Haem 3 - CML and myeloproliferative disorders

Question 1

What are dilution studies/ Fick’s principle?

Answer 1

To assess whether polycythaemia is relative (lack of plasma) or true (excess erythrocytes)

a)take components out – b) radiolabel them – c) reinfuse + measure dilution

o Red cell mass: 51Cr labelled RBC
o Plasma Vol: 131I labelled albumin

Question 2

Give causes of relative polycythaemia

Answer 2

Lack of plasma

• Alcohol, obesity, diuretics, dehydration, burns, D, V, cigarette smoking

Question 3

Give causes of true polycythaemia

Answer 3

Primary - reduced EPO
PV
Familial Polycythaemia

Secondary - raised EPO
Appropriate EPO
o	High altitude
o	Hypoxic lung disease (COPD)
o	Cyanotic heart disease
o	High affinity Hb 

Inappropriate EPO
o Renal disease (cysts, tumours, inflammation)
o Uterine myoma
o Other tumours (liver, lung)

Question 4

Myeloproliferative disorders

Ph -ve
Ph +ve

Answer 4

Ph -ve (JAK2 positive - JAK2 is a TK - TK promote cell growth)
PV
ET
PMF

Ph +ve
CML

Question 5

What happens in a JAK mutation

Answer 5

• JAK2 is a tyrosine kinase that is normally bound to the inactive EPO receptor
• EPO binds to the EPO receptor  receptor dimerises + autophosphorylates  phosphorylates JAK2
• Activation of the JAK2 signalling pathway  normal response to EPO
• JAK2 mutation  JAK2 signalling pathway is constitutively active  EPO response even in the absence of EPO
• JAK2 V617F mutation – most common
o Single point mutation (V617F) in 100% cases of PV
• JAK2 exon 12 mutation

Question 6

MDP associated gene mutations in

PV
ET
PMF

Answer 6

PV
100% JAK2 V617F

ET - essential thrombocythemia
60% JAK2 V617F
30% Calreticulin
5% MPL

PMF
60% JAK2 V617F
30% Calreticulin

Question 7

PV clinical features

Answer 7

• JAK2 V617F mutation
• High RBC, Hb, plt, WCC
• Low MCV, EPO, ferritin
• Splenomegaly
• Sx of hyperviscosity – headaches, light-headedness, stroke, visual disturbances, fatigue, dyspnoea, plethoric (red nose), gout, thrombosis, retinal vein engorgement, erythromelalgia
• Sx of histamine release - aquagenic pruritus, peptic ulceration
• Sx of hypervolaemia/hypermetabolism

Question 8

PV diagnosis

Answer 8

1 major + 2minor
2 major + 1 minor

Major
Hb >185g/l (m), >165g/l (f)
JAK2V617F mutation

Minor

• BM biopsy - Hypercellularity, prominent erythroid. granulocytic, megakaryocytic proliferation
• low EPO
• endogenous erythroid colony formation in vitro

Question 9

PV mx

Answer 9

reduce HCT (HCT <45%)
•	Venesection 
o	Only suitable in younger/healthy pt
•	Hydroxycarbamide /hydroxyurea 
o	Cytoreductive therapy  less DNA synthesis in RBCs

Reduce risk of thrombosis
• Aspirin
o Keep plts <400x10^9/L

Question 10

ET clinical features + diagnostic/confirming critetria

essential thrombocythemia

Answer 10

CLINICAL FEATURES
• JAK2, calreticulin, MPL mutations
• Increased plt – sustained thrombocytosis >600 x 10^9/L
• Splenomegaly (modest)
• Erythromelalgia
• Signs of thrombosis (arterial or venous) – CVA, gangrene, TIA, MI, DVT/PE
• Signs of bleeding – mucous membranes and cutaneous
• Symptoms of hyperviscosity (headaches, light-headedness, stroke, visual disturbances, fatigue, dyspnoea)

