Chronic Myeloid Leukaemia

Question 1

Genetics and pathogenesis

Answer 1

Clonal disorder of pleuripotent stem cells

TRANSLOCATION 9:22 (long arms)
==> oncogene ABL1 moves to BCR gene on chr 22
==> Ph chromosome BCR-ABL1 – codes for fusion protein with increased tyrosine kinase activity and confers proliferative and anti-apoptotic effects

Question 2

Natural history

Answer 2

Chronic phase
Accelerated phase
Blast phase

(over a period of 5-10 yrs if untreated)

Question 3

Epidemiology and aetiology

Answer 3

Slight male predominance, possible at all ages but commonly 40-60 yrs old and rare in children
400-500 new cases/yr

Higher incidence with heavy radiation exposure

Question 4

Chronic phase - symptoms, CBC, survival

Answer 4

90% of patients present in this phase
Up to 50% no physical complaints and discovered incidentally
Typically remains stable for 3-4 yrs; 5-7 yrs survival

Symptoms due to hypercatabolic effects, fatigue, weight loss, nocturnal sweats (anaemia)

SPLENOMEGALY ALWAYS PRESENT - can be massive and cause LUQ pain

Hyperleucocytosis of relatively mature cells = no clear symptoms

Plt may be increased

Question 5

Accelerated phase - pathogenesis, survival

Answer 5

Acquisition of additional molecular lesions, genomic instability and progressive impairment of differentiation

Accumulation of immature precursors and undifferentiated blasts in BM, blood and extramedullary tissue

Median survival (w/o treatment) = 12-18 months

Question 6

Blast phase - features, survival

Answer 6

Progression of CML to acute leukaemia

Blasts >20%!
- >50% myeloid, up to 1/3 lymphoid

Additional cytogenetic aberrations in 60-80% cases

Median survival: 3-8 months

Question 7

Lab features - peripheral blood, BM and molecular

Answer 7

Peripheral blood:

• LEUCOCYTOSIS (may reach >200; marked increase in buffy coat)
• Complete spectrum of myeloid cells (including precursors)
• Increased circulating basophils
• Plt variable (most frequently increased)

BM:

• Hypercellular
• Granulopoietic dominance
• Megakaryocyte increased and hypolobated

RT-PCR analysis: BCR-ABL1 gene fusion
Cytogenetic: Ph chromosome

(serum uric acid raised due to increased cell turnover)

Question 8

Diagnosis

Answer 8

Demonstration of Ph chromosome

• cytogenetic analysis
• FISH
• RT-PCR for BCR-ABL1 fusion gene

Cytogenetics and FISH performed together

• karyotyping: identify Ph (95% cases), look for further chromosomal abnormalities (clonal evolution)
• FISH: variant translocation, 5% cases that aren’t detected by karyotyping, abnormal chromosomal deletion

Question 9

FISH method and results

Answer 9

Dual colour dual fusion probe

• 2 fusion signals and 1R and 1G
• der(22) BCR-ABL1 and der (9) ABL1-BCR

Question 10

Molecular genetics method - qualitative vs quantitative, gel electrophoresis results

Answer 10

Qualitative RT-PCR and quantitative real-time PCR to check presence of transcript and tumour load

Gel multiplex results
- e14a2 and e13a2 as the most common gene transcripts

Question 11

Treatment of CML - chronic and accelerated phase (mechanism, examples)

Answer 11

Tyrosine kinase inhibitors as standard
Bind close to ATP binding site of BCR-ABL1 –> inhibit enzyme activity of the protein

=> prevent phosphorylation of substrate and prevent substrate binding to decrease proliferative and anti-apoptotic features of protein

Imatinib (1st gen)
Nilotinib or Dasatinib (2nd gen)
Ponatinib (3rd gen; T315I mutation tumours)

(oral cytotoxics can be used before diagnosis to reduce leucocytosis)

Question 12

Treatment of CML - blast phase

Answer 12

TKI alone or with chemotherapy

Followed by allogeneic SCT

Question 13

Side effects of TKI

Answer 13

Imatinib: myelosuppression, fluid retention

Nilotinib: myelosuppression, prolonged QT interval (risk of arrhythmia)
Dasatinib: prolonged QT, pleural effusion

Question 14

Monitoring of treatment response - possible methods and their adv/disadv

Answer 14

CBC - WBC returns to normal but not sensitive enough

Cytogenetics and FISH

• cytogenetics: Ph chr, low sensitivity, labour intensive
• need BM examinations every 3 months which is inconvenient
• *Real-time PCR for BCR-ABL1 transcripts
• mainstay for monitoring
• much more accurate and sensitive
• report as ratio of transcripts to control gene transcript numbers (use international scale - IS for comparibility)

Question 15

Quantitative PCR monitoring

Answer 15

Residual tumour burden described as log10 reduction from standardised baseline

• <10% = 1log reduction
• <1% = 2log reduction = complete cytogenetic remission (CCyR)
• <0.1% = 3log reduction = major molecular response
• <0.01% (MR4)
• 0.0032% (MR4.5)
• 0.001% (MR5)

To ensure quality of the sample:
For MR4 and MR4.5 to be valid, need to analyse >32,000 copies
MR5 need >100,000 copies

Question 16

Classifying treatment response in CML at different time points

Answer 16

Optimal, warning (increase monitoring and may change dose/drug) or failure (change TKI or give SCT)

3 months: <10% = optimal; >10% = warning or failure

6 months: <1% = optimal; 1-10% = warning; >10% = failure

12 months: <0.1% = optimal; 0.1-1% = warning; >1% = failure

Anytime:

• loss of major molecular response (<0.1%) = warning
• resistance mutations, high risk chr abnormalities e.g. chr17 deletion = failure

Question 17

Causes of treatment failure and management

Answer 17

10-20% with treatment resistance

30-50% of these due to kinase domain mutation

• Point mutations resulting in decreased binding affinity of TKI to protein
• T315I mutation resistant to all except Ponatinib

Other causes of resistance: compliance, absorption and metabolism

Question 18

Treatment discontinuation

Answer 18

If:
- major molecular response with undetectable transcripts after >2 yrs of TKI treatment

Stable treatment free remission was 50% at 2 yrs and all patients sensitive to TKI if relapse

Stopping TKI is feasible and some patients may be cured

Overall prognosis very good (90% have long term control)