20.06.15 Genetic of CML

Question 1

What is CML

Answer 1

-Chronic myeloid leukemia -A myeloproliferative neoplasm originating from pluripotent bone marrow stem cells. -15% of adult leukemias. -Median age of onset is in 50s -No clear heritability, exposure to radiation increases incidence.

Question 2

Symptoms of CML

Answer 2

-Fatigue joint pain, fever, splenomegaly, weight loss and loss of appetite. -Gradually worsen to include recurrent infections, easy bruising, swellings in neck, armpit and groin. -FBC shows increased granulocytes and immature myeloblast cells.

Question 3

Incidence of CML

Answer 3

1-2 per 100,000

Question 4

What are the phases of CML (if untreated)

Answer 4

1. Chronic phase: where 90% are diagnosed, usually by blood test. Slow development of symptoms. Mean length is 3-4 years. Good response to TKI therapies (imatinib) 2. Accelerated phase. As mutant cells persist in bone marrow, they eventually accumulate further mutations and disease enters accelerated phase. Cell proliferation increases and symptoms become more severe. Lasts from months to a year. 3. Blast crisis. >20% of blast (immature) cells in blood or bone marrow. Cells infiltrate other tissues (skin and lymph nodes) . Hard to treat, so poor patient outcome.

Question 5

What is the common reciprocal translocation see in CML

Answer 5

• Philadelphia (Ph) chromosome
• t(9;22)(q34;q11) (BCR-ABL1)
• 95% CML patients

Question 6

Review of Philadelphia chromosome

Answer 6

• t(9;22)(q34;q11) (BCR-ABL1)
• 5’ end of BCR (break point cluster region) from chr 22 is fused with 3’end of ABL1 (Abelson murine leukemia viral oncogene homolog 1)
• Causes increased tyrosine kinase activity, leading to uncontrolled cellular proliferation and inhibition of apoptosis.

Question 7

How many common products are derived from Ph translocation

Answer 7

-3 -M-bcr (major break point cluster) in exons 12-16 of BCR. -Usually fuse to exon 2 of ABL1 (exon 1 a and b are usually splice out). -Resultant protein is 210kDa fusion protein (p210Bcr-Abl)

Question 8

Methods to diagnose CML

Answer 8

• Presence of BRC-ABL1 fusion is diagnostic for CML
• Chromosome analysis detects Ph in 95%. Can also detect atypical translocations and other secondary chromosomal abnormalities (indicative of blast/ accelerated phase).
• FISH using a BCR-ABL1 dual probe.
• Real-time PCR. Measures level of BCR-ABL transcript (RNA isolated from BM or PB). Break points need to be determined first. Levels can be compared against subsequent samples.

Question 9

What is minimal residual disease (MRD)

Answer 9

When small numbers of leukaemic cells remain in the person during treatment, or after treatment

Question 10

Why is it important to monitor MRD

Answer 10

-Determine effectiveness of treatment, below phenotypic level. -Taken every 3-6 months after diagnosis. -qPCR on blood cells to get % BCR-ABL (not available for rare transcripts)

Question 11

What is the Haematological response (HR)

Answer 11

-Presence of leukemic cells visible in bone marrow aspirate. -Complete haematological response (CHR)= WBC and platelet counts normalise and splenomegaly is reversed -Sensitivity= 5%.

Question 12

What is early molecular response (EMR)

Answer 12

amount of BCR-ABL gene in your blood is 10% (or 1 fold less) of what’s expected in someone with untreated CML. Done by qPCR

Question 13

What is the cytogenetic response (CyR)

Answer 13

• Number of cells with Ph chromosome (as a percentage)
• G-banding on metaphase chrom= sensitivity of 1-5%
• FISH= sensitivity of 0.1-1%
• Complete CyR= no Ph+ chr detectable using cytogenetic methods.

Question 14

What is major molecular response (MMR)

Answer 14

-amount of BCR-ABL gene in your blood is 0.1% (or 3 fold less) of what’s expected in someone with untreated CML using molecular techniques -Predicts ~100% survival, disease progression is uncommon once this level is achieved.

Question 15

How often are patients tested after MMR is achieved

Answer 15

every 3 months to assess eligibility for treatment discontinuation

Question 16

How is qPCR data normalised

Answer 16

-BRC-ABL: ABL1 ratio -ABL1 copy numbers vary considerably on sample age and size, level of disease burden. Therefore, ABL1 copy numbers indicate RNA quantity and quality in the test.

Question 17

When is ABL1 sequencing recommended

Answer 17

If mile stones are not reached or secondary resistance arises.

Question 18

What are the two types of resistance to TKI treatment

Answer 18

• Primary: Initial failure to achieve a haematological response.
• Secondary: Initially respond to treatment but then have subsequent loss of response

Question 19

Why does resistance increase in later phases of disease

Answer 19

• Accumulation of further ABL1 mutations.
• Also due to increased expression of PTGS1 (metabolises imatinib), low levels of OCT1 (drug intake protein).

Question 20

TKI used for treatment of CML

Answer 20

-1st generation TKI= Imatinib -2nd generation= dasatinib and nilotinib (affected by fewer ABL1 mutations so can overcome imatinib resistance) sensitive to T315I mutation -3rd generation= ponatinib

Question 21

Review of imatinib

Answer 21

-Tyrosine kinase inhibitor. Competes with ATP for binding with ABL1. Without ATP, ABL1 can’t phosphorylate downstream targets. Cells then undergo apoptosis. -First line treatment for CML in chronic phase. -Less effective in blast/accelerated phases. -If no response to imatinib or milestones are lost, treatment is switched to different TKIs.

Question 22

Review of second generation TKIs

Answer 22

-Created after BCR-ABL crystal structure determined. Drugs are more specific and potent. 1. Dasatinib: binds activate conformation of ABL1 kinase, so can inhibit all BCR-ABL kinase domain mutants (except p.T315I) 2. Nilotinib: Binds inactive protein with improved specificity.

Question 23

What is the only cure for CML

Answer 23

-Allogenic haematopoietic stem cell transplantation. -Bone marrow transplant from healthy donor -Offered if patients are unrepsonvie to TKIs or have entered accelerated/ blast phases. -Difficulties: finding matched donors, all Ph+ cells must be removed prior to transplant otherwise relapse is possible.