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Cytogenetics > AML > Flashcards

AML Flashcards

1
Q

What is AML?

A

Disease which results from the clonal expansion of myeloid blasts in the blood, marrow or other tissue.

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2
Q

What is the key thing that happens during haematopoeisis in an acute leukemia?

A

Maturation block - immature cells overwhelm marrow that can’t function

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3
Q

What does the WHO classification do?

A

Incorporates morphological, immunophenotypic, genetic and clinical features into classification of disease.

Using this allows the clinician to give the patient info on their subtype such as prognosis and predict how they will respond to treatment.

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4
Q

According to BPG, what percentage of adult AMLs and paediatric AMLs show a karyotypic abnormality?

A

~55% of adult (approx half)

~78% of paediatric (majority)

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5
Q

What sample type is appropriate for investigation of AML?

A

Bone marrow preferable.

Blood sample can be used as long as there are circulating blasts.

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6
Q

Are there any mandatory FISH tests according to BPG?

A

KMT2A must be carried out if case is either biphenotypic leuk or infant leuk.

CBFB-MYH11 must be carried out if either morphology suggests inv(16) or a suspicious secondary abn is seen such +22 or del 9q.

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7
Q

According to BPG how many cells should we look at in an AML referral?

A

Abnormal - minimum 5 analysed and 5 scored

Normal - 20 cells, 10 analysed and 10 scored

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8
Q

What is the minimum number of cells that need to be looked at in a normal case? What must go on the report?

A

A minimum of 10 cells to report the case but this must be accompanied by a caveat on the report.

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9
Q

What is the minimum number of cells that can be used to report an abnormal?
What else should we try to do in this situation?

A

Minimum of 2 cells with a gain/structural abnormality.
Minimum of 3 cells with a chromosome loss.

We should try to confirm any abnormalities using FISH wherever possible.

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10
Q

What is the average age of adult AML at diagnosis?

What is the typical age of a child with AML?

A

~65 yrs

Peaks at 1 yrs with a median age on onset of 2 yrs.

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11
Q

What should be considered in failed AMLs?

A

FISH for specific rearrangements, especially if an abnormality which has an association with a diagnostic rearrangement is spotted e.g. a +22 might suggest inv(16)

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12
Q

If a previously abnormal AML sample arrives for testing what can we offer?

A

We can either screen 30 cells for the previous abnormalities or FISH 100 if more appropriate.

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13
Q

If a previously normal karyotype AML comes in for follow-up testing, what can we offer?
Are there any exceptions?

A

We would just culture and store the sample. Karyotyping prev normal AMLs is rarely of value (BPG).
Exception would be if the patient was ?relapse or the HODS info suggests relapse.

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14
Q

What are therapy related AMLs associated with?

A

Treatment with alkylating agents/irradiation (5s and 7s)

Treatment with topoisomerase II (KMT2A)

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15
Q

What percentage of blasts are required for a diagnosis of AML?
Are there any exceptions?

A

20% blasts

Yes, such as if an AML specific abnormality is detected such as a t(8;21), t(15;17), inv(16)/t(16;16) is detected

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16
Q

What are the main WHO sub-category’s for AML?

A 
AML with recurrent genetic abnormalities
AML with MDS related changes
Therapy related myeloid neoplasms
AML, NOS
Myeloid proliferation’s related to DS
Myeloid Sarcoma
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17
Q

If you find a single cell with an abnormality, would you report it?

A

Depends if clinically significant and/or matches the reason for referral!

  • could confirm with FISH
  • if no more material remaining then we could keep topline as FAIL but put a comment /rider on the report saying a single cell was seen with an abnormality but we were unable to confirm this due to insufficient material.
  • example would be a t(9;22) rearrangement
  • could also speak to clinician and ask if they think it would fit? E.g. does the patient have CML
  • check HODS.
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18
Q

What is BPG for processing/analysing AML’s Vs our local policy?

A

BPG:

  • karyotype 20 mets and in poor cases FISH 16s
  • infant AML or biphenotypic AML FISH for KMT2A rearrangement

?Local policy:

  • new AMLs get full FISH panel if requested?
  • rest get karyotype and all normals get FISH for KMT2A and CBFB/MYH11
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19
Q

When reporting an abnormal AML what must be included on the report?

A
  • comment linking it to appropriate WHO subgroup of AML and use exact wording e.g. AML with PML-RARA
  • comment on risk group according to ?Grimwade et al for 16-59yrs or Harrison et al for children.
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20
Q

What symptoms might you see in an AML patient and why?

