20.06.14 Genetic of AML

Question 1

What is Acute myeloid leukemia (AML)

Answer 1

A heterogenous disease resulting from the clonal expansion of meyloid progenitors (blasts) in peripheral blood (PB), bone marrow (bm) or other tissue.

Question 2

How is a diagnosis of AML made

Answer 2

• At least 20% blasts are present in PB or bm,
• If <20% then also if a myeloid sarcoma is present, an AML-related chromosomal abnormality is detected, or erythroid leukemia is present

Question 3

Incidence of AML

Answer 3

• Adult= 2.5 in 100,000. 25% acute leukemias

- Pediatric= 0.7 in 100,000.15-20% of acute leukemias

Question 4

Median age of AML diagnosis

Answer 4

67yrs

Question 5

Clinical symptoms of AML

Answer 5

• Fatigue, shortness of breath, easy bruising/bleeding, increased risk of infection, splenomegaly.
• Progresses rapidly and is fatal within weeks or months if left untreated.

Question 6

What chromosomal abnormality involves RUNXIT1 and RUNX1

Answer 6

• t(8;21)(q22;q22)
• 5% AML.
• Often have secondary chromosomal abnormalities (70%)

Question 7

What chromosomal abnormality causes acute promyelocytic leukemia (APL)

Answer 7

• t(15;17)(q24.1;q21.2)
• PML-RARA genes.
• In 5-8% of AML.
• Detected by FISH or RT-PCR
• Characterised by excess promyelocyte accumulation in bone marrow

Question 8

What chromosomal abnormality involves MLLT3 and KMT2A

Answer 8

• t(9;11)(p21.3;q23.3)

- 9-12% paed AML

Question 9

What is a therapy-related myeloid neoplasm (t-MN)

Answer 9

• 10-20% AML
• In patients who have had alkylating agents, topoisomerase inhibitors and/or radiation
• 90% have cytogenetic abnormalities.
• Worse patient outcomes

Question 10

Myeloid proliferations related to Down syndrome

Answer 10

• Transient abnormal myelopoiesis. In 10% DS newborns. In 20-30% it becomes non-transient AML
• Myeloid leukemia associated with DS: Children with DS are 50 fold increased risk of developing AML in first 5 years.
• Due to mutations in GATA1 (on X chromosome)

Question 11

Best practice guidelines for AML (2012) state which tissue is preferred for testing

Answer 11

• Bone marrow

- PB can be used when circulating blasts are seen in pb film

Question 12

Best practice guidelines for AML (2012) state how many abnormal metaphases needed for an abnormal karyotype

Answer 12

• 5

- Score another 5 for same or additional changes

Question 13

Best practice guidelines for AML (2012) state how many abnormal metaphases needed for a normal karyotype

Answer 13

-20 metaphases (10 fully the others for obvious abnormalities)

Question 14

What happens when karyotyping, if 1 potentially significant abnormality is identified

Answer 14

-Screen a further 10 cells or do FISH

Question 15

When should FISH/ RT-PCR be considered

Answer 15

• When karyotyping fails (<10 analyzable cells)
• Where common additional abnormalities are found.
• Expected abnormality not found

Question 16

What is the average number of driver mutations identified in AML cases

Answer 16

3 driver mutations

Question 17

What is required for development of AML

Answer 17

• Collaboration of at least two classes of mutation.

- Mutations in the same category are mutually exclusive

Question 18

What are the functional groups for recurrent AML mutations

Answer 18

• Signalling and kinase pathway
• Epigenetic modifiers (DNA methylation and chromatin modification)
• Nucleophosmin (NPM1)
• Transcription factors
• Tumour suppresors (TP53)
• Spliceosome complex
• Cohesin complex

Question 19

What are class 1 mutations

Answer 19

• Mutations that activate signal transduction pathways and confer proliferative advantage on haematopoietic cells.
• Present in 2/3 of AML patients
• Mutations in this class are frequently identified in subclonal cellular fractions

e. g.
- FLT3 gene: receptor tyrosine kinase expressed on hematopoietic progenitors. Mutated in 1/3 of AML cases. Lead to constitutive signalling.
- PTPN11: cytoplasmic tyrosine phosphatase. Highly expressed in haemopoietic cells. Gain of function mutations.
- cKIT: protooncogene. Tyrosine kinase. Gain of function mutations.

