplev S et al. Advances in the pathologic diagnosis and biology of acute myeloid leukemia (figure 8).
Type II and III
 Type II
◦ Nuclear and cytoplasmic features are similar to
type I
◦ Delicate azurophilic granules in the cytoplasm
(up to 20)

 Type III Konoplev S et al. Advances in the pathologic diagnosis and biology of acute myeloid leukemia (figure 9).

◦ Numerous azurophilic granules in the cytoplasm

http://www.pathologyoutlines.com/leukemia.html
Auer Rods
 One characteristic feature of myeloblasts in
AML
◦ Presence of Auer rods with abnormal azurophilic
granules
◦ (60% - 70% of all cases)
◦ (faggot cells)

Konoplev S et al. Advances in the pathologic diagnosis and biology of acute myeloid leukemia (figure 10).
Monoblast
 Derived from myelocytic-monocytic
progenitor cells in BM
 Round, sometimes folded, large early
nucleus with 1 or 2 nucleoli
 Finely dispersed linear chromatin
 Small indentation (nuclear creases)
 Basophilic cytoplasm with no granules
(sometimes fine granules & occasional
vacuoles)
Erythroblast
 0% - 1% in normal bone marrow of adults

 Round nucleus, Sl condensed nuclear

chromatin

 Variable prominent nucleoli

 Moderate amount of deeply basophilic

cytoplasm.
Megakaryoblast
 Moderately large cell with 1 or 2 nuclei (round
nucleus)
 Nucleus begins to indent with lobes start to
form and nucleus increases in size
 Nucleoli often demonstrable
 Non-granular cytoplasm, may have blunt
pseudopods or blebs, stain bluish
 In AML, very significantly in appearance from
one case to another
 Promyelocyte
http://www.healthsystem.virginia.edu/internet/hematology/hessidb/normal-hematopoietic-cells.cfm

 Primary granules (azurophilic or dark blue), no

secondary granules

 1% - 5% in normal bone marrow, not seen in normal

blood

 Its size varies (may exceed 20µm)

 Nucleus is round and large in relation to the cytoplasm

 Chromatin is sl courser than myeloblast

 Nucleoli may be visible (often indistinct)

 Cytoplasm is dark blue with a relatively light area

adjacent to the nucleus
 Neoplastic Promyelocyte (Blast
Equivalent)
 Eccentric, often folded and lobulated
nucleus

 Sl condensed nuclear chromatin

 Intense cytoplasmic granulaity

 An apparent Golgi zone

Konoplev S et al. Advances in the pathologic diagnosis and biology of acute myeloid leukemia (figure 10).
Cytochemical Stains
 Since the early 20th century, cytochemical
staining of cells has been a useful tool in
differentiating hematopoietic diseases.

 Smears and imprints made from bone marrow,

lymph nodes, spleen, or peripheral blood are
preferred.
◦ In enzymatic techniques, fresh smears are used
to ensure optimal enzyme activity

 Certain elements may be inhibited during the

fixation of smears and imprints
Myeloperoxidase (MPX/MPO)
 The proxidase enzyme reacts with H2O2 & release
O2, which oxidizes the indicator dye and produce
orange-brown granules in the cells (3-amino-9-
erythrocarbazol)

 Enzyme MPX is found in the 1o granules of

granulocytes, neutrophils and precursors (from the
promyelocyte stage on) & eosinophils
 Monocytes may be weakly pos
 Leukemic myeloblasts are usually pos and Auer
rods stain very strongly
 Used for differentiating AML (+) from ALL (-)
 Normal bone marrow smear <5 days old used for
control slides (promyelocyte - neutrophils)
http://www.aquinaspathology.com/images/sp_MyeloperoxidaseStainAcut.jpg

http://www.dfhcc.harvard.edu/core-facilities/specialized-histopathology-services-pathology/services/
Non-Specific Esterase (NSE)
 Nonspecific esterase liberates alpha-naphthyl
from the substrate alpha-naphthyl acetate.
Alpha-naphthyl is couples with the dye
molecule to form dark reddish-brown granules

 Monocytes, monblasts, macrophages,

histiocytes, megakaryocytes and some
carcinomas are NSE pos
 Abnormal erythroblasts are strongly pos
 Lymphocytes are neg or may show dot
positivity
 NSE continued
 Used for differentiating myelomonocytic and
monocytic leukemia (+) from granulocytic
leukemia (-)
 Monocyte NSE are fluoride sensitive

