MYELOPROLIFERATIVE NEOPLASMS (MPN)

Diseases

of the pluripotent hematopoietic stem cell

Characterized by exaggerated proliferation of hematopoietic
tissue
Characterized by abnormalities of tyrosine-kinase (TK) genes
The proliferating cells are normally differentiated, for long
periods of time
Potential of transformation into acute leukemias
Several entities are described:
CHRONIC MYELOID LEUKEMIA (CML)
POLICYTHEMIA VERA (PV)
ESSENTIAL THROMBOCYTHEMIA (ET)
PRIMARY MYELOFIBROSIS (PMF)
Systemic mastocytosis (SM)
Chronic eosinophilic leukemia (CEL)
Chronic neutrophilic leukemia (CEN)
The first four entities (CML, PV, PMF and ET) are more frequent

Pluripotent hematopoietic stem cell

(Hemocytoblast)

Hematopoiesis in Humans

Lymphoid
stem cell

Myeloid stem cell

Proerythroblast
(Pronormblast)

Megakaryoblast

Myeloblast

B. promyelocyte

Basophilic erythroblast

N. promyelocyte

Lymphoblast

E. promyelocyte

Monoblast

Promegakaryoblast

Prolymphocyte

Polychromatic erythroblast

B. myelocyte

N. myelocyte

E. myelocyte

Orthochromatic
erythroblast
(Normoblast)

B. metamyelocyte

N. metamyelocyte

E. metamyelocyte

Promonocyte

Megakaryocyte
Small lymphocyte
Polychromatic
erythrocyte
(Reticulocyte)

B. band

N. band

Natural killer cell

E. band

T lymphocyte B lymphocyte

Thrombocytes

Erythrocyte

Basophil

Mast cell

Neutrophil

Eosinophil

Monocyte

Myeloid

Lymphoid

TK

Tyrosine
residue

Tyrosine-kinase (TK) mediated intracellular signalling.

Substrates are activated by phosphorilation of tyrosine residues.
Phosphate (P) groups originate from ATP and are transferred by tyrosine
kinases (TK)

CHRONIC MYELOID LEUKEMIA (CML)

Definition:
MPN characterized by exaggerated proliferation of
the granulocytic lineage
Is characterized by the abnormality of the ABL TK
Natural history - biphasic evolution in 100% of
cases
Chronic phase 3-20 years (median 5 years)
Acute (blastic) phase terminal phase, usually <1 year
resembles acute leukemia

 Epidemiology:
Incidence: 1.5/100,000/year
Peak of incidence 30-50 years

Etiology:
Unknown
Role of radiation, organic solvents

Pathogenesis of CML

 t(9;22) ABL (9) + BCR (22) = BCR-ABL

(22) gene bcr-abl P210 protein
The translocated 22 chromosome is also
known as the Philadelphia chromosome
(Ph1)
P210 increased tyrosine-kinase activity
increased message transduction from
membrane to nucleus

1. CML in chronic phase

Clinical Picture
10% of patients: non-symptomatic
General symptoms - fatigue, weight loss,
sweating, anorexia, abdominal pain
Pallor
Splenomegaly sometimes huge
Hepatomegaly
Priapism
Dyspnea

 Laboratory Data
Peripheral blood

Leukocytosis often > 100,000/l

Anemia usually mild
Thrombocytosis, sometimes >1000,000/l
Peripheral smear
Neutrophilia
Left shift: bands, metamyelocytes, myelocytes,
promyelocytes, myeloblasts
Basophilia

Normal peripheral smear

Leucocytosis and left shift in chronic phase

CML

 Bone marrow
increased granulopoiesis (G:E ratio >4:1)
left shift, basophilia

Cytogenetics and molecular genetics

karyotype t(9;22) Philadelphia chromosome
(Ph) ~ 90-95%
molecular cytogenetics (rqPCR) BCR-ABL ~ 100%

