20.06.14 Genetic of AML Flashcards
What is Acute myeloid leukemia (AML)
A heterogenous disease resulting from the clonal expansion of meyloid progenitors (blasts) in peripheral blood (PB), bone marrow (bm) or other tissue.
How is a diagnosis of AML made
- 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
Incidence of AML
- Adult= 2.5 in 100,000. 25% acute leukemias
- Pediatric= 0.7 in 100,000.15-20% of acute leukemias
Median age of AML diagnosis
67yrs
Clinical symptoms of AML
- Fatigue, shortness of breath, easy bruising/bleeding, increased risk of infection, splenomegaly.
- Progresses rapidly and is fatal within weeks or months if left untreated.
What chromosomal abnormality involves RUNXIT1 and RUNX1
- t(8;21)(q22;q22)
- 5% AML.
- Often have secondary chromosomal abnormalities (70%)
What chromosomal abnormality causes acute promyelocytic leukemia (APL)
- 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
What chromosomal abnormality involves MLLT3 and KMT2A
- t(9;11)(p21.3;q23.3)
- 9-12% paed AML
What is a therapy-related myeloid neoplasm (t-MN)
- 10-20% AML
- In patients who have had alkylating agents, topoisomerase inhibitors and/or radiation
- 90% have cytogenetic abnormalities.
- Worse patient outcomes
Myeloid proliferations related to Down syndrome
- 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)
Best practice guidelines for AML (2012) state which tissue is preferred for testing
- Bone marrow
- PB can be used when circulating blasts are seen in pb film
Best practice guidelines for AML (2012) state how many abnormal metaphases needed for an abnormal karyotype
- 5
- Score another 5 for same or additional changes
Best practice guidelines for AML (2012) state how many abnormal metaphases needed for a normal karyotype
-20 metaphases (10 fully the others for obvious abnormalities)
What happens when karyotyping, if 1 potentially significant abnormality is identified
-Screen a further 10 cells or do FISH
When should FISH/ RT-PCR be considered
- When karyotyping fails (<10 analyzable cells)
- Where common additional abnormalities are found.
- Expected abnormality not found
What is the average number of driver mutations identified in AML cases
3 driver mutations
What is required for development of AML
- Collaboration of at least two classes of mutation.
- Mutations in the same category are mutually exclusive
What are the functional groups for recurrent AML mutations
- Signalling and kinase pathway
- Epigenetic modifiers (DNA methylation and chromatin modification)
- Nucleophosmin (NPM1)
- Transcription factors
- Tumour suppresors (TP53)
- Spliceosome complex
- Cohesin complex
What are class 1 mutations
- 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.
What are class 2 mutations
- 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)
Unclassified mutations
- 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
Are there germline predispositions to AML
- 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.
What diagnostic procedures are used in AML
- 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).
What are the benefits to analysing RNA for gene fusions.
- 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.