 Peripheral smear with appreciable # of

monocytes or a normal BM smear used for
control slides

http://www.healthsystem.virginia.edu/internet/hematology/hessedd/malignanthematologicdisorders/leukemias/aml-m4.cfm
Periodic Acid Schiff (PAS)
 Periodic acid oxidizes glycogen, mucoproteins,
and other high-molecular weight carbohydrates
to aldehydes.
 Aldehydes react with colorless Schiff reagent,
staining them a bright red-pink
 Megakaryocytes and platelets stain strongly
pos
 Normoblasts will stain Pos
 Lymphoblasts in ALL show course and granular
(block) positivity
 PAS Continued
 Myeloblasts are Neg
 Aids in diagnosis of ALL, erythroleukemia, and
megakaryoblastic leukemia
 Normal bone marrow smear used for control
slides

http://www.pathologyoutlines.com/leukemia.html
What about those coag tests?
 The coagulation tests performed were very
abnormal and are suggestive of
disseminated intravascular coagulation
(DIC).
 80% of APL cases present with
hemorrhagic manifestations

http://www.hoslink.com/haematology/purp.jpg
 Pathophysiology of DIC
 A widespread systemic activation of
coagulation resulting in diffuse fibrin
deposition in the microvasculature.
 This can lead to multi-organ dysfunction;
red blood cell shearing; consumption of
coagulation factors and platelets; and
bleeding.

http://www.pathologystudent.com/wp-content/uploads/2009/07/DIC_With_Microangiopathic_Hemolytic_Anemia_301920983.jpg
The APL connection
 When promyelocytes release the contents
of their primary granules, their pro-
coagulant activity initiates DIC.

 Aside from increased expression of

procoagulant activity, there is also an
activation of primary fibrinolysis and
inflammation.
DIC and the lab
 Consumption of coagulation factors leads to
prolonged PT and PTT, and a decrease in
fibrinogen.
◦ Factors consumed: fibrinogen, factor V, factor
VIII and factor XIII.
 Fibrinolysis of cross-linked fibrin leads to
the formation of fibrin degradation products,
notably D-Dimer.
Fibrinogen
 The thrombin clotting time of dilute plasma
is inversely proportional to the
concentration of fibrinogen.
 The method for measuring fibrinogen
involves testing dilutions of patient and
control plasma with excess thrombin.
 Results are calculated using a standard
curve.

http://labsystems.roche.com/content/products/sta_r/benefits.html
Diagnosing DIC
 Aside from the prolongation of the PT and
PTT, the decreased fibrinogen, and CBC
results, there are a number of tests used to
diagnose DIC including:
◦ Antithrombin Levels
◦ D-Dimer
◦ Prothrombin Fragment F1.2
Antithrombin Assay
 Antithrombin III will complex with thrombin
and activated factor X, resulting in
diminished plasma levels in DIC.
 Micro-latex particles are coated with
antibodies against antithrombin. Light is
passed through the cuvette of a wavelength
much greater than the diameter of the
particles.
 An increase in absorbance at 570 nm is
directly proportional to the concentration of
antithrombin.
Antithrombin Levels
 Antithrombin levels provide an indirect
measurement of thrombin activation.
 Antithrombin levels may be decreased due
to a hereditary deficiency or one of many
acquired deficiencies including:
◦ DIC
◦ nephrotic syndrome
◦ liver disease
◦ oral contraceptive use,
◦ post-surgery
◦ prolonged heparin therapy.
http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=coffeebrk&part=A22
D-Dimer Assay
 Plasmin will lyse stabilized fibrin clots and
form D-dimers as well as other fibrin
degradation products.
 Micro-latex particles are coated with
monoclonal antibodies against human D-
dimer. Light is passed through the cuvette
of a wavelength much greater than the
diameter of the particles.
 An increase in absorbance at 570 nm is
directly proportional to the concentration of
D-dimer.
http://www.vet.uga.edu/VPP/clerk/ayoob/fig02_adj.jpg
D-Dimer Levels
 D-dimer levels are elevated in situations
where active thrombosis is occurring,
notably:
◦ DIC
◦ Deep Vein Thrombosis
◦ Pulmonary Embolism
 D-dimer levels will not be increased in
primary fibrinolysis (only cross-linked fibrin
will lead to D-dimer formation)
 The assay has a great negative predictive
value, but a positive result must correlate
with other lab values in the diagnosis of DIC
Prothrombin Fragment F1.2
 The conversion of prothrombin to thrombin
involves the cleavage of a small molecule
off the parent molecule.
 The measurement of this fragment reflects
thrombin activation and active thrombosis.
 One of the methods of measurement
employs a monoclonal antibody against the
fragment, in an ELISA sandwich assay.
Prothombin F1.2 Levels
 Increased levels reflect thrombin activation
and active thrombosis, which may occur in
DIC or other hypercoagulable states.
 The use of prothrombin fragement F1.2 is
not often used in the diagnosis of DIC.
 Insufficient experience with the
measurement of prothrombin F1.2 may limit
it’s usefulness relative to other diagnostic
tests for DIC.
Treatment: All-Trans Retinoic
Acid (ATRA)
 Considered as a first line therapeutic drug in
the treatment for APLs
 Acts by promoting terminal differentiation of the
abnormal promyelocytes to mature neutrophils
 Highly effective in induction of complete
remission
 Safe, convenient and cheap
 Prevent the fatal bleeding caused by DIC,
reduction of early mortality (major advantage)
 ATRA is an isomer of retinoic acid (RA)
Retinoic Acid
 Retinoic acids (RA) are signalling molecules
that play important roles during embryonic
development, also influence physiological
functions like organogenesis, organ
homeostasis and growth, differentiation or
death of adult cells.