A

Infections - white cells responsible for immune reaction might be compromised, immature cells can’t do their job properly.

Splenomegaly - because the spleen is involved in removing abnormal cells or may be infiltrated by the circulating blasts

Fatigue - red blood cells carry oxygen and if the numbers drop as a knock on effect then patient will feel tired. Also energy used by body to fight the disease may be a factor.

Bruising and bleeding - thrombocytes may be involved or just lowered so the body can’t deal with bleeding as effectively e.g. patient might have bleeding gums.

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21
Q

List some of the recurrent cytogenetic rearrangements that are seen in AML? What genes are involved in them?

A

t(15;17)(q24;q21) involving PML-RARA
t(8;21)(q22;q22) involving RUNX1-RUNX1T1
inv(16)(p13.1q22) or t(16;16)(p13.1;q22) involving CBFB-MYH11
t(9;11)(p21.3;q23.3) involving MLLT3-KMT2A
t(6;9)(p23;q34.1) involving DEK/NUP214
inv(3)(q21.3q25.2) or t(3;3)(q21.3;q26.2) involving GATA2, MECOM
Megakaryoblastic AML with t(1;22)(p13.3;13.3) involving RBM15-MKL1

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22
Q

Briefly talk about the t(8;21)(q22;q22) rearrangement seen in AML:

  • what genes are involved?
  • where does the key event lie?
  • who gets it?
  • prognosis?
  • any secondary abs of note?
  • any variants?
  • typical FISH signal?
A

5% of AML

Seen in younger patients, median age of ~30yrs

Involves RUNX1 (21) and RUNX1T1 (8) - the key fusion lies on the der(8) where RUNX1 is fused onto the remainder of RUNX1T1

Associated with the presence of auer rods on morphology

Secondary abs in 70% including loss of a sex chr or del9q

Prognosis is favourable in both adults and children irrespective of additional abnormalities

Rare variant trans can occur involving another chromosome

FISH probe is dual fusion so typical abnormal would be 2F(R/G) 1R 1G.
Normal pattern would be 2R2G.

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23
Q

Briefly all about the inv(16)(p13q22) / t(16;16)(p13;q22) rearrangement:

  • what genes are involved?
  • what is the key event?
  • who gets it?
  • prognosis?
  • any secondary abs of note?
  • any variants?
  • typical FISH pattern?
A

Involves the genes CBFB (q) and MYH11 (p) - 95% of cases are the inversion

Key event is break and fusion of 5’ CBFB onto 3’ MYH11

Associated with eosinophilia so watch out for this on referral card

Generally affects younger patients with median age of 35yrs

Occurs in 5-8% of AML

Prognosis is good irrespective of secondary abs

Secondary abs in 40% and a +22 is VERY specific for this rearrangement! May also see +8 or del7q.

Typical FISH pattern would be splitting of probe when it inverts so 2F(R/G) 1R 1G.
Normal pattern would be 2R 2G.

24
Q

Briefly talk about the t(15;17)(q24;q21) rearrangement:

  • what genes are involved?
  • where does the key event lie?
  • variants?
  • who gets it?
  • what is particular about this leukemia?
  • prognosis?
  • any secondary abs of note?
  • any variants?
A

Involves the genes PML (15) and RARA (17) - 90% have classic trans

Crucial event lies on chr15 which receives part of RARA.

5-10% have variants - can include a third chromosome or be cryptic. Some variants are undetectable even by FISH (Insertion of RARA into PML) - PGM3 will catch these though

<1% have variants involving other chromosome such as 11 - these don’t respond to ATRA. FISH probe will tell us RARA disrupted but not where it’s gone without mets. Poor prognosis. If no mets available would have to investigate onward referral to another lab.

Seen in all age groups but overall younger, median age in 40yrs.

Treatment is an EMERGENCY and FISH result needs to be out ASAP. We have a special probe called PML-RARA FAST which gets them a rapid result the same day.

If treated with ATRA then prognosis is GOOD - an example of where cyto abn defines treatment. Irrespective of additional abs. ?intermediate in childhood as not listed at all in table.

Secondary abs include +8 in 10-15%
also del7q, ider(17q) del(9q) +21

Cells with multiple auer rods seen by morphology - faggot cells

25
Q

Why is the treatment of APML an emergency?

