PART 1

Coverage:
The World Health Organization (WHO) has classified the myeloproliferative neoplasms or
disorders into four (4) predominant hematologic disorders:

POLYCYTHEMIA ESSENTIAL
VERA THROMBOCYTHEMIA

CHRONIC MYELOID PRIMARY

LEUKEMIA MYELOFIBROSIS
Learning Outcomes:
1. Name the hematologic disorders included in the chronic myeloproliferative disorders.
2. Describe the relationship of CML to the other chronic myeloproliferative disorders.
3. List the risk factors for the development of CML.
4. Describe the relationship between the Philadelphia chromosome, the translocation between
chromosomes 9 and 22, and the BCR-ABL fusion gene.
5. Describe the role of the BCR-ABL fusion gene and its protein product in the pathogenesis
of CML.
6. Describe the clinical presentation of CML, including the common signs and symptoms.
7. List the three phases of CML, along with the diagnostic criteria for each.
8. List the common laboratory findings in CML.
9. List the important differential diagnoses of CML.
10. Describe the cytogenetic analysis of CML, including the analytical techniques used, and
the advantages of each.
Learning Outcomes – continuation
11. Describe the origin of chronic myeloproliferative disorders.
12. Identify the most common chronic myeloproliferative disorders.
13. List the characteristics of polycythemia vera (PV).
14. Identify the major and minor Polycythemia Vera Study Group (PVSG) and 2008 World
Health Organization (WHO) diagnostic criteria for PV.
15. List the laboratory findings in PV.
16. Identify the major and minor Polycythemia Vera Study Group (PVSG) and 2008 World
Health Organization (WHO) diagnostic criteria for essential thrombocythemia (ET).
17. List the characteristics of ET.
18. List the laboratory findings in ET.
19. List the features of primary myelofibrosis (PMF).
20. List the laboratory findings in PMF.
Myeloproliferative vs Myelodysplastic Disorders
MYELOPROLIFERATIVE DISORDERS
 Clonal hematopoietic stem cell disorder.
 Characterized by bone marrow
hyperplasia related to excessive
proliferation of one or more hematopoietic
cell lines, with overproduction of the
corresponding cell(s) in the peripheral
blood. Rate of apoptosis is decreased.
‘spleno’ (spleen) + ‘-megaly’ (enlargement of)
 Common findings include splenomegaly
and extramedullary hematopoiesis.
‘extra-’ (outside) + ‘medullary’ (bone marrow medulla)
‘dys-’ (abnormal, difficult) + ‘plasia’ (growth)
MYELODYSPLASTIC DISORDERS
 Clonal hematopoietic stem cell disorder.
 Characterized by peripheral blood
cytopenias, dysplastic blood cells, and a
tendency to transform into acute
leukemia.
‘cyto’ (cell) + ‘-penia’ (decrease in number)
 Increased rate of apoptosis, ineffective
hematopoiesis, and cytopenias are key
biological features. Splenomegaly is rare.
‘apoptosis’ (programmed cell death)
‘ineffective hematopoiesis’ (premature death of developing cells)
Overview of Myeloproliferative Disorders/Neoplasms

Myelo + Proliferative + Neoplasm

new and
bone marrow abnormal growth
of tissue
growing and increasing
in number rapidly

The myeloproliferative neoplasms (MPNs) are clonal hematopoietic disorders caused

by genetic mutations in the hematopoietic stem cells (HSCs) that result in expansion,
excessive production, and accumulation of terminally differentiated peripheral blood
cells (e.g., erythrocytes, granulocytes, and platelets).
Overview of Myeloproliferative Disorders/Neoplasms

Major proliferative components:

Hematopoietic Stem Cell

Common Myeloid Progenitor

EPO TPO, IL-3
G-CSF M-CSF

Rubriblast Myeloblast Megakaryoblast

EPO G-CSF M-CSF, IL-3, IL-5 TPO IL-11

Erythrocyte Granulocyte / Thrombocyte

Monocyte
Chronic Myeloid Leukemia
Definition:
 CML is an MPN arising from a single genetic
translocation mutation in pluripotential hematopoietic
stem cells producing a clonal overproduction of the
myeloid cell line, resulting in a great number of
immature cells in the neutrophilic line.