Biochemistry
Hyperuricemia
High LDH

Ph1

Karyotype in CML showing the t(9;22) translocation

2. CML in blastic phase

Natural history: blastic phase occurs after 4-8 years
of chronic phase
Sometimes preceded by the ACCELERATED PHASE:

Decreased effectiveness of therapy

Increased symptoms
Progressive splenomegaly
Increased leukocytosis
Basophilia >20%
Anemia and/or thrombocytopenia unrelated to therapy
Increased blasts 15-29%
New cytogenetic abnormalities: Ph-Ph, 8+, 19+, etc

 Full blown BLASTIC PHASE is defined by:

Blasts > 30% (peripheral blood or bone marrow)

Clinically and hematologically, CML in blastic

phase resembles to acute leukemias:
Symptoms of anemia
Bleeding due to thrombocytopenia
Infections due to neutropenia

The phenotype of the blasts is:

Myeloid in 85% of cases myeloid acutisation
Lymphoid in 15% of cases lymphoid acutisation

CML blastic phase

Treatment of CML
Response criteria:
Hematologic response (blood count, blood smear)
Complete hematologic response (CHR)
No splenomegaly
Normal blood count, normal differential count

Cytogenetic response (karyotype):

Complete cytogenetic response (CCyR): Ph1= 0%
Major cytogenetic response (MCyR): 1-35% Ph1)
Minor cytogenetic response (nCyR): 35-95% Ph1)

Molecular response (rqPCR):

Major molecular response (MMR): <0.1% BCR-ABL (>1000x
reduction)
Complete molecular response (CMR): BCR-ABL undetectable

 Treatment of chronic phase CML:

Tyrosine kinase inhibitors (TKI)
Imatinib (1st generation TKI) - current standard
first line therapy
90-95% CHR
70-90% CCyR
30-50% MMR
Dasatinib, Nilotinib (2nd generation TKI)
Active in most cases of resistance to imatinib
Responses are quicker and deeper than
imatinib may become standard treatment in
the near future

Imatinib blocks the ATP-binding site on the BCR-ABL protein

Treatment of chronic phase CML

(continued)
Other drugs (older):
Hydroxiurea may induce CHR, no cytogenetic
response
- Interferon (IFN) mai induce CCyR in 10-20% of pts

Allogeneic stem cell transplantation

Currently the only cure
Still plagued by high transplant-related
mortality (TRM)

 Treatment of blastic phase CML

Combination chemotherapy
AML/ALL type regimens
High toxicity
Low response rate

Allogeneic stem cell transplant

High toxicity
Low response rate

Imatinib, Dasatinib, Nilotinib

Low toxicity
Transitory hematological response in 50% of patients,
sometimes lasting years
Sometimes also CCyR, MMR

POLYCYTHEMIA VERA
Definition:
MPN characterized by increased red cell number
(polyglobulia) due to autonomous, non-stimulated
proliferation of erythrocytes.
Characterized by the abnormality of the JAK2 TK

Epidemiology
incidence: 5-15/1,000,000/year
incidence peak 50-60 years
predominance in males

 Etiopathogenesis
etiology unknown
80-90% of PV point mutation of the
JAK2 gene V617F: increased tyrosine
kinase activity (2005)
autonomous proliferation of erythroid
progenitors, not stimulated by EPO
also increased production of
granulocytes and platelets
hyperviscosity and thrombotic risk
the risk of acutisation is relatively low,
5-10%
Janus, the two-faced
god

 Clinical Picture
Dizziness, vertigo, blurred vision
Sometimes stroke, heart attacks,
thromboembolism
Rarely bleeding
Pruritus
Red skin, especially face and palms
Splenomegaly, rarely hepatomegaly

