Cytogenetic and molecular studies - Janus kinase 2 (JAK2) V617F
JAK2 and JAK-STAT signalling
- JAK2 - a non-receptor (cytoplasmic) tyrosine kinase
- Transduces signalling from receptors that have no intrinsic kinase activity: EPO receptor, thrombopoietin receptor (MPL), G-CSF receptor
- -> JAK-STAT pathway
- V617F = valine to phenylalanine at codon 617, exon 14, in the JH2 pseudokinase domain
- The pseudokinase domain normally autoinhibits the kinase domain
- -> loss of autoinhibition -> constitutive, cytokine-independent JAK-STAT signalling
- -> erythroid, megakaryocytic and granulocytic proliferation without EPO or TPO
The driver mutation triad in MPN
| Mutation | PV | ET | PMF |
|---|---|---|---|
| JAK2 V617F | ~95% | ~50-60% | ~50-60% |
| JAK2 exon 12 | ~3% (the remaining PV) | - | - |
| CALR (type 1/2) | - | ~20-25% | ~25-30% |
| MPL | - | ~3-5% | ~5-8% |
| "Triple negative" | - | ~10-15% | ~10% |
- *PV is essentially always JAK2-mutated* - a JAK2-negative, exon 12-negative erythrocytosis is not PV
Epidemiology
- MPN incidence ~1-2 per 100,000/yr each for PV and ET
- Median age at diagnosis 60-65 (ET has a second, younger, female peak)
- JAK2 V617F detectable in ~0.2-3% of the general population as clonal haematopoiesis, rising with age
- -> a positive result without cytoses is CHIP, not an MPN
- Germline 46/1 (GGCC) haplotype predisposes to acquiring V617F
Mechanism and allele burden
- Acquired somatic mutation in a haematopoietic stem cell
- Allele burden determines phenotype
- Heterozygous, low burden -> ET (thrombocytosis)
- Homozygous (mitotic recombination, 9pLOH), high burden -> PV and myelofibrosis
- The same mutation, three diseases - dose and cellular context decide
- Downstream: STAT5/STAT3, PI3K-AKT, MAPK
- inc proliferation, dec apoptosis, inc inflammatory cytokines (IL-6, TNF, TGF-beta)
- -> constitutional symptoms and, via TGF-beta from megakaryocytes, marrow fibrosis
- Thrombosis mechanism - why MPN clots
- inc haematocrit -> inc viscosity, inc red cell margination
- Activated, hyperreactive platelets and leucocytes; neutrophil extracellular traps
- JAK2-mutant endothelium (derived from the same clone) is prothrombotic
- -> JAK2 V617F is an independent thrombosis risk factor, over and above the blood counts
- Additional mutations (ASXL1, EZH2, SRSF2, IDH1/2, TP53, U2AF1) drive fibrotic and leukaemic progression
When to test
- Unexplained erythrocytosis (Hb >165 g/L men / >160 women, or Hct >0.49/0.48)
- Unexplained persistent thrombocytosis >450 after excluding reactive causes
- Splanchnic vein thrombosis (Budd-Chiari, portal, mesenteric) - *test even with normal counts*; portal hypertension and haemodilution mask the cytosis
- Unexplained leucocytosis or splenomegaly; marrow fibrosis
- Do not order it as part of a "thrombophilia screen" in ordinary DVT
How
- Allele-specific quantitative PCR on peripheral blood - sensitive to ~1% allele burden
- Sequencing alone is not sensitive enough at low burden
- If negative and erythrocytosis persists -> JAK2 exon 12
- If negative and thrombocytosis/fibrosis -> CALR then MPL
- Serum EPO: low/subnormal supports PV; normal or high argues for secondary erythrocytosis
WHO diagnostic use
WHO diagnostic use
- PV major criteria: raised Hb/Hct or red cell mass; trilineage marrow hypercellularity with pleomorphic mature megakaryocytes; JAK2 V617F or exon 12
- Minor: subnormal EPO
- ET: platelets >=450; megakaryocyte proliferation with large hyperlobulated forms; not meeting criteria for another MPN; a clonal marker (JAK2/CALR/MPL) or exclusion of reactive causes
- PMF: megakaryocytic atypia with reticulin/collagen fibrosis; clonal marker; plus minor criteria (anaemia, leucocytosis, splenomegaly, LDH, leucoerythroblastosis)
