Haem 3 - CML and myeloproliferative disorders Flashcards
What are dilution studies/ Fick’s principle?
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
Give causes of relative polycythaemia
Lack of plasma
• Alcohol, obesity, diuretics, dehydration, burns, D, V, cigarette smoking
Give causes of true polycythaemia
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)
Myeloproliferative disorders
Ph -ve
Ph +ve
Ph -ve (JAK2 positive - JAK2 is a TK - TK promote cell growth)
PV
ET
PMF
Ph +ve
CML
What happens in a JAK mutation
• 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
MDP associated gene mutations in
PV
ET
PMF
PV
100% JAK2 V617F
ET - essential thrombocythemia
60% JAK2 V617F
30% Calreticulin
5% MPL
PMF
60% JAK2 V617F
30% Calreticulin
PV clinical features
- 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
PV diagnosis
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
PV mx
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
ET clinical features + diagnostic/confirming critetria
essential thrombocythemia
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)
ET mx
essential thrombocythemia
- Aspirin – thrombosis prevention
- Hydroxycarbamide – antimetabolite that suppresses cell turnover
- Anagrelide – inhibits platelet formation from megakaryocytes, not commonly used (SE of palpitations + flushing)
PMF clinical features
- Pancytopenia related symptoms (anaemia, thrombocytopenia)
- Thrombocytosis
- Extra-medullary haemopoiesis –> massive heptosplenomegaly
- Hypermetabolic state – WL, fatigue, dyspnoea, night sweats, hyperuricaemia, fever
PMF features
Blood film
Bone marrow
O/E
DNA analysis
• 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
PMF mx (6)
- 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
Bad prognostic signs in PMF
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
Explain the pathophysiology of CML
• 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)
CML clinical + laboratory features (FBC, blood film)
- 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
CML
diagnosis
monitoring response to therapy
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
Advanced phases of CML
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
CML mx plan
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
CML mx how do you assess/ monitor response?
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)
Lymphadenopathy in a leukaemic picture - lymphoid or myeloid lineage?
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)
name 2 haematological cancers causing massive hepatosplenomegaly with no lymphadenopathy
CML
PMF
Also look at menti Q
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Ph translocation BCR ABL1 rearrangement is oncogenic via
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
How does JAK2 become the oncogenic JAK2 V617F
• JAK2 V617F
o Single DNA substitution in the JAK2 gene
Oncogenesis via dysregulated expression of an intact oncogene is mostly found in
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
Look at menti q
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