From chromosomes to targeted therapy

Question 1

What was the first targeted medication against CML?

Answer 1

Hematinib,

Question 2

Who first described CML?

Answer 2

Original descriptions by Cragie, Virchow, Bennett 1840

Question 3

When was the Philadelphia chromosome first described?

Answer 3

First description of the Philadelphia chromosome - 1960 (main cause)

Question 4

What was the chromosome worked out to be 10 years later?

Answer 4

You have a reciprocal translocation - part of C22 goes onto C9 and bit of C9 goes onto C22. Its balanced - don’t lose any genetic material.

Question 5

How have historical treatment options developed over time for CML?

Answer 5

Development was slow until 2001 where tyrosine kinase inhibitors were discovered.

Question 6

What was the survival of CML patients in the pre-imatinib era?

Answer 6

The curve that plateaus are the patients that had a bone marrow transplant. But those who did not have a transplant - 8yrs survival rate = 20% survival.

Question 7

Describe CML epidemiology.

Answer 7

1-2/100000. Scotland 0.9/100000 (2008). Increases with age. Virtually none below 8 years old (rare in children).

Question 8

What are chronic myeloproliferative diseases and what do they have in common?

Answer 8

A group of diseases where you have excess of myeloid cells: Primary polycythaemia - too many red cells; Primary thrombocythemia - too many platelets; Idiopathic myelofibrosis - too much marrow fibrosis; Chronic myeloid leukaemia (CML) - too many white cells (neutrophils). All of these diseases can transform into one another.

Question 9

What is CML?

Answer 9

Type of myeloproliferative disease. Accumulation of myeloid progenitors. Has a high white blood cell count. Normally has a large spleen.

Question 10

What can CML transform into?

Answer 10

Acute leukemia.

Question 11

What was it previously treated with?

Answer 11

Myelosuppressive therapy (hydroxycarbamide) and transplantation.

Question 12

What can you see on a CML blood film?

Answer 12

See an increase in white blood cells and lots of precursors, e.g. metamyelocytes, myelocytes, promyelocytes and final blasts. Also get lots of basophils, eosinophils.

Question 13

What will you find in bone marrow aspirate?

Answer 13

See a lot of cells, particularly a lot of precursors - ‘left shifted’. You take this from the pelvis and look at the biopsy on a blood film.

Question 14

Bone marrow trephine histology?

Answer 14

Take from pelvis and you can see fat spaces, in between the fat spaces are bone marrow. The marrow is packed with cells.

Question 15

How can we be sure a patient has CML?

Answer 15

Almost every patient with CML will have a Philadelphia chromosome (90%). The rest have an alternative BCR-ABL gene fusion.

Question 16

What genes does CML involve?

Answer 16

2 genes: BCr which lies on C22 and ABL which lies on C9. Part of BCR fuses onto part of the ABL gene in frame, leading to BCR-ABL fusion gene which has tyrosine kinase activity.

Question 17

Describe the activity of the ABL gene and what occurs when it fuses.

Answer 17

A tyrosine kinase that converts ATP to ADP to phosphorylate other proteins. ABL not always expressed in normal cells. When fused with BCR → becomes constructively active inside the white blood cell.

Question 18

What protein is formed from this gene and what are its functions?

Answer 18

P210. Increased proliferation, decreased apoptosis, disturbed interaction with the cells extracellular matrix.

Question 19

How can we see the fused genes on FISH?

Answer 19

You can label the genes using fluorescent probes. E.g. green is ABL and Red = BCR. You have got a normal C9 and normal C22 but you also get Philadelphia chromosome so BCR and ABL have fused and so you get a yellow signal.

Question 20

Is the Philadelphia chromosome specific to people with CML?

Answer 20

No, normal people can have it too but they don’t get CML.

Question 21

Is the Philadelphia chromosome the only fusion you can get of the BCR and ABL gene?

Answer 21

No, you can get various different fusions depending on where these are joined together. P190 → shorter; Most are P210 → the longer protein.

Question 22

How does CML present?

Answer 22

Variable. Some patients are asymptomatic. Hyperviscosity may be discovered incidentally by an unrelated blood test showing a very high white blood cell count.

