CML pt1

Question 1

Example of Myeloproliferative Neoplasm

Answer 1

Chronic myeloid leukaemia

Question 2

What is the genetic abnormality in Chronic Myeloid Leukaemia

Answer 2

Characterised by fusion of the BCR gene (22q) and the ABL1 gene (9q) in 100% of cases.

95% of patient fusion occurs via reciprocal translocation event, forming the Philadelphia Chromosome

5% of patients,
BCR-ABL1 fusion gene is generated via more complex rearrangements including translocations of more than 2 chromosomes and submicroscopic insertions.

Question 3

How many cases of CML are diagnosed in the general population?

State the symptoms

Answer 3

1-2/100,000 new cases are diagnosed in general population (slight male predominance)

Can occur at any age, most common in 5th / 6th decade of life
- 20-40% cases are asymptomatic at time of diagnosis
- Symptoms include: Weight loss, night sweats, fatigue, anemia, splenomegaly

Question 4

What does the funsion of BCR gene and ABL1 gene create in 95% of cases?

Answer 4

This rearrangement generates an abnormal chromosome 22 which harbors the fusion-oncogene. The abnormal chromosome 22 is called the Philadelphia Chromosome

Question 5

How many phases does CML have?

Answer 5

3 phases
(1) Chronic Phase
(2) Acceleration Phase (10-19% blasts in PB or BM)
(3) Blast Phase (more that 20% blasts in the PB)

Question 6

What would happen is patient is resistant to treatment for doesnt get any at all?

Answer 6

. If these patients become resistant to treatment – or if the patients were to receive no treatment at all – they would progress to an acute phase of the disease known as Blast Phase or blast crisis – when there 20% or more blasts in the peripheral blood.

Question 7

What is acceleration phase?

Answer 7

Acceleration phase is an intermediate stage of the disease, which is not always obvious in some patients This is mainly because when the disease starts to progress, it tends to do so very rapidly, this makes the acceleration phase so short lived that it’s often missed.

Question 8

How is it thought the disease it caused?

Answer 8

It’s thought that the disease is caused by the formation of a BCR-ABL1 fusion oncogene in one of the myeloid stem cells in the bone marrow.

This oncogene reprograms the stem cell with a deregulated and higher rate of proliferation.

As a consequence, leukocytosis (which means too many cells) of the granulocyte lineage is observed.

Question 9

What happens in chronic phase of CML

Answer 9

Platelet count is high: Normal 150,000-200,000/μl of PB

Bone becomes hyper-cellular and crowded
- Cellularity of the bone marrow is increased due to the increased numbers of granulocytes.
- Leukocytosis of granulocytes, at different stages of maturation:

Question 10

What happens when leukamias progress?

Answer 10

The chronic features of the disease tend to persist and we also start to observe involvement of less mature cells i.e. BLAST CELLS.. This is observed in acceleration phase

Question 11

WHats the genetics of CML in chronic phase

Answer 11

BCR/ABL1 Fusion

Question 12

Whats the genetics of accelerated phase

Answer 12

BCR/ABL1 + Evolution

Question 13

How can progression of CML be prodicted?

Answer 13

Observation of karyotype - clonal evolution and clonal expansion

Question 14

Acceleration phase of CML

Answer 14

Importantly in this phase of the disease, we start to see blast cells in the peripheral blood accounting for between 10 and 19% of cells.

Persistant high platelet count

Cellularity of the bone marrow is increased due to increased number of granulocytes

Persistant or increasing Leukocytes of granulocytes at different stages of maturation.

Question 15

Whats the genetics of blast phase CML?

Answer 15

BCR/ABL1 + Evolution

Question 16

Blast phase of CML

Answer 16

In Blast phase or blast crisis, the immaturity of the disease becomes even more prominent.

Higher blast counts - 20% or more in peripheral blood (acute form of disease).

Observe involvement of other lineages due to de-differentiation of malignant myeloid stem cells.

Further clonal evolution and clonal expansion is observed.

Question 17

What do the additional genomic changes impact in blast phase? what is the consequence?

Answer 17

The ability of malignant cells to fully mature.

As a consequence, many of the granulocytes such as the basophils, neutophils, eosinophils and monocytes fail to completely mature.

Question 18

What happens in reult of unmature granulocytes.

Answer 18

This means the blood is filling up with cells that have altered or no function.

Question 19

How can CML be picked up?

Answer 19

Routine blood tests with elevated white cell counts

Question 20

As elevated white blood count could be due to different reasons, what else must be observed?