DIAGNOSIS/CONFIRMING CRITERIA
• Normal/slightly increased BM cellularity
• Platelet count >600 x 10^9
• Blood film – large platelets and megakaryocyte fragments
• BM – increased megakaryocytes (not reactive)
• No/minimal reticulin fibrosis
• Splenomegaly
• Normal ESR + CRP (non-reactive)

Question 11

ET mx

essential thrombocythemia

Answer 11

• Aspirin – thrombosis prevention
• Hydroxycarbamide – antimetabolite that suppresses cell turnover
• Anagrelide – inhibits platelet formation from megakaryocytes, not commonly used (SE of palpitations + flushing)

Question 12

PMF clinical features

Answer 12

• Pancytopenia related symptoms (anaemia, thrombocytopenia)
• Thrombocytosis
• Extra-medullary haemopoiesis –> massive heptosplenomegaly
• Hypermetabolic state – WL, fatigue, dyspnoea, night sweats, hyperuricaemia, fever

Question 13

PMF features

Blood film
Bone marrow
O/E
DNA analysis

Answer 13

•	Blood film
o	Leucoerythroblastic picture (primitive cells - nucleated RBC, immature WBC)
o	Tear drop poikilocytosis (dacrocyte)
o	Giant platelets
o	Circulating megakaryocytes

• Bone marrow
o Dry tap
o Trephine biopsy
 increased reticulin or collagen fibrosis
 increased prominent megakaryocyte hyperplasia and clustering with abnormalities
 New bone formation

• Liver + spleen – massive hepatosplenomegaly – extramedullary haematopoiesis
• DNA analysis – JAK2 (60%) or calreticulin mutation, MPL

Question 14

PMF mx (6)

Answer 14

• Supportive – RBC/platelet transfusion (often ineffective bc splenomegaly  rapid breakdown of RBCs)
• Cytoreductive therapy – hydroxycarbamide (for thrombocytosis, may worsen anaemia)
• Splenectomy – symptomatic relief, dangerous operation followed by worsening of condition
• Jak2 inhibitor – Ruxolitinib (only used in high prognostic score cases)
• Thalidomide, steroids
• Allogenic HSCT – potentially curative

Question 15

Bad prognostic signs in PMF

Answer 15

o	Bad prognostic signs
	Severe anaemia <100g/L
	Thrombocytopenia <100 x10^9 /L
	Massive splenomegaly  
	High DIPPS score*
•	Score 0 – median survival 15 y
•	Score 4-6 – median survival 1.4 years

DIPPS score - prognostic scoring system for PMF

Question 16

Explain the pathophysiology of CML

Answer 16

• Ph Chr +ve
• BCR-ABL Ph Chr mutation t(9;22)  Ph Chr
o Breakage and translocation of part of Chr 9 on Chr 22  newly rearranged chromosome = Philadelphia chromosome
o Abnormal Ph Chr  abnormal protein BCR-ABL – TK activity greater than the normal ABL protein
• DNA of Philadelphia chromosome forms mRNA which contains a 5’ portion of the BCR gene + a 3’ portion of the ABL gene

 normal ABL is highly repressed and only expressed when the cells need to proliferate
 when ABL is related to the BCR region upstream, it is constituently expressed  switched on TK activity driving cell proliferation
o BCR-ABL fusion gene  constitutively expressed + constitutively activated  drives cell replication in cells containing the Philadelphia chromosome
o BCR-ABL  fusion oncoprotein with constitutive TK activity drives myeloid proliferation

o BCR – Breakpoint cluster region (constitutively expressed by itself as a housekeeping gene) // ABL – Abelson Tyrosine Kinase (not constitutively expressed, only in stimulated cells)

Question 17

CML clinical + laboratory features (FBC, blood film)

Answer 17

• Lethargy
• Hypermetabolism
• Thrombotic event (mono-ocular blindness, CVA, bruising, bleeding)
• Massive hepatosplenomegaly

• FBC
o N/raised Hb + plt (contrast with acute leukaemia which presents with bone marrow failure)
o Massive leukocytosis (50-200 x 10^9/L)