Blood picture (peripheral blood smear):

 Leukocytosis and a dramatic ‘left shift’ is noted
 Segmented neutrophils, bands, metamyelocytes,
and myelocytes predominate
 Myeloblasts: ~1%
 Promyelocytes: ~5%
Chronic Myeloid Leukemia
According to the 2016 WHO Definition:

(1) Chronic Phase

 Blasts comprising <10% (0% to 9%) of white
blood cells (WBCs) in the peripheral blood and/or
nucleated bone marrow cells
 Extramedullary disease: none

VS

Monocytes Myeloblast
Chronic Myeloid Leukemia (2016 WHO Definition)

(2) Accelerated Phase

 Intermediate metamorphosis phase (before blastic transformation)
 Blasts comprising 10% to 19%
 Peripheral blood myeloblasts and promyelocytes combined is Myeloblast
>30%
 Peripheral blood basophils is >20%
 Persistent thrombocytopenia (<100 x 109/L) unrelated to
therapy, or persistent thrombocytosis (>1000 x 109/L)
unresponsive to therapy. Promyelocyte
 Increasing WBC counts and spleen size unresponsive to therapy
 Additional clonal cytogenetic abnormalities in Philadelphia
chromosome-positive cells
 Persistent or increasing splenomegaly
Basophil
Chronic Myeloid Leukemia (2016 WHO Definition)
(3) Blast Phase or Blast Crisis Granulocytic sarcoma: extramedullary blast proliferation

 Blasts comprising >20% of total WBC in peripheral blood and nucleated bone marrow cells
 Extramedullary blast proliferation; involves peripheral blood, bone marrow, and extramedullary tissues
 Large foci (or clusters) of blasts on bone marrow biopsy (demonstrated by CD34 immunohistochemistry)

Bone marrow aspirate smear (Wright stain) Peripheral blood smear (Wright stain)
Chronic Myeloid Leukemia

Immunohistochemistry:
 It is helpful, especially for demonstrating blasts by
CD34 immunostaining.

 CD34 is an important marker for adult hematopoietic

stem cells (HSCs).

 Dendritic interstitial cells, endothelial cells, embryonic

fibroblasts, and some cells in the fetal and adult
nervous tissue also express this marker and will stain
positive.

 Positive result: brown/dark brown colored product

Blast cells positive for CD34 immunostaining
 HRP: horseradish peroxidase
Chronic Myeloid Leukemia
Indirect Immunohistochemistry (Principle):

HRP-labeled

Anti-CD34
Y Y Y Y secondary
antibody against
Anti-CD34
CD34
Specimen

Diaminobenzidine (DAB) chromogen + Hydrogen Peroxide

Anti-CD34
Y Y Y Y Brown-colored
product (+)

CD34
Specimen
Blast cells positive for CD34 immunostaining
 Oncogene: a gene that has the potential to cause cancer
Chronic Myeloid Leukemia Proto-oncogene: a normal gene which, when altered by mutation,
becomes an oncogene that can contribute to cancer.

Molecular Genetics: BCR1 (Breakpoint Cluster Region)

 Normal BCR1 gene is located on chromosome 22
 Codes for p160, which exhibits serine and threonine kinase
activity
Normal
 Functions in the regulation of cell growth
chromosome 22

ABL1 (Abelson Murine Leukemia Viral Oncogene)

 A proto-oncogene
BCR1  Normal ABL1 gene is located on chromosome 9
q11.2  Codes for p125, which exhibits tyrosine kinase activity
 Functions in cell differentiation, cell division, cell adhesion, and
q34.1 stress response
 The kinase activity of the ABL1 protein is expressed and regulated
ABL1
by the its three primary domains: SH1, SH2, and SH3
Normal
 Deletion of the SH3 domain turns ABL1 into an oncogene
chromosome 9
Chronic Myeloid Leukemia
Molecular Genetics:
 CML is characterized by the chromosomal reciprocal translocation t(9;22) (9q34.1;22q11.2),
resulting in the BCR-ABL1 fusion gene and formation of the Philadelphia chromosome, which
causes an increase in blood granulocytes and bone marrow myeloid precursors as the major
proliferative component.
Mutated
Normal Abnormal chromosome 22
chromosome 22 chromosome 9
BCR
ABL1