Question 23

What are the specific symptoms of CML?

Answer 23

1) Bleeding - there is not enough space for megakaryocytes so platelet levels drop significantly; 2) Anaemia (because you are making WBC at the expense of other cells); 3) Tiredness; 4) Infection (recurrent) - as WBC may not function properly.

Question 24

What are the common signs and symptoms of chronic phase CML?

Answer 24

1) Fatigue (due to anaemia); 2) Weight loss (as making lots of cells at an expense); 3) Sweating (due to infection); 4) Haemorrhage - e.g. easy bruising, discrete ecchymoses; 5) Splenomegaly - as WBC go into spleen.

Question 25

What are the expected blood values demonstrating this?

Answer 25

Hb: 7.9 g/dL (anemia); WBC: 467 x10^9/L; Plts: 600 x 10^9 /L (Can be high or low, even if they are high they do not function properly).

Question 26

What can splenomegaly result in?

Answer 26

Can result in rare symptoms because WBC need to go somewhere to get into your spleen: Splenic infarction, Leucostasis, Gout, Retinal haemorrhages, Priapism, Fever.

Question 27

Describe the diagnostic pathway of CML.

Answer 27

1) Blood count & film: White Cell Count, Low platelets & Haemoglobin, Blood film; 2) Biochemistry: Abnormal Liver function can occur, Impaired renal function – due to Raised urate, Raised Lactate Dehydrogenase (LDH).

Question 28

What are the haematological investigations?

Answer 28

Blood counts, Blood Film morphology, Bone Marrow diagnosis, Cytogenetics, Molecular diagnostics.

Question 29

What is the haematological diagnosis?

Answer 29

1) Clinical; 2) Blood tests; 3) Blood Film morphology; 4) Bone Marrow diagnosis; 5) Cytogenetics; 6) Molecular diagnostics (FISH).

Question 30

Describe what is seen on a karyotype analysis (G banding - Giemsa).

Answer 30

Here is the 9-22 translocation so we have the diagnosis. We can also confirm this via FISH.

Question 31

What type of molecular diagnosis are there?

Answer 31

FISH, QPCR.

Question 32

Describe molecular karyotyping and how it is used.

Answer 32

You can label the genes using fluorescent probes. E.g. green is ABL and Red = BCR. You have got a normal C9 and normal C22 but you also get Philadelphia chromosome so BCR and ABL have fused and so you get a yellow signal.

Question 33

Describe PCR and when it is used.

Answer 33

Used when you want to find out the sequence of a bit of DNA. 1) Easily done by putting one primer on BCR and one primer on ABL where you think the break point is going to be.

Question 34

Describe PCR and when it is used

Answer 34

PCR is used when you want to find out the sequence of a bit of DNA. It involves putting one primer on BCR and one primer on ABL where you think the break point is going to be, heating up your DNA, annealing the primer, then polymerizing it, cooling it down, and then they join together. Every time you get this, you double the product to see if there is a BCR-ABL fusion.

Question 35

What is real time quantitative PCR?

Answer 35

Real time quantitative PCR (RTQPCR) tells us how much of a gene is present. Each time the gene is copied, a reporter fluorophore is released, giving a signal. The intensity of the signal rises exponentially. It is compared with a known gene (ABL) to determine the amount of BCR/ABL present.

Question 36

Describe how acute treatment of CML is conducted

Answer 36

1) First reduce WBC count by giving hydroxyurea or performing leukapheresis. 2) Prevent hyperuricaemia using allopurinol and IV fluids. 3) Provide analgesics for pain relief, particularly from the spleen.

Question 37

Describe leukapheresis

Answer 37

Leukapheresis uses a machine that takes blood from you via a cannula, centrifuges it to spin off the WBC, and puts back everything else. This treats symptoms but won't treat the underlying cause.

Question 38

What is the chronic treatment of CML?

Answer 38

1) Tyrosine kinase inhibitors such as Imatinib, Dasatinib, Nilotinib, Bosutinib, and Ponatinib. 2) Interferon (10-20%) was used in the past but is not commonly used anymore. 3) Allogeneic transplantation is used if other drugs do not work.

