Myeloproliferative Disorders

Question 1

What are the WHO criteria for the accelerated phase of CML?

Answer 1

1) Blasts 10- 19% in the peripheral blood or bone marrow
2) Peripheral blood basophils >20%
3) Persistent leukocytosis, >10, despite treatment
4) Persistent thrombocytosis, >1000, unresponsive to therapy
5) Persistent thrombocytopenia, <100, unrelated to therapy
6) Persistent splenomegaly despite therapy
7) Presence of additional chromosomal abnormalities (both at diagnosis and acquired during therapy (clonal evolution)

Question 2

What are the WHO criteria for blast phase of CML?

Answer 2

1) Blasts ≥20% in blood or bone marrow

2) Presence of extramedullary proliferation of blasts

Question 3

What parameters are used to calculate the sokal and Eutos scores?

Answer 3

1) Sokal: age, spleen size, platelet count, blasts

2) Eutos: spleen size, basophil count

Question 4

What is the Philadelphia chromosome?

Answer 4

Defective and abnormally short chromosome 22 caused by a reciprocal translocation between chromosomes 9 and 22.
Results in the generation of a fusion protein/ tyrosine kinase BCR-ABL

Question 5

What are the most common genetic aberrations seen in CML, other than the Philadelphia chromosome?

Answer 5

Major route cytogenetics:

• Duplication of the Ph chromosome (amplification of BCR-ABL)
• +8, +19, iso17(q)
• Indicate accelerated phase of disease, poor prognosis

Question 6

What different BCR-ABL breakpoints can be seen?

Answer 6

• P190 breakpoint e1a2: most common in ALL
• P210 breakpoint e13a2 (prev b2a2) and e14a2 (previously b3a2): most common in CML
• P230 and other rare variants also exist

Question 7

What are the different response criteria to TKIs used for CML disease monitoring?

Answer 7

1) Complete haematological response
- Normal FBC, bone marrow morphology and spleen size
- Aim to achieve by 3 months of treatment
2) CCyR
- Absence of Ph+ metaphases on conventional cytogenetics
- Performed on a BM sample.
3) Using qPCR/ IS standardised baseline
- Log 1= 10% IS
- Log 2= 1% IS (equivalent to a CCyR)
- Log 3= 0.1% IS= MMR
- Log 4= 0.01% IS= MR4
- Log 4.5= 0.0032% IS= MR4.5, sometimes called a complete metabolic response
- Log 5= 0.001% IS= MR5

Question 8

What are the targets in the treatment of CML with TKIs

Answer 8

1) Complete haematological response/ BCR-ABL <10% IS at 3 months
2) CCyR/ BCR-ABL 1% IS at 6 months
3) MMR at 12 months

Question 9

What is the definition of treatment failure in CML?

Answer 9

· At 3 months: Ph+ >95% and/or no CHR
· At 6 months: Ph+ >35% and/ or BCR-ABL >10%
· At 12 months: BCR-ABL >1% and or Ph+ >0%

Anytime: loss of CHR, loss of CCyR, loss of MMR, CCA in Ph+ cells

Question 10

What mutation in CML confers resistance to all TKIs?

Answer 10

T315I (at the TKI binding site)

Question 11

How is erythropoietin measured?

Answer 11

• ELISA, enzyme immunoassay and radioimmune kits available

- Use a pure form of human erythropoietin from recombinant DNA

Question 12

What are the differences between essential thrombocytosis and pre-fibrotic myelofibrosis?

Answer 12

Bone marrow cellularity
- Normal in ET, increased in pre-fibrotic MF
M:E
- Normal in ET, increased in pre-fibrotic MF
MK tight clusters
- Rare in ET, common in pre-fibrotic MF
MK size
- Normal- large- giant in ET, variable in pre-fibrotic
MF
MK nuclei
- Hyperlobulated in ET, variable in pre-fibrotic MF:
bulbous, hypolobulated, high N:C ratio, condensed
chromatin, “cloud-like” in pre-fibrotic MF
Reticulin grade
- 0-1 in both ET and pre-fibrotic MF

Question 13

What is the definition of a small and large cluster in pre-fibrotic MF and PMF

Answer 13

• Small cluster= >3 cells

- Large cluster= >7 cells

Question 14

At what proportions are JAK2, CALR and MPL mutations seen in ET and PMF?