A

A symptom of APML is Disseminated Intravascular Coagulation (DIC)

It is caused by a combination of failed platelet production and expression of activators of coagulation by leukemic cells.

Basically it is widespread activation of the clotting cascade resulting in the formation of blood clots throughout the body and damage to organs (blood clots can still form despite low platelets because fibrin makes blood thick and sticky and this cascade is what is activated.)

Patient is at risk of haemorrhage/stroke e.g. bleeding severely causing death. This is what is the most common cause of death in APML and occurs during induction.

26
Q

What does normal PML do?

A

Provides instructions for making a protein which acts as a tumour supressor

  • prevents cells from growing and dividing too rapidly or in an uncontrolled way
  • in combination with other proteins it can block proliferation and induce apoptosis
27
Q

What does normal RARA do?

A

Protein that controls the activity of genes that are important for maturation of immature white blood cells (promyelocyte)

28
Q

What does the resulting PML-RARA fusion do?

A

Inhibits differentiation
Increases cell self-renewal
Fails to block proliferation and doesn’t induce apoptosis

29
Q

What is ATRA and why does it work so well?

A

All Trans Retinoic Acid

Removes differentiation block, cells continue down haematopoiesis pathway and then die as they should

30
Q

Briefly talk about the t(9;11)(p22;q23) rearrangement seen in AML:

  • what genes are involved?
  • who gets it?
  • prognosis?
  • secondary abs?
  • visible cytogenetically?
A

Involves MLLT3 (9) and KMT2A (11).

Most common MLL/KMT2A translocation in AML.

Occurs in infants, children and young adults - median age is 20yrs
Much more prevalent in paediatric AML. Equally affects males v females.

Often sole change but secondary abs can include +8 (most common)
Also +6, +19, +21

Associated with MONOBLASTIC presentation (monoblasts and promonocytes)

It is STRONGLY associated with prior treatment with a topoisomerase II inhibitor and its prevalence in t-AML is much higher than de novo AML

Can be difficult to see in a poor prep. If AML with +8 as sole change, consider FISH for t(9;11)

Locally we FISH all normal AMLs for KMT2A now

Prognosis: intermediate in both groups - superior to other KMT2A rearrangements

31
Q

Briefly talk about the inv(3)(q21q26) / t(3;3)(21;q26) / ins(3;3)(q21;q26) rearrangement:

  • what genes are involved?
  • who gets it?
  • prognosis?
  • secondary abs?
  • seen in just AML?
A

These 3 are all variants of one another and all involve the movement of the GATA2 enhancer close to MECOM. This isn’t a fusion, it is a position effect.

Seen in AML but can also be seen in blast crisis CML

Very rare in childhood AML - usually adult AML.

Secondary abs can be seen and is usually -7 (up to 50% - clear link!)

Associated with trilineage dysplasia - disease course is very aggressive and survival is short

POOR according to Grimwade but not mentioned in childhood table?

32
Q

What is different about the inv(3)/t(3;3)/ins(3;3) rearrangements seen in AML?

A

The driver isn’t a fusion gene, it is a position effect.

An enhancer of GATA2 is moved close to MECOM and inappropriately activates it’s expression.

Simultaneously this causes GATA2 haploinsufficiency.

33
Q

What is the t(6;9)(p23;q34)?
Prognosis?
Genes involved?

A 
Seen in AML
Genes: DEK and NUP214
Basophilia is common
Seen in both children and adults
Strongly associated with FLT3 internal tandem duplication (ITD)
35
Q

What is t(1;22)(p13;13) RBM15 (1) MKL1 (22) associated with?

A

Acute Megakaryoblastic Leukemia - very rare!

Seen in infants <1yr old

Intermediate prognosis

Big translocation - easily detectable even in poor cells.

36
Q

What abnormalities are you more likely to identify in older patients e.g. elderly or those who have possibly had prior treatment

A

More likely to have unbalanced karyotype

-5/del(5q) or add(5q)
-7/del(7q) or add(7q)
-17
abn 17p

All poor prognosis in adult AML.

37
Q

What is the most likely finding in an infant AML?

A

KMT2A rearrangement 11q23

t(1;22)

38
Q

What is the most likely finding in either a child or a relatively young adult?

A 
Balanced rearrangements such as:
t(8;21)
inv(16) or t(16;16)
t(15;17)
inv(3) or var
39
Q

What is the most likely finding in an elderly patient with AML?

A

Unbalanced karyotype, losses or structural issues with chr 5 and 7.
Complex karyotype.
An MDS in transformation.