 Laboratory findings
Increased red cell mass
Hgb>18.5g/dl () or >16.5g/dl (), Hct->51% () or
>48% (), often >60%, RC >6 mil/l
Leucocytosis, usually <50,000/l with left shift
Thrombocytosis, sometimes > 1000,000/l
Biochemistry
High LDH
High cobalamin
Low iron
High histamin
Blood gas assay: normal oxygen saturation
Low/normal erythropoietin levels
JAK2 V617F in 95% of cases

 Diagnostic criteria (WHO 2008):

Major criteria:
1. Hgb>18.5g/dl in men or >16.5g/dl in women
2. Presence of the JAK2 V617F mutation

Minor criteria
1. Bone marrow tri-lineage proliferation
2. Low serum erythropoietin (EPO) levels
3. Endogenous erythroid colony (EEC) growth

Diagnostic algorithm:

2 major criteria + 1 minor criterion

Major criterion 1 + 2 minor criteria

 Differential diagnosis:
Secondary polycythemia
Living at altitude - >3000m
Diseases associated with chronic hypoxia
chronic bronchitis
chain smokers
cor pulmonale
congenital cardiopathies
renal artery stenosis
EPO secreting tumors

 Treatment of PV
Phlebotomy
300-400ml per phlebotomy until Hct 40-45%,
then one/2 weeks - 1 month

Antiplatelet drugs:
Aspirin 75-150mg/day
Ticlopidine, clopidogrel

Hydroxyurea 1-2g/day
Antihistamine drugs
IFN
JAK2 specific inhibitors possibly in the near
future

 Evolution, Complications
When treated correctly, most cases have a
good prognosis
Most frequent complications stroke,
myocardial infarction, DVT, Budd-Chiarri
510% acutisation secondary AML
5-10% progress towards myelofibrosis

ESSENTIAL THROMBOCYTHEMIA
Definition:
Persistent thrombocytosis (>450,000/l)
without other causes.
It is a difficult diagnosis: requires
exclusion of other conditions that may
display thrombocytosis
Epidemiology
Incidence: 7/1,000,000/year
Peak of incidence: 50-70 years

 Etiopathogenesis

Etiology unknown
Autonomous growth of megakaryocytes
About 50% have the JAK2 V617F mutation (2005)
25-30% : mutations of the CALR (calreticulin)
gene (2013)

Clinical picture
Most patients non-symptomatic
20-40% present with thrombosis
10-15% present with hemorrhage

Necrotic lesions due to repeated thrombosis in a case

of essential thrombocythemia

 Laboratory findings

Plt > 450,000, often >1,000,000/

WBC sometimes high
Left shift, basophilia
Normal serum iron, ferritin
Absence of Ph1
Absence of polycythemia
Megakaryocytic hyperplasia
JAK2 V617F mutation in 50-60% of cases
CALR mutations in 25-30% of cases

Peripheral thrombocytosis in a case of ET

Megakariocytic hyperplasia in ET

 Diagnostic criteria (WHO 2008)

1. Platelets >450,000/l
2. Megakaryocyte hyperproliferation with large,
lobulated megakaryocytes
3. Exclusion of other myeloid proliferations (CML,
PV, PMF, MDS)
4. Presence of the JAK2 V617F mutation or other
clonal mutation (CALR?) or exclusion of reactive
thrombocytosis

Diagnostic algorithm: for ET diagnosis, all 4

criteria must be present

 Treatment
Low risk: Plt<1,500,000, <40 years, no thrombosis, no
hemorrhage
aspirin low-dose (75-150mg/day)

Intermediate risk: Plt<1,500,000, 40-60 years

aspirin low-dose (75-150mg/day)
If other risk factors for thrombosis (HT, diabetes, heart disease, etc)
are present, cytoreductive treatment is added - hydroxiurea or
anagrelide

High-risk: Plt>1,500,000 and/or history of thrombotic events

hydroxiurea or anagrelide + low-dose aspirin

If hemorrhage avoid aspirin

In case of very high platelet count and acute hemorrhage or
thrombosis platelet apheresis