Answer 14

• JAK2 in 50-60%
• CALR in 25-30%
• MPL in 5%

Question 15

What are the two different types of CALR mutations? What is the prognostic implications of CALR mutations in ET and PMF

Answer 15

Type 1 CALR= Deletional. Most common.
Type 2 CALR= Insertional.

Type 1 CALR: more indolent course in PMF. Increased risk of transformation to MF in ET.

Question 16

List the variables present in the MIPSS70 score for PMF

Answer 16

Clinical:
1) Constitutional symptoms

FBC:

1) Hb <100
2) Plts <100
3) WCC >25
4) Circulating blasts ≥2%

BM:
1) Reticulin fibrosis grade ≥2

Molecular:

1) Absence of a CALR mutation
2) Presence of a HMR
- ASXL1, EZH2, SRSF2, IDH1/2
3) Presence of ≥2 HMRs

Question 17

What are the 5 HMRs in PMF?

Answer 17

ASXL1, EZH2, SRSF2, IDH1/2

Question 18

Acquisition of which three mutations are associated with the blast phase of MF?

Answer 18

RUNX1, TP53 and ETV6 are rarely seen in the chronic phase of MF but become increasingly common in the accelerated and blast phase.

Question 19

What mutations are commonly seen in ET and PCV

Answer 19

• JAK2, CALR and MPL= most common driver mutations
• Mutational burden: PMF»PCV>ET
• More mutations= higher risk of disease progression
• Mutations in epigenetic regulators most common: DNMT3A, TET2, ASXL1 and IDH1/2
• Mutations in TP53 rare

(DTA + I)

Question 20

Is JAK2 VAF important?

Answer 20

Can help with determining the diagnosis:

• Homozygous mutations associated with PCV over ET
• Higher VAF seen in pre-fibrotic MF (VAF >50%) vs ET

Can predict phenotype in PCV
- Higher VAF= increased Hct, WCC and splenomegaly

Can predict outcomes

• Increased risk of fibrotic transformation in ET and PV with increased VAF/ homozygous mutation
• Increased risk of thrombrosis in ET and PCV (when VAF >75% in PCV)
• Increased risk of leukaemic transformation with low VAF in PMF (esp if high TP53 VAF)
• Inferior overall survival with low VAF in PMF (esp if high TP53 VAF)

Question 21

Discuss chromatin/ spliceosome mutations in MPN

Answer 21

• Poorer prognosis when present (increased MF transformation in ET and PCV, reduced EFS, reduced OS)
• Enriched in MF and MPN/ MDS.
• More common in those with JAK2 and MPL mutations than in those with CALR

Question 22

What are the diagnostic criteria for chronic neutrophilic leukaemia?

Answer 22

Peripheral blood:
· WCC >25 with persistent neutrophilia for ≥3 months
· Segmented and band neutrophils ≥80% of WCC
· Neutrophil precursors <10% of the WCC
· Monocyte count <1
· Myeloblasts rarely observed
· No dysgranulopoiesis

Bone marrow:
· Hypercellular bone marrow
· Increased neutrophil granulocytes (M:E >20:1)
· Normal neutrophil maturation
· Myeloblasts <5%

Molecular:
· No PDGFRA, PDGFRB, FGFR1 or PCM1-JAK2
· CSF3R or another activating CSFR mutation

Question 23

What are the diagnostic criteria for chronic eosinophilic leukaemia?

Answer 23

• Eosinophilia ≥1.5 for >6 months.
• No PDGFRA, PDGFRB, FGFR1, PCM1-JAK2, ETV6-JAK2 or BCR-JAK2 present.
• Blast cells <20% and no recurrent cytogenetic abnormalities diagnostic of AML.
• Clonal cytogenetic abnormality OR blast cells ≥2% in blood or ≥5% in the bone marrow.

Question 24

What is the LIS?

What are the important aspects of LIS functionality for a Haem lab?

Answer 24

• Test ordering (can it incorporate questionnaires/ prompts about appropriateness: i.e. requests for thrombophilia tests, flags that the test has been ordered previously (molecular tests such as JAK2)
• Interface with analyser, middleware and automation: rules for slides to be made, interface with cellavision
• Result entry and validation: critical limits, reflex testing, detection of errors such as clotted sample, expert systems for automatic comments
• Result notification: automatic notification to the cancer registry when a new malignant diagnosis is confirmed
• Data mining: ease of assessing TAT for critical tests such as molecular tests