BCR-ABL1 fusion gene

Normal BCR
chromosome 9 q11.2 The resulting chimeric BCR-ABL gene is
transcribed to produce a 210-kDa
q34.1 BCR protein with tyrosine kinase activity
ABL1 (called p210BCR-ABL)  leukemogenic
ABL1
cell growth and prolonged apoptosis.
Chronic Myeloid Leukemia The presence of t(9;22) (q34.1;q11.2) or BCR-ABL1
abnormality could be demonstrated by karyotype
analysis, FISH, or PCR-based methods.

Molecular Genetics: Chromosome 22 Chromosome 9

q34.1

SH3
Normal q11.2 BCR1
chromosome 22
SH2 ABL1

The BCR-ABL1 protein is a dysregulated

SH1
tyrosine kinase that causes CML cells to
BCR1 grow and divide out of control.
q11.2
ABL1 has 3 domains:
q34.1
BCR SH1: ATP binding site
ABL1 ABL1 SH2: docking point for phosphate receiver proteins
Normal SH3: controls the rate and timing of the
chromosome 9 BCR-ABL1 fusion gene phosphorylation activity
Chronic Myeloid Leukemia
Pathogenic Mechanism:
 BCR-ABL expression inappropriately prolongs the growth factor-independent survival of CML
myeloid progenitors and granulocytes by inhibiting apoptosis, a genetically programmed process
of active cell death.
 In the case of CML, the BCR-ABL1 translocation occurs next to the SH3 domain of the ABL1
moiety.
 BCR-ABL1 tyrosine kinase loses the ability to shut off kinase activity.
 The BCR-ABL1 enzyme continuously adds phosphate groups to tyrosine residues on cytoplasmic
proteins, activating several signal transduction pathways, which result in the following:
 Stimulation of gene expression
 Increase in growth factor–independent myeloid cellular proliferation
 Reduction of cell differentiation
 Reduction of adhesion of cells to bone marrow stroma
 Decrease in or resistance to apoptosis
 Chronic Myeloid Leukemia
Laboratory Features:
Bone marrow from a CML patient
stained with reticulin (silver) stain to
show reticulin fibrosis
Reticulin is a normal component of the bone marrow stroma and can be detected with a reticulin stain in 73% to 81% of
healthy subjects. Increased reticulin staining (reticulin fibrosis) is associated with many benign conditions as well as some
malignant diseases.
Peripheral Blood: Bone Marrow:
 Neutrophilic leukocytosis with  Myeloid hyperplasia
immature forms (from  Blasts <10% (chronic); 10-19% (accelerated);
segmented neutrophils to >20% (blast crisis)
occasional blasts)  Minimal or no dysplasia
 Basophilia / eosinophilia  Typically increased megakaryocytes, clustered in
 Thrombocytosis groups of three or more
 Anemia  Mild or moderate reticulin myelofibrosis
 Blasts <10% (chronic phase)  Monocytes usually <3%
 Blasts 10-19% (accelerated)  Gaucher-like histiocytes (sea blue histiocytes),
 Blasts >20% (blast crisis) having blue pigment in the cytoplasm) can be
 Decreased LAP score seen in one-third of patients
 Increased lactate
dehydrogenase (LDH) Genetic Studies:
 Increased uric acid  Philadelphia chromosome – positive (90-95%
 Increased vitamin B12  BCR-ABL – positive (>95%)
Chronic Myeloid Leukemia Leukemoid reaction: an increase in the white blood
cell count (leukocytosis), which can mimic leukemia.
The reaction is actually due to an infection (“left-shift”)
or another disease and is not a sign of cancer.
Differential Diagnosis:

Leukemoid Chronic Myeloid

Diagnostic Criteria
Reaction Leukemia
Leukocyte count (x 109/L) 30,000 to 50,000 >100,000
Toxic vacuoles / granules 2 – 4+ 0 – 1+
Döhle bodies Frequent Rare
Eosinophilia / Basophilia 0 1 – 3+
Pseudo-Pelger-Huët 0 – 1+ Occasional
Karyorrhexis 0 – 1+ 1 – 2+
Giant bizarre nuclei 0 – 1+ 1 – 3+
Myelocyte bulge 0 Present
LAP score High Low
Philadelphia chromosome Negative Positive
Splenomegaly None Prominent
Transformation to AML No Yes
Chronic Myeloid Leukemia
Cytochemical Staining: Leukocyte Alkaline Phosphatase (LAP)
Chronic Myeloid Leukemia
Cytochemical Staining: Leukocyte Alkaline Phosphatase (LAP)

Reaction Principle: (according to Kaplow, 1955)

Alkaline phosphatase
Naphthol AS-BI Phosphate Aryl naphthylamide
(buffered at pH 9.6)
(substrate) (hydrolyzed substrate)

Aryl naphthylamide + Fast Violet B Salt Insoluble precipitate (red azo-dye)

(hydrolyzed substrate) (azo-coupling diazonium salt) at the site of enzymatic activity

Counterstain: Mayer’s Hematoxylin or Fast Blue RR Salt: stains the granules blue
Methylene Blue (stains the nucleus blue) Counterstain: Neutral Red (red nucleus)
Chronic Myeloid Leukemia
Cytochemical Staining: Leukocyte Alkaline Phosphatase (LAP)

Negative / no stain Fine / faint stain Moderate stain Strong stain Brilliant stain

0 1+ 2+ 3+ 4+

Procedure:
 One hundred (100) neutrophils are counted and graded (from 0 to 4+) according to staining intensity of the
red dye and number of granules stained in the cytoplasm of the neutrophil.
 The sum of the grades or ratings is the LAP score; cell ratings and the total LAP score are reported.
 Quality control: the acceptable LAP score of the known positive sample is >140
 The normal score is established by the laboratory: 15 to 130 (Wintrobe’s Clinical Hematology, )
 LAP is found predominantly in mature neutrophils and the metamyelocyte stage.
Let’s Review Chronic Myeloid Leukemia
1. The _______ is the most immature cell of the _______ myeloid lineage and is the major
proliferative component in CML.

2. CML progresses into three phases, namely ________, ________, and ________ phase.

3. The normal BCR1 gene is located on chromosome ____ and the ABL1 gene is located on
chromosome ____.

4. The translocation mutation _____ results in the formation of the aberrant _______.

5. The chimeric _______ gene codes for the protein with a dysregulated ___________ activity.

6. CML must be differentiated from ________ by determining the _________, detection of the
__________, and examination of the _________.
Polycythemia Vera
Definition: Pan- (means ‘all’ or ‘everything’)

 Also known as polycythemia rubra vera

 PV is a neoplastic clonal MPN that commonly
manifests with panmyelosis in the bone marrow and
increases in erythrocytes, granulocytes, and platelets
in the peripheral blood. Splenomegaly is common.
The disease arises in a HSC and is clonal in nature.

Blood picture (peripheral blood smear):

 RBC count, hemoglobin, and hematocrit: elevated
 RBC morphology: normocytic, normochromic
 Total WBC count: elevated
 Granulocyte count: elevated
 Platelet count: elevated
Polycythemia Vera Erythropoiesis: Process and Regulation
Red blood cell production (erythropoiesis) takes place in
the bone marrow under the control of the hormone
erythropoietin (EPO). Juxtaglomerular cells in the
kidney produce EPO in response to decreased oxygen
delivery to the tissues (hypoxia).

Overview of the JAK2 Protein:

 It is coded by the JAK2 gene, which is found on
chromosome 9 (band 9p24.1)
 Normal JAK2 protein is similar to the ABL1 protein
(they have tyrosine kinase enzymatic activity).
 JAK2, together with the STAT proteins, is involved in
the control of cellular growth and proliferation.
 JAK2 is closely associated with the cytokine
receptors and functions near the cell membrane.
 Binding of the cytokine (e.g., EPO) to its receptor on
the cell membrane will result in the activation of the
Red blood cell (RBC) production (erythropoiesis) JAK2 protein.
Polycythemia Vera
Erythropoiesis: Process and Regulation
Red blood cell production (erythropoiesis) takes place in
the bone marrow under the control of the hormone
erythropoietin (EPO). Juxtaglomerular cells in the
kidney produce EPO in response to decreased oxygen
delivery to the tissues.

