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