40
Q

What is the definition of a complex karyotype in AML?

A

According to Grimwade 2010 its 4 or more unrelated abnormalities.

41
Q

Why is FISH critical in the diagnosis of AML?

A

Samples can have a high failure rate and repeat sample often isn’t an option (patient may have started treatment, painful to have done) - FISH can be done easily on interphase

Normal results - normal cells might outgrow leukaemia cells and give a false normal result by K (FISH can look at more cells)

Some rearrangements are cryptic or are subtle and will be missed when morphology is poor - FISH can detect these and quality is generally good regardless (cryptic PML RARA, subtle 11q23)

42
Q

What is the key prognostic indicator in AML?

A

Age - young patients do much better overall.

43
Q

What abnormality is seen in AML and is associated with previous exposure to alkylating agents? What is the prognosis?

A

+1, der(1;7)(p10;q10)

Poor.

44
Q

What is a myeloid sarcoma?

A

Extra-medullary presentation of AML.

Can occur in conjunction with AML or de novo.

Associated with t(8;21)

45
Q

What is Down Syndrome associated with?

What mutation is know to occur in blast cells?

A

AML 50 fold increased risk of developing - particularly before 5yrs old

GATA1 mutations (X chr)

Acute megakaryoblastic leukemia - (but surprisingly not associated with the t(1;22))

Don’t see the typical rearrangements that you see in childhood AML

Transient abnormal myelopoiesis (TAM) - preleukaemic syndrome which often goes into remission by itself.

46
Q

What is a core binding factor leukemia (CBF-AML)?

A

AML caused by primarily by either t(8;21) or the inv(16) and it’s variants.

47
Q

What does normal core binding factor (CBF) do?

A

Key role in transcriptional activation of a number of genes required for normal haematopoietic differentiation.

Simply: attaches to regions of DNA and switches on genes involved in the development of blood cells.

48
Q

How does CBF bind to target genes?

Which 2 proteins heterodimerise to form CBF?

A

Via the DNA binding domain on RUNX1.

CBFB (16q22) and RUNX1 (21q22).

49
Q

What happens to the Core Binding Factor heterodimer if there is a RUNX1-RUNX1T1 rearrangement affecting the cell?

A

The 2 proteins still heterodimerise and the protein can still bind to a target gene but:

  • the usual function of RUNX1T1 (8q) is to switch genes OFF/block activity
  • therefore the abnormal fusion gene switches genes OFF instead of on!

Result: maturation of blood cells switched OFF = immature blast cells

50
Q

What happens to the Core Binding Factor heterodimer if there is a CBFB-MYH11 rearrangement affecting the cell?

A

CBFB-MYH11 fusion protein can still heterodimerise with RUNX1

Mutant protein (caused by MYH11) can’t activate transcription/can’t control gene activity

Again it can no longer control the maturation of blood cells and immature myeloid cells (blasts) are the result

51
Q

In summary then what do both rearrangements that affect the CBF cause?

A

Both result in alteration to Core Binding Factor so that it can no longer control maturation of blood cells

= maturation arrest

52
Q

What mutations are CBF leukaemias associated with?
Do they change prognosis?
What other disorder are these mutations seen in?

A

KIT mutations.
Unclear at the moment.
Mastocytosis

53
Q

What other mutations can occur in AML?
Are they associated with any particular rearrangement?
What is the prognosis?

A

Biallelic CEBPA

  • good prog
  • usually normal K

NPM1

  • good
  • mutually exclusive of rearrangements

FLT3

  • poor
  • very very poor if -13/del(13q) deletes wild type
  • seen with t(15;17) or t(6;9) (strong association with latter)
54
Q

If we find an abnormality in a patient whose age falls outside of the studies in the BPG, how would we determine the prognosis?

A

Could still quote the studies from BPG but put a line in to say something like:
‘the prognosis of patients aged 16-59 is XXXXX according to Grimwade et al, however we can not be certain of the prognosis in this case due the patients age being outside of this group’. Basically just a caveat.

55
Q

Are there any clinical trials relevant to AML at the moment? Do we have to do anything specific for this?

A

Yes, MyeChild trial.

Yes - AML18/19

56
Q

What do we use for prognosis in AML?

A

Still best practice guidelines even though they’re old.

Grimwade et al, 2010 for ages 16-59yrs.

Harrison et al, 2010 for children.

Cytogenetics (44 decks)

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