Acute myeloid leukaemia

Question 1

How does the age of diagnosis differ between acute myeloid leukaemia and acute lymphoblastic leukaemia?

Answer 1

AML = across all ages but sees an increase in incidence as the population gets older
CML = high incidence in early age groups which goes down over time
essentially they are the opposite of each other

Question 2

How is acute leukaemia classified?

Answer 2

Malignant cells that have an undifferentiated phenotype
A disease that generally progresses quickly
Often bone marrow failure at presentation which is mainly due to clinical features

Question 3

What is the biggest single group of people that develop AML (that’s not due to chance)

Answer 3

People that have undergone cytotoxic agent therapy (chemo/radiotherapy)

Question 4

what types of chemo drugs give a predisposition to AML? Why?

Answer 4

1. Alkylating agents
2. Topoisomerase inhibitors
   both induce DNA damage, if in a bone marrow stem cell which survives, it may predispose to AML

Question 5

What are the biggest group of patients that develop secondary AML related to chemotherapy?

Answer 5

Women treated with chemo for breast cancer (usually given topoisomerases)

Question 6

What inherited conditions are associated with AML? Why?

Answer 6

Down syndrome, Fanconi anaemia, congenital neuropenia, Li-Fraumeni (TP53). Diseases that have abbarent DNA damage repair

Question 7

How is AML diagnosed and classified>

Answer 7

1. Morphology - microscopy of cells in the blood. Or if in the bone marrow perform an aspirate and trephine biopsy.
2. Immunophenotype by flow cytometry potentially with immunohistochemistry
3. Cytogenics - analysis of metaphases by banding by FISH
4. Molecular genetics

Question 8

How does flow cytometry work for AML diagnosis?

Answer 8

Take the BM sample or blood if there are sufficient blasts. The sample is incubated with antibodies to specific cell surface antigens. Each of these antibodies are conjugated to different fluorescent molecules. Then run through the FC, the sample is channelled through a flow chamber into a single cell suspension so a single cell passes through several lasers of different wavelengths. If the cell has bound the antibody then sensors will pick up that fluorescent emission. This produces a profile of the antigens being expressed by cells in the sample. If they are those that are specific to AML then AML…

Question 9

What are the typical antigens for AML diagnosis?

Answer 9

high CD34 and CD117 (both are hematopoietic stem/progenitor cell markers)
CD13 and CD33 which are generally only expressed on myeloid cells.

Question 10

what is G banding?

Answer 10

a technique used in cytogenetics to produce a visible karyotype by staining condensed chromosomes that are in metaphase. This can be used to identify if any cytogenic abnormalities have developed which is often the case in AML.

Question 11

What is FISH

Answer 11

Fluorescent in situ hybridisation. Again this is to find any cytogenic abnormalities for diagnosis as well as holding prognostic value.

Question 12

What mutational analysis is undertaken in the diagnosis of AML and why?

Answer 12

Standard gene panel looks for mutations in FLT3 (around 25%), NPM1 (around 30%), CEBPA (around 10%)
also a 52 gene NGS panel mainly looking at ASXL1 (prognostic), TP53 (poor predictive chemo response) and RUNX1 (prognostic)

Question 13

What are the consequences of being neutropenic for sustained periods of time?

Answer 13

For 1-3 weeks, you pick up a fungal infection, generally these are found in the lungs of AML patients. Also can get fungal infections in the liver, spleen and brain.

Question 14

How is AML treated?

Answer 14

1. Remission induction by cytotoxic chemotherapy

2. Further therapy which is further chemotherapy with potentially an allogeneic bone marrow transplant.

Question 15

What are drugs used commonly in AML

Answer 15

1. Anthracyclines - topoisomerase II inhibitors
2. Cytarabine - inhibits DNA replication in S phase
3. Etoposide - Topoisomerase II inhibitor
4. Gemtuzumab - moAb targeting CD33 coupled to the cytotoxic agent caliceamicin

Question 16

What are the benefits of using allogeneic BMT in leukaemia?

Answer 16

1. Allows you to give high dose chemotherapy as you aren’t concerned with killing the normal stem cells.
2. reconstitution with donor lymphoid system. Donor T-cells recognise residual leukaemia cells left in the patient. More potent in some patients than others as well as in different diseases.

Question 17

What are the techniques used to assess minimal residual disease in AML?

Answer 17

1. qRT-PCR for fusion gene transcripts eg RUNX1-RUNX1T1
2. qRT-PCR for mutant gene products eg NPM1
3. Flow cytometry for leukaemia-specific immunophenotypes.

Question 18

Why is an RNA based detection technique better than a DNA based technique when assessing minimal residual disease?

Answer 18

Because we want to detect viable cells. Dead/dying cells will still have DNA that can be detected. This is not the case in mRNA based techniques.

Question 19

How is qRT-PCR used for the assessment of minimal residual disease?

Answer 19

reverse transcription of mRNA into cDNA then perform PCR. Use a fluorescent probe that fluoresces proportionately to the amount of PCR product that you have. After multiple cycles, the fluorescence will pass beyond the background fluorescence giving a Cq value. The number of cycles it takes to produce detectable fluorescence (Cq) can be used to quantify how many copies of mRNA were in the original sample.

Question 20

How is flow cytometry used for MRD detection? Patient example

Answer 20

leukaemic cells expressing both CD34 and CD117. At diagnosis they are also analysed with T cell markers CD3 and CD7 (neither of which should be detected on myeloid blasts)
There is inappropriate expression of CD7 on myeloid blasts which makes it possible to distinguish them from normal T cells. Get an overall % of the number of bone marrow cells that are the CD7 positive myeloid blasts.
After one round of chemo this % goes down.
After the second cycle we would be looking for disease to be undetectable but sometimes a small % is still present

Question 21

What factors are shown to influence outcome in AML?

Answer 21

Age, performance status, white cell count (higher = worse), secondary AML (previous chemo or another malignancy, previous MDS or MPN)
cytogenetics (varies)
response to initial chemotherapy
Mutations in several genes

Question 22

What is the difference in survival when comparing FLT3 and NPM1 mutations?

Answer 22

When FLT3 is WT and NPM1 is mutant there is a much greater survival compared to those that are mutant FLT3 (internal tandem duplication) and WT NPM1

Question 23

Describe clonal heterogeneity of AML cells

Answer 23

Firstly there is a pre-leukaemic initiating mutation which is followed by clonal expansion of these HSCs. Then further hits occur to develop AML with subclones within that. When treated, a certain clone that offers survival benefit for the tumour becomes selected and drives relapse. \