Overview of the JAK2 Protein:

 Neoplastic clonal stem cells in PV are hypersensitive
to erythropoietin (EPO) or function independently of
EPO for cell growth.
 The specific JAK2 mutation, JAK2 (V617F), is
detected in more than 95% of patients with PV and is
found on chromosome band 9p24.

Red blood cell (RBC) production (erythropoiesis)

Polycythemia Vera
Initiation of the JAK2/STAT Signaling Pathway:
JAK2/STAT signaling begins with the activation of JAK2 by
binding of a ligand such as growth factors, interferons, or
interleukins to specific transmembrane receptors.
Signaling Process:
1. EPO binds to membrane EPO receptor.
2. Conformational change in EPO receptor.
3. JAK2 docks or binds itself to the EPO receptor.
4. JAK2 gets activated and becomes a tyrosine kinase.
5. JAK2 phosphorylates its main target, the STAT proteins, in a
cascade of reactions.
6. Phosphorylation, activation, and the stimulation of STAT,
MAPK, and PI3K/AKT signal transduction pathways.
7. STAT translocates into the cell’s nucleus.
8. In the nucleus, the STAT dimers bind to specific DNA targets
and induce gene transcription that effects appropriate
physiological responses (e.g., proliferation and apoptosis)

STAT: signal transducer and activator of transcription

Polycythemia Vera
Pathogenic Mechanism:
 Mutation of the JAK2 gene (JH2 domain) will code for an
abnormal, dysregulated JAK2 protein (inhibitory function is
lost).

 Constitutive tyrosine kinase activity of the mutated JAK2

protein causes continuous activation of several signal
transduction pathways that are normally activated after
erythropoietin stimulation via the erythropoietic receptor.

 Mutated JAK2 is active and will phosphorylate STAT

proteins in the absence of erythropoietin or will over-
phosphorylate in its presence.

 HSCs that bear the JAK2 mutation are resistant to

erythropoietin deprivation apoptosis by upregulation (to
increase the response) of BCL-xL, an anti-apoptotic protein
in erythropoiesis.
Polycythemia Vera Normalized blood /
tissue oxygen levels
Negative
feedback
Inhibition / Regulation of Apoptosis:

In the presence of EPO  apoptosis is inhibited

 Normally, EPO protects erythroid cells from apoptosis.
 EPO activates ERK1 and ERK2, which upregulate or increase
the expression of BCL-xL (an anti-apoptotic protein)
Kidneys stop
 Caspase-3-dependent cleavage of the BCL-xL protein will be
producing EPO
inhibited, thus resulting in the protection of erythroid cells
from apoptosis.

In the absence of EPO  apoptosis is initiated

 EPO deprivation induces the activation of caspase-3, leading
to apoptosis of erythroblasts.
 Activated caspase-3 will cleave BCL-xL.
 Cleavage of BCL-xL will result in apoptosis of erythroid cells.
EPO deprivation to
ERK: extracellular signal-regulated kinase erythroid precursors
BCL-xL: B-cell lymphoma-extra large (a mitochondrial transmembrane molecule) in the bone marrow
Polycythemia Vera

Clinical Features, Signs and Symptoms:

 PV is a chronic disease that usually has an insidious onset. Many patients are asymptomatic
and are diagnosed incidentally on routine blood work drawn for other evaluations.
 Patient may present with thrombosis and/or bleeding secondary to erythroid expansion
 Blood hyperviscosity (blood becomes too sticky or viscous)
 Thrombocytosis (elevated platelet count)
 Symptoms may include headache, epistaxis, ischemic or hemorrhagic stroke, angina,
myocardial infarction, acute gouty arthritis, and cramps or pain in leg muscles.
 Hepatic vein thrombosis, leading to Budd-Chiari syndrome, has been reported in up to 10%
of patients diagnosed with PV. Characterized by: abdominal pain, ascites, and liver enlargement