- Marrow biopsy remains required - genotype alone cannot separate ET from early prefibrotic PMF, and that distinction changes prognosis
Traps
- A positive JAK2 does not by itself diagnose an MPN - it can be CHIP
- A negative JAK2 does not exclude ET or PMF (CALR, MPL, triple-negative)
- Allele burden is not routinely used for monitoring outside trials
Risk-adapted, aimed at preventing thrombosis
- All PV and ET: low-dose aspirin unless contraindicated (bd dosing in high-risk ET)
- PV: venesect to Hct <0.45 - the single most important intervention (CYTO-PV)
- Manage conventional cardiovascular risk factors; smoking cessation
Cytoreduction - when
| Indication | |
|---|---|
| PV | Age >60, prior thrombosis, poor venesection tolerance, progressive splenomegaly, symptomatic thrombocytosis/leucocytosis |
| ET | Age >60 with JAK2 mutation, prior thrombosis, platelets >1500 (bleeding risk - acquired von Willebrand) |
- First line: hydroxyurea
- Interferon (peg-IFN alfa-2a, ropeginterferon alfa-2b) - preferred in younger patients and in pregnancy; can reduce JAK2 allele burden
- Anagrelide - ET second line (headache, palpitations, fluid retention; more arterial events and fibrotic progression than hydroxyurea in PT-1)
- Ruxolitinib (JAK1/2 inhibitor) - hydroxyurea-resistant/intolerant PV; splenomegaly and symptoms in myelofibrosis
- Reduces spleen volume and symptoms; does not reliably eradicate the clone
- Busulfan - elderly only (leukaemogenic)
Myelofibrosis
Myelofibrosis
- JAK inhibitors: ruxolitinib, fedratinib, pacritinib (usable with platelets <50), momelotinib (also improves anaemia - useful in the cytopenic phenotype)
- Allogeneic HSCT is the only curative option - for DIPSS intermediate-2/high risk and fit patients
- Anaemia: transfusion, danazol, luspatercept, erythropoiesis-stimulating agents
Thrombosis and surgery
- Full anticoagulation for an index thrombosis; indefinite in splanchnic vein thrombosis
- Optimise Hct and counts before elective surgery; VTE prophylaxis
- Check von Willebrand activity before procedures if platelets >1000 - extreme thrombocytosis causes acquired vWD and bleeding, and aspirin then becomes dangerous
Associations
- Polycythaemia vera, essential thrombocythaemia, primary myelofibrosis
- Splanchnic vein thrombosis - Budd-Chiari, portal and mesenteric vein thrombosis (JAK2 positive in ~25-45% of Budd-Chiari)
- Refractory anaemia with ring sideroblasts and thrombocytosis (MDS/MPN-RS-T - SF3B1 + JAK2)
- Clonal haematopoiesis of indeterminate potential
- Erythromelalgia; aquagenic pruritus (PV)
- Acquired von Willebrand syndrome at extreme platelet counts
- Pregnancy - inc miscarriage, placental insufficiency, thrombosis
- Post-PV and post-ET myelofibrosis; secondary AML
Survival
- PV median survival ~14-20 years; ET approaches that of the general population
- Thrombosis is the dominant cause of morbidity and death - arterial > venous
Transformation
| Myelofibrosis | AML | |
|---|---|---|
| PV | ~10-20% at 15-20 yr | ~5-10% at 20 yr |
| ET | ~5-10% at 15 yr | ~2-5% |
| PMF | - | ~10-20% |
- Post-MPN AML has a very poor prognosis - median survival <6 months, chemoresistant
- Myelofibrosis prognosis by DIPSS-plus / MIPSS70+: median survival <2 yr (high risk) to >15 yr (low risk)
- Adverse molecular markers: ASXL1, SRSF2, EZH2, IDH1/2, U2AF1 Q157, TP53
- Triple-negative ET/PMF and CALR type 1 differ markedly: CALR type 1 myelofibrosis has the best prognosis, triple-negative the worst
Monitor for
- Thrombosis and bleeding, progressive splenomegaly and symptoms (MPN-SAF TSS), falling Hb or rising blasts, secondary malignancy (non-melanoma skin cancer with hydroxyurea)
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