Question 39

What is imatinib?

Answer 39

Imatinib is a Tyrosine Kinase Inhibitor (TKI) introduced in 1992. It inhibits Abl-K, c-kit, and PDGF-R, is soluble in water, orally bioavailable, and not mutagenic.

Question 40

How does Imatinib work?

Answer 40

Imatinib fits into the ATP binding pocket of the BCR/ABL protein, inhibiting the binding of ATP to the ABL tyrosine kinase. This prevents phosphorylation of target molecules, allowing for downstream signaling that leads to cell proliferation and survival, contributing to leukemia.

Question 41

Explain the IRIS trial and its results

Answer 41

The IRIS trial was a randomized controlled trial testing imatinib vs interferon alpha with 550 patients in each arm treated over 36 months. Progression-free survival on imatinib was much better than on previous therapy, and patients on imatinib lived significantly longer, with a 5-year survival rate of 90%.

Question 42

How do we keep track of response milestones to imatinib?

Answer 42

We use ELTS to risk stratify long-term scores, monitor haematological responses by checking normal blood counts at 1 month, and measure molecular levels of BCR/ABL via PCR with specific percentage reductions at 3, 6, and 12 months.

Question 43

What is MRD?

Answer 43

MRD stands for Minimal Residual Disease, which involves monitoring BCR-ABL PCR transcript levels. It is integral for the management of CML patients on TKI inhibitors.

Question 44

Compare how many cells one has at the time of diagnosis and after treatment

Answer 44

At diagnosis, there are around 1x10^12/13 cells. After treatment, there are still some chromosomes remaining, with around 10^11 cells. Once undetectable by PCR, the count is down to around 10^7/8.

Question 45

In order to get an adequate response from the drug, what must you do?

Answer 45

You must take the drug more than 90% of the time. If you maintain this adherence, your life expectancy is the same as everyone else's.

Question 46

What are the most common mechanisms for resistance to imatinib?

Answer 46

Resistance is often due to BCR/ABL mutations, with kinase domain mutations occurring in 30-50% of cases of acquired resistance.

Question 47

What are the effects of resistance?

Answer 47

Resistance manifests through changes to the stability of the BCR-Abl conformation and a reduction in the binding efficiency of imatinib.

Question 48

What is used when there is imatinib resistance?

Answer 48

Next generation BCR-ABL inhibitors such as Nilotinib, Dasatinib, Bosutinib, Ponatinib, and ASciminib are used when the disease is resistant to imatinib.

Question 49

How does Nilotinib compare to imatinib?

Answer 49

Nilotinib works better and is more effective for newly diagnosed CML, showing deeper responses and a greater reduction in the number of cells compared to imatinib.

Question 50

Second generation TKIs vs Imatinib, which is better?

Answer 50

Second generation TKIs are better than imatinib for progression to accelerated phase or blastic crisis, although mortality rates are similar between the two.

Question 51

Is imatinib a cure?

Answer 51

No, imatinib stops Ph+ CD34+ cells from proliferating but does not eradicate them; leukemic stem cells remain, necessitating lifelong treatment.

Question 52

Can patients ever stop taking imatinib?

Answer 52

Around 50% of patients with a very deep response can come off the drug long-term without relapse.

Question 53

When is HSCT resorted to?

Answer 53

HSCT is resorted to if TKIs fail.

Question 54

Describe the process of HSCT.

Answer 54

1) Administer high dose (myeloablative) chemotherapy or chemo-radiotherapy for conditioning. 2) Re-infusion of haematopoietic stem cells using autologous or allogeneic cells. 3) Post-transplant, patients are extremely vulnerable to complications. 4) After 6 months to a year, patients improve significantly.

Question 55

Define chimera.

Answer 55

Chimera refers to a situation where you are yourself, but your blood is from someone else.

Question 56

What are the pros and cons of allogenic HSCT?

Answer 56

Pros: it is the only curable option and has good survival chances in young patients. Cons: only 30% of patients are eligible due to age, morbidity, and donor availability.