Question 20

What are class 2 mutations

Answer 20

• Affect transcription factors and primarily serve to block haemopoietic differentiation.
• Mutated in 20-25% of adult AML cases.

e. g.
- Mutations in CBF (cor binding factor)- heterodimeric transcription factor. Comprised of RUNX1 and CBFB. Essential for haematopoietic development.
- CEBPA: transcription factor involved in myelopoiesis.
- KMT2A: histone methytransferase (silences tumour suppressor genes)

Question 21

Unclassified mutations

Answer 21

• NPM1: 1/3 of AML cases. Nucleophosmin, a chaperone. Mutations lead to delocalisation to the cytoplasm.
• WT1. Transcription factors. Mutated in 10% of AML
• DNMT3A. Methyltransferasae. LOF mutations in 14-18% of AML
• Acquired UPD: renders cells homozygous for an existing mutation

Question 22

Are there germline predispositions to AML

Answer 22

• Generally thought to be somatic events
• RUNX1 mutations cause familial platelet disorder with propensity to AML. Patients don’t develop leukemia until later in life, when they acquire a chromosomal abnormality.
• Often a germ line sample is not taken so sequencing cannot distinguish between germline and somatic variants.

Question 23

What diagnostic procedures are used in AML

Answer 23

• Morphology using microscopic blood film (Wright-Giemsa stain). AML= a marrow or blood blast count of >20% (AML regardless of blast count if recurrent genetic abnormality detected)
• Immunophenotyping using flow cytometry to determine lineage (examines cell surface expression of various markers)
• Cytogenetics. Chromosomal abnormalities in 55% of adult AML
• Molecular genetics. Sequencing of RUNX1, NPM1 and CEBPA (define disease categories), FLT3 (determine efficacy of TKIs) or TP53 and ASXL (associated with poor prognosis).

Question 24

What are the benefits to analysing RNA for gene fusions.

Answer 24

• Gene fusions are too large to detect by PCR of DNA due to introns.
• Often multiple gene fusions due to alternative splicing
• Detects transcription of gene fusions not just their presence.
• Can detect cryptic fusions that are not present or where karyotyping fails.

Question 25

What is minimal residual disease monitoring

Answer 25

• Molecular based monitoring of blood/bone marrow samples received at regular intervals following diagnosis.
• Relative level of mutation in comparison to a housekeeping gene (ABL), done by qPCR.
• Need to know specific variant or breakpoint
• Certain mutations cause faster doubling time of mutated clones so intervals between blood sampling may vary.
• Flow cytometry can be used for MRD monitoring, less sensitive.

Question 26

What molecular-based therapies exist for AML

Answer 26

• ATRA (all-trans-retinoic acid) therapy in APL (Acute Promyelocytic Leukemia)
• Inhibitors of FLT3 (receptor tyrosine kinase). Often a transient effect, used in combination with conventional chemotherapy.
• AML due to multiple genetic/epigenetic lesions, so therapies that target a single aberrant protein are unlikely to eradicate all leukemic clones. Combination therapy required.

Question 27

Future of AML testing

Answer 27

• Gene expression profiling: subcategorise leukemias, prognostic stratification based on expression profiles.
• MicroRNA analysis: using microarrays to demonstrate expression of certain miRNAs. Could allow prognostic stratification
• Epigenetic profiling: genome wide screening for methylated CpG islands (significant increase in methylation at relapse). Hypomethylating agents at remission could prevent emergence of hypermethylated clones.