 Pruritus (itching) is especially noted after a warm bath. ( histamine from mast cells)
 Erythromelalgia (erythema and painful burning sensation in the hands and feet) ( platelet
thromboxane secretion)
 Polycythemia Vera
*cyanosis may occur in PV unless RBC count reaches critically high levels.
Differential Diagnosis:
Clinical Features Polycythemia Vera Secondary Erythrocytosis Relative Erythocytosis
Cyanosis Absent* Present May be present
Heart or lung disease Absent Present Absent
Splenomegaly Present in 75% Absent Absent
Hepatomegaly Present in 35% Absent Absent
Laboratory Features
Red cell mass Increased Increased Normal
Erythropoietin levels Decreased Increased Normal
Arterial oxygen sat. Normal Decreased Normal
Leukocyte count Increased in 80% Normal Normal
Platelet count Increased in 50% Normal Normal
nRBCs, poikilocytes Often present Absent Absent
LAP score Increased in 70% Normal Normal
Bone marrow picture Hypercellular, panmyelosis, fibrosis Increased erythropoiesis Normal
Serum B12 / Uric acid Increased in 75% / 40% Normal Normal
Polycythemia Vera Diagnosis of PV requires meeting either:
• Both major criteria and one (1) minor criterion OR
Diagnostic Criteria (WHO, 2008):
• First major criterion and two (2) minor criteria

 Major criteria:
1. Hemoglobin/Hematocrit:
 Hemoglobin: >18.5 g/dL (men) or >16.5 g/dL (women); or
 Hemoglobin or hematocrit: >99th percentile of reference range for age, sex or altitude of residence; or
 Hemoglobin: >17 g/dL (men) or >15 g/dL (women) if associated with a sustained increase of >2
g/dL from baseline that cannot be attributed to the correction of iron deficiency; or
 Elevated red cell mass >25% above the mean normal predicted value.
2. Presence of JAK2 mutation
 The specific JAK2 mutation, JAK2 (V617F), is detected in more than 95% of patients with PV and is
found on chromosome band 9p24.
 Minor criteria:
1. Bone marrow trilineage myeloproliferation
2. Subnormal (low) serum erythropoietin level
3. Endogenous erythroid colony (EEC) growth or formation
Polycythemia Vera
Coagulation Testing for PV Patients:

Important points to remember:

1. The correct blood-to-anticoagulant ratio
must be strictly followed.

2. PV patients have decreased plasma volume

relative to the increased hematocrit; hence,
the amount of anticoagulant MUST be
adjusted.

3. Adjust the volume of the anticoagulant IF the

patient’s hematocrit is HIGHER than 55%
Polycythemia Vera For example: supposing you have polycythemia
vera patient with a hematocrit value of 65%. What
would be the volume of citrate anticoagulant to
Coagulation Testing for PV Patients:
remove?
Polycythemia Vera For example: supposing you have polycythemia
vera patient with a hematocrit value of 65%. What
would be the volume of citrate anticoagulant to
Coagulation Testing for PV Patients:
remove?
Polycythemia Vera For example: supposing you have polycythemia
vera patient with a hematocrit value of 65%. What
would be the volume of citrate anticoagulant to
Coagulation Testing for PV Patients:
remove?

0.18)

1.62 = 0.10 mL
Let’s Review Polycythemia Vera
1. PV has characteristically _____ erythropoietin (EPO) levels.

2. Diagnosis of PV is considered if the following laboratory findings are observed: high ____, ____, and ____;
detection of ______ mutation; ____ EPO levels; _______ in the bone marrow; _____ platelet count; and
______ leukocyte count.

3. The correct blood-to-anticoagulant ratio for coagulation testing is ______.

4. ________ and _________ must be ruled out to arrive at an accurate diagnosis of PV.

5. EPO is a _____ secreted by the ______ of the _______.

6. Too much red blood cells in your circulation will result in venous ______ and ______ blood flow.

7. The original volume of citrate anticoagulant the 1.8 mL light blue top tube is ____ mL.

8. The sample type used for coagulation testing is _____________________.