 Prognosis
If complications (mainly thrombotic) are
avoided, most patients have normal life
span
3-5% develop acute leukemia
Often neurologic complications and
sequelae from stroke

PRIMARY MYELOFIBROSIS (PMF)

Definition
MPN characterized by myelofibrosis, hepatosplenomegaly,
progressive cytopenias

Epidemiology
Incidence : 1/100,000/year
Peak of incidence 60 years

Etiopathogenesis
Etiology unknown
Proliferation of dysplastic megakaryocytes production of
cytokines, especially platelet derived growth factor (PDGF)
that stimulate fibroblasts myelofibrosis
JAK2 V617F mutation in 50-70% (2005)
CALR mutations in 25-30% (2013)

 Clinical Picture
Symptoms of anemia
Weight loss, sweating, fever
Abdominal pain, bloating
Splenomegaly huge
Hepatomegaly
Hemorrhage

Huge splenomegaly in a case of primary myelofibrosis

 Laboratory Findings
Anemia, sometimes severe
Leucoerythroblastic picture on the peripheral
smear: left-shift + erythroblasts
Presence of tear-drop red cells (dacriocytes)
Sometimes autoimmune hemolytic anemia, AHAI
(reticulocytosis, jaundice, positive Coombs test)
Moderate leukocytosis, usually <50,000
Thrombocytopenia
Bone marrow biopsy Gomori stain
myelofibrosis of various degrees
JAK2 V617F in 50-70% of cases
CALR mutations in 25-30% of cases

Dacryocytes (tear drop shaped red cells)

Myelofibrosis Gomori stain

 Diagnostic criteria (WHO 2008)

Major criteria:
1. Proliferation with atypical megakaryocytes +
reticulin/colagen fibrosis
2. Exclusion of other myeloid proliferations (CML, PV, ET, MDS)
3. The presence of the JAK2 V617F mutation, (CALR?) or
exclusion of reactive myelofibrosis

Minor criteria:
1.
2.
3.
4.

Leucoerythroblastic peripheral smear

High serum LDH
Anemia
Splenomegaly

Diagnostic algorithm: 3 major criteria + 2 minor criteria

 Treatment
Mostly transfusions for anemia
Hydroxiurea and IFN can sometimes reduce
splenomegaly
Corticosteroids if AHAI
Splenectomy: if huge, painful splenomegaly
Recently: JAK2 inhibitors (ruxolitinib) were found
to be beneficial in some cases, reducing
splenomegaly and symptoms

Prognosis
Median survival 5-6 years (2-15)
Acutisation in 10-20% of cases

MPN

Peripheral blood

Bone marrow

Fibrosis

Splenome
galy

TK defect

TKI response

CML

Leucocytosis, left
shift, basophilia

Granulocytic
lineage
hyperplasia

Variable

++

BCR/ABL
(100% of
cases)

+++

PV

Poliglobulia,
leucocytosis,
trombocytosis,
left shift,
basophilia

Hiperplazie
eritroida,
megacariocite
atipice

Variable

JAK2 V617F
(95% of cases)

PMF

Anemia,
leucoerythroblastic
picture, dacriocytes

Dysplastic
megakaryocytes

Increased

+++

JAK2 V617F
(50-70%)
CALR (30-40%)

ET

Trombocitosis,
leucocytosis,
basophilia

Megakaryocytic
hyperplasia,
atypical
megakaryocytes

Minimal

-/+

JAK2 V617F
(50% of cases)
CALR
(30-40%)

CNL

Leucocytosis,
neutrophilia

Granulocytic
hyperplasia

Variable

++

JAK2 V617F
(sometimes)

CEL

Eozinophilia>1500/l,
>6 months

Eozinophilia

Variable

+/-

FIP1L1PDGFR
(most cases)

++

SM

Left shift, eosinophilia,

basophilia

Mast cell
infiltration

Minimal

++

KIT D816V
(most cases)