• Blood film
Biphasic peak in neutrophils and myelocytes
o Neutrophils
o Myelocytes (not blasts if chronic phase)
o Basophilia

Question 18

CML

diagnosis
monitoring response to therapy

Answer 18

diagnosis
conventional karyotyping
FISH - probe it with 9 and 22 probes to look for the fusion gene
rt-PCR amplification + detection
normal cells - no PCR product to amplify
cells with fusion gene - fusion transcript can be amplified by the primers - abnormal band indicates presence of fusion gene
quantification of BCR-ABL fusion transcripts through rt-PCR also helps determine response to therapy

Question 19

Advanced phases of CML

Answer 19

o Disease transforms from a chronic phase through an accelerated phase into an acute leukaemia (blast crisis)
(Myeloid>lymphoid)
 Myeloblasts in the bone marrow

Accelerated phase 10-19% blasts in BM

Blast crisis >20% blasts in BM

if Hb + plt coutn are preserved - chronic, not accelerated or crisis

Question 20

CML mx plan

Answer 20

Imatinib

ABL TK inhibitor

blocks constitutive activation of ABL TK

o 1st gen – Imatinib
o 2nd gen – Dasatinib, Nilotinib
o 3rd gen – Bosutinib

Summary: TKI 1st gen –> TKI 2nd/3rd gen –> HSCT

• Commence on an oral TKI 1st gen
• Monitor response FBC, Cytogenetics, RQ-PCR
o CCyR (Complete Cytogenic Response) at 12 mo

• Failure 1 – switch to 2nd gen or 3rd gen TK inhibitor
o If no CCyR at 1 year or
o If they respond but acquire resistance

• Failure 2 – consider allogenic stem cell transplantation
o If inadequate response or intolerant to 2nd generation TKIs or
o If the disease progresses to accelerated or blast phase

Question 21

CML mx how do you assess/ monitor response?

Answer 21

response can be assessed in 3 ways
haematological response
cytogenic response
molecular response

o FBC – haematological response
 Complete haematological response (WBC < 10X10^9/L)
 WBC count starts to normalise

o Cytogenetics – cytogenic response
 At diagnosis – 100% of the cells are Philadelphia positive
 Partial cytogenetic response – 1-35% Philadelphia positive
 Complete cytogenetic response (CCyR)– 0% Ph positive

o RQ-PCR – molecular response (reduction in % BCR-ABL transcripts) – most sensitive
 BCR-ABL transcripts reduce 100%>10%>1%>0/1%
 Major molecular response (MMR) <0.1% (3 log duration)

Question 22

Lymphadenopathy in a leukaemic picture - lymphoid or myeloid lineage?

Answer 22

Lymphoid cause (not myeloid)  white cells circulate through lymph nodes + so will gorge them (myeloid cells will not circulate through those)

• haematological disorders with hepatosplenomegaly + no lymphadenopathy  myeloid disorder
• haematological disorders with hepatosplenomegaly + lymphadenopathy  lymphoproliferative disorder)

Question 23

name 2 haematological cancers causing massive hepatosplenomegaly with no lymphadenopathy

Answer 23

CML

PMF

Question 24

Also look at menti Q

Answer 24

x

Question 25

Ph translocation BCR ABL1 rearrangement is oncogenic via

Answer 25

novel fusion protein (not a mutation, just a chromosomal translocation that results in novel fusion oncoprotein)
o Translocation rearranges the DNA which is then transcribed as a novel mRNA which is then translated into a new protein  chimeric protein
o Chimeric protein has got 5’ portion of the BRC gene and 3’ portions of the ABL gene

Question 26

How does JAK2 become the oncogenic JAK2 V617F

Answer 26

• JAK2 V617F

o Single DNA substitution in the JAK2 gene

Question 27

Oncogenesis via dysregulated expression of an intact oncogene is mostly found in

Answer 27

more relevant in lymphoid malignancies - you get an intact proto-oncogene which becomes over expressed because it is translocated under the influence of an Ig promotor

Question 28

Look at menti q

Answer 28

x