 RA isomers are a group of active metabolites of

vitamin A and their physiological effects are
mediated through two families of RA receptors:
RARs and retinoid X receptors (RXRs).
Retinoic Acid Continued
 There are 3 members in each receptor family,
encoded by different genes, namely RARα, β,
and γ, and RXRα, β, and γ
 Promotes myeloid differentiation

http://www.cisreg.ca/cgi-bin/tfe/articles.pl?tfid=337
Leukemogenesis of APL
 The PML–RARα chimeric protein acts as a
dominant negative mutant over wild-type RARα by
forming a homodimer and prevents activation of
key RA target genes
 The leukemia-specific fusion proteins display a
higher avidity for corepressors of RARα.
 As a result, the RARα/RXR pathway necessary to
the granulocytic differentiation is abolished.

http://www.bioscience.org/2009/v14/af/3333/figures.htm
Leukemogenesis of APL
• PML-RARα forms homodimer through the
coiled-coil motif of PML and competes with
RARα for binding to RARE of target genes
Mechanism of ATRA
 Pharmacologic dosage of ATRA directly
modulates PML-RARα and its interaction
with the nuclear receptor co-repressor
complex
◦ Restores the wild-type RARα/RXR regulatory
pathway and induces the transcriptional
expression of downstream genes

 ATRA can induce degradation of the PML–

RARα oncoprotein, leading to activation of
repressed target genes
Mechanism of ATRA

http://rstb.royalsocietypublishing.org/content/362/1482/959.full
Modulation of the interaction of the receptor
with CoR or CoA
 At physiological concentration (0.01 mM), ATRA can
dissociate CoR from wild-type RARα/RXRα and recruit
CoA for transcriptional activation
 PML-RARα is less sensitive to ligand-induced
modulation, 0.1 ~ 1 mM (pharmacological
concentrations) of ATRA is needed

http://www.bioscience.org/2009/v14/af/3333/figures.htm
Limitations of ATRA
 Patients with another translocation involving RARα
that results in expression of the PLZF-RAR α
protein, are insensitive to ATRA and arsenic
trioxide (CT)
 ATRA will ↑ the WBC count and cause leukocytosis
rapidly which may lead to lethal consequences
 Causes retinoic acid syndrome
 Long term use may induce ATRA-resistance
 1/3 to 1/2 of patients still relapsed probably due to
a selection of clones resistant to ATRA (Relapse is
the major subject of concern at present)
 Combining ATRA with
Chemotherapy
 ATRA exerted its effect by inducing terminal
myeloid differentiation, but could not prevent
the occurrence of malignant transformation in
myeloid progenitor cells

 The combination of ATRA with a

chemotherapeutic agent yields a higher CR
rate and a longer overall survival

 Arsenic Trioxide (ATO) combined with ATRA

further improved the 5-year overall survival
Combining ATRA with
Chemotherapy

http://rstb.royalsocietypublishing.org/content/362/1482/959/F2.large.jpg
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