Acute Promyelocytic Leukaemia

Question 1

What is APML caused by?

Answer 1

Fusion of PML-RARA

Question 2

What % of AMLs does it account for?
Who is it mainly diagnosed in?

Answer 2

5-8%
Adults but can occur at any age

Question 3

What is it associated with?

Answer 3

Disseminated Intravascular Coagulation (DIC)

Question 4

What is DIC

Answer 4

Massive stimulation of coagulation in the blood.
Massive internal bleeding

Question 5

Describe DIC

Answer 5

Deregulation of homeostasis: widespread activation of the clotting cascade

Formation of small blood clots throughout body (cause damage to vital organs)

Mutiple organ damage

Coagulation process also consumes clotting factors and platelets - normal clotting is disrupted and severe bleeding can occur from various sites.

10-50% mortality
Urgent treatment required

Question 6

Does RARA always fuse with PML?

Answer 6

No
RARA can also fuse with 8 other genes causing 5% of APML

Question 7

Where is RARA
WHere is PML

Answer 7

RARA = 17q12
PML = 15q22

Question 8

What are breakapart probes used for?

Answer 8

Detect if rearrangement is present

Done in combination of DUal fusion for PML-RARA

Question 9

How is breakapart different to Dual fusion FISH?

Answer 9

Dual usion looks for two signals coming together
Breakapart looks for two signals breaking apart

Question 10

How do we anaylse PML-RAR fusion using breakapart FISH

Answer 10

Upstream RARA (close to centromere) painted red, then RARA gen pained in green.

In normal individual - two normal copies of 17. SHould seen two red-green signals together.

In mutated - A red and green signal will be broken apart. Greeen signal will be on the abnormal chromosome 15.

Question 11

What is the classic marker for PML-RARA in karytype analysis?

Answer 11

3 chromosome 14 and a missing chromosome 15.
Need to do further genetic analysis to characterise at the molecular level

Question 12

Why do we need to do a dual fusion as well?

Answer 12

To know which gene RARA has fused to

This is so the correct rational therapy can be recomended. As therapy used for PML-RARA does not work as well for other RARA gene fusions.

Could also mean RARA gene has just moved to another region on chromosome.

Question 13

Where does the fusion gene PML-RARA sit?

Answer 13

On abnormal 15

Question 14

Limitation of FISH

Answer 14

Looking at 3D nucleus.
Could get colocalisation event of red and green signal in 1% of nuclei (if you rotate the red and green signal will eventually overlap)

However if you do this with 2 red spots or 2 green spots

Question 15

What is used to treat APML RARA-PML gene fusion?

Answer 15

ATRa and ATO

Question 16

mechanism of PML_RARA fusion gene

Answer 16

On the one hand, PML-RARA produces a block of myeloid differentiation at the promyelocytic stage

In this case, PML-RARA represses the transcription of several genes implicated in myeloid differentiation, such as those involved in the differentiation towards the granulocyte lineage, in a manner insensitive to physiological levels of retinoic ligands

On the other hand, PML-RARA confers a survival and proliferative advantage to leukemic cells, resulting in the progressive accumulation of promyelocytes in the bone marrow of APL patients

In this case, PML-RARA-RXR complexes mainly recognize atypical RAREs and interact with genomic regions characterized by a distinct pattern of chromatin modifications

(Liquori, 2020)

Question 17

Mechanism of ATRA and ATO

Answer 17

These induce the PML-RARA degradation by binding to the RARA and PML parts of the fusion protein, respectively. Therefore, ATRA turns PML-RARA into a transcriptional activator, as a consequence of the release of several corepressors, including epigenetic enzymes (e.g., HDACs and DNA methyltransferases) and the interaction with a series of coactivators (e.g., coactivator-1, multi-protein complex including the cellular coactivator p300), leading to a more accessible chromatin

On the other side, ATO induces different posttranscriptional modifications at the second B-box (B2) domain of the PML moiety, resulting in the change of the PML-RARA organization from microspeckles to NBs

(Liquori, 2020)