Answer 20

Observation of abnormal nuclear and cellular morphology and abnormal tissue architecture in the bone marrow

Question 21

When are samples sent for genetic investigation?

Answer 21

Often when leukocytosis of the myeloid lineage and hyper-cellularity of the bone marrow is observed, which is not thought to be the result of infection

Question 22

How are the samples processed?

Answer 22

And these samples are processed to generate G-Banded metaphase chromosomes – and slides ready for FISH investigation.

Question 23

When is CML diagnosed?

Answer 23

CML can only be diagnosed by the presence of the BCR-ABL1 fusion oncogene. And this is most commonly generated via reciprocal translocation between chromosomes 9 and 22

Question 24

How many copies of ABL2 gene and BCR gene?

Answer 24

One copy of ABL2
Two copies of BCR

Question 25

What is CML most likely caused by and how?

Answer 25

CML is most usually caused by a translocation event, that generates a break in the DNA between the ABL1 gene and the centromere (this is described as a proximal break, i.e closer to the centromere with respect the marker of interest).

A distal break is also generated in chromosome 22, i.e. between the BCR gene and the telomere of the chromosome arm.

Question 26

How is Philadelphia chromosome formed?

Answer 26

Segments are then exchanged so that the ABL1 gene and the BCR gene fuse. This rearrangement means that the new oncogene is located on the abnormal 22 – otherwise known as the Philadelphia chromosome.

Question 27

Whats the cytogenetic Nomenclature for CML?

Answer 27

t(9;22)(q34;q11.2)

Question 28

What needs to be done in a karyotype analysis to diagnose CML?

Answer 28

All I want you to understand is that multiple cells need to be analysed in order to detect or exclude the presence of clonal evolution, and quantify the prevalence of each abnormal clone identified – so that clonal expansion may be monitored in follow up investigations.

Question 29

What can happen in unusual cases of CML?

Answer 29

An unusually short chromosome 4, a very large chromosome 9 and what appears to be a Philadelphia chromosome. This karyotype is actually the product of a three way translocation.

Question 30

How does a three way translocation occur?

Answer 30

The first 2 breaks have translocated the ABL1 gene on to one of the 22’s, thereby generating the BCR-ABL1 fusion oncogene
The distal part of chromosome 22 has then fused to the long arm of chromosome 4 which generates the abnormal – very small chromosome 4 in the patient.

The chromosome 4 segment is then fused to the bottom of 9q, and generates the abnormally long derivative 9

Question 31

Whats the ISCN to describe the three way translocation patient?

Answer 31

46,XX, t(4;9;22)(q21;q34;q11.2)

Question 32

What happens once rearrangement has been observed in the karyotype analysis?

Answer 32

FISH - to confirm fusion of BCR and ABL1 fusion

Question 33

How is FISH used to confirm BCR and ABL1 fusion?

Answer 33

Using dual fusion probes specific of BRC and ABL1 regions.
After translocation, the red and green signals are split: “Dual Fusion Probe”, designed to reduce false positive results

After translocation, the red and green signals are split: “Dual Fusion Probe”, designed to reduce false positive results

Question 34

What would be seen on the FISH analysis

Answer 34

The metaphase chromosome are shown at the to of the slide. Chromosome 9 is shown on the left. The abnormal G-banded chromosome 9 is on the left.

Once hybridised to the dual fusion probe, this chromosome would have both green and red signals, as shown on the far left of the slide.

The Philadelphia chromosome should also have green and red signals and these correspond to the actual fusion oncogene.

Question 35

What is expected to happen in FISH analysis?

Answer 35

2 probes would be expected to colocalise by chance in approximately 1% of human interphase nuclei. No more than 200 interphase nuclei are scored.

Question 36

What happens to patient after diagnosis?

Answer 36

Disease load of patients are monitored via FISH, until only 1% of nuclei are abnormal.

Patients are then monitored by quantitative PCR of the transcript. So once the disease load in the bone marrow drops to 1% or below, we then switch to a more sensitive monitoring technology, and we stop quantifying the genomic copies of the fusion gene, and start to monitor it’s expression.

Quantitative PCR is far more sensitive than FISH. It is also cheaper and this allows us to monitor patients more frequently – which allows us to detect signs of relapse very rapidly and early on..

Question 37

What are the benefits to quantitative PCR?

Answer 37

Quantitative PCR is far more sensitive than FISH. It is also cheaper and this allows us to monitor patients more frequently – which allows us to detect signs of relapse very rapidly and early on.