Chronic Myeloid Leukaemia

Question 1

CML - what is special about this cancer?

what type of cancer is it (and what two kinds look similar)?

Answer 1

1st cancer ever linked to a specific genetic abnormality, the philadelphia chromosome

Type of myeloproliferative neoplasm (self-explanatory, it affects myeloid cells and is characterised by overproliferation). There are 8 MPNs, both chronic neutrophilic leukaemia and essential thrombocythaemia can look a bit like CML

Question 2

what is the specific genetic abnormality associated with CML?

is this genetic abnormality alone diagnostic of CML?

in more detail - what is the commonest abnormality seen?

Answer 2

ALL cases have fusion of ABL1 gene on Chr 9q (a tyrosine kinase, phosphorylates a bunch of stuff to do with cell survival), and BCR gene on Chr 22q (a TF, binds to DNA)

the fusion is only diagnostic of CML IF there is abnormal proliferation of the myeloid lineage. It is also seen in lymphoblastic leukaemia, a disease only seen in children

Type of fusion - 95% basic translocation of end of Chr 9, including ABL, to q arm of Chr 22 to form Philadelphia gene (and end of Chr 22 goes to end of 9, it’s a swapsies - reciprocal translocation). The other 5% = more complex rearrangement, possibly cryptic (unidentifiable, often insertion)

Question 3

what is the occurrence like for CML?

Answer 3

Occurrence - 1-2 cases/100,000 people/year

This is rare in general (breast cancer = 100/100,000) but not crazy rare for a leukaemia (e.g. acute promyelocytic leukaemia = 1/1.5 million)

typically occurs 5-7th decade of life, tho has a slight peak at toddler age

Question 4

when is CML mostly diagnosed?

what are some symptoms?

what is meant by CML being ‘triphasic’?

Answer 4

Diagnosed - 20-40% of time without symptoms, just in routine blood work in elderly

Symptoms - weight loss, night sweats, fatigue, anaemia, splenomegaly (enlarged spleen as its site of leukocyte maturation, of which you have overproliferation)

It’s triphasic -
Chronic phase (BC count in PB low), acceleration phase (BC 10-19% in PB) super transient, quickly moves to last phase, blast phase (>20% BCs in PB). not always seen as three phases now due to effective treatments

Question 5

you get the fusion, how does this look in terms of cells in the blood?

Answer 5

BCR-ABL1 fusion in a myeloid stem cell (says myeloblast, but somehow platelets are affected?)

this is an oncogene - the stem cell is now deregulated and over proliferates

this results in luekocytosis of granulocyte lineage - granulocytosis. also see too many platelets (thrombocytosis)

so in terms of histology, you’ve got an irregularly high cellularity

and of course, cytosis of one lineage causes cytopenia of others because their stem cells won’t have the space to divide

Question 6

give some numbers to contextualise the cytosis (x2) seen in CML

Answer 6

Normal levels -
Platelets: 150,000-200,000/μl of PB

granulocytes, at different stages of maturation: 4,500-11,000/μl of PB

But in CML you get granulocytosis and thrombocytosis -
Platelets: over 1 million/ul of PB
granulocytes, at different stages of maturation: 1-12 million/ul of PB

DIAGNOSIS - this is visible in a centrifuged blood sample,
granulocytes normally form a thin cobwebby layer hard to see, but in CML it is this very obvious large white-ish layer

Question 7

as the CML progresses, more and more of the cells involved in the disease are seen to be i____?
why is this?

Answer 7

Immature.

this is because of clonal evolution - as a cell gains more abnormalities, they begin to block maturation, cells don’t become functional units/do what they are supposed to, which is toxic

Question 8

briefly, in terms of the test conducted, how is CML diagnosed?

Answer 8

First, high white cell count detected, infection ruled out as cause by observing cellular and nuclear morphology
Next = genetic investigation, G-banded metaphase chromosomes observed, and also samples prepared for FISH

Question 9

in depth explain how the BCR-ABL1 fusion occurs in the 95% of cases

include the nomenclature

Answer 9

We each have 2 copies of the ABL1 gene and 2 copies of the BCR gene, and these are located on the long arms of chromosomes 9 and 22 respectively.

CML is 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 to 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.

These 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.

BCR and ABL1 fusion via this mechanism (which is seen in roughly 95% of CML cases) generates 2 derivative chromosomes - a chromosome 9 with an abnormally long q arm, and a particularly small and pale 22

And the bottom/distal bit of 22q gets stuck onto Chr9q, this is the abnormal long Chr 9

nomenclature - t(9;22)(q34;q11.2). note - The numbers for the break sites are based on chromosome maps (tho these differ for a chromosome depending on how condensed they are)

Question 10

what kind of translocation could be seen in the remaining 5%? why is it rare?

Answer 10

a three way translocation, e.g. between Chrs 4, 9 and 22

rare because: the breaksites of all three Chrs would have to be in close proximity in mitosis at time of DNA damage

Question 11

explain how FISH is used to confirm a CML diagnosis

Answer 11

The more conclusive tool - fusion of the genes must be confirmed to give CML diagnosis. test 200 nuclei, 90-100% are positive at diagnosis.

Uses a probe for Chr 9 in one colour, in two parts/two regions of Chr 9 covered, one part including the ABL1 gene, and the other sitting proximal/closer to the centromere
Another colour is used, again two probes of e.g. green, one part close to centromere, the other further away. The gap between the probes = where the rearrangement happens……

So when things are normal, you get both red signals together on Chr 9, and both green signals together on Chr 22

When there has been a translocation - you get TWO signals indicating fusion, i.e. two overlaps of red and green, one on the Philadelphia chromosome, and the other on the abnormal Chr 9

Question 12

now, why do they use a ‘dual probe’ (as described) unlike before when just one for Chr9 and one for Chr 22 was used?

Answer 12

They used to use one probe for ABL on Chr 9 and one for BCR on Chr 22 - which would give just one overlapping (yellowish) signal on the Philadelphia chromosome (you wouldn’t get the one on the abnormal Chr 9).

This is a problem as colocalisation - when the fluorescent signals overlap by chance as a nucleus is 3D - occurs 1% of the time (depending on size of probes - foci area - and size of nucleus).

This is 1 in 100 cells. Using a dual probe as we do now reduces chances of colocalisation happening fort both fusions to 1 in 10,000 cells

Question 13

how is FISH used to monitor the effectiveness of treatment?

Answer 13

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 down to approximately 0.001%

rtPCR is more sensitive. You’ve stopped quantifying the genomic copies of the fusion gene (in FISH), and start to monitor its expression. This is called MRD (minimal Residual Disease)

It is also cheaper and so allows us to monitor patients more frequently – which allows us to detect signs of relapse very rapidly and early on. BOTH ARE FAST SO GOOD FOR URGENT CLINICAL SERVICES AND EARLY DETECTION OF RELAPSE

Question 14

what are the three classes of chronic- phase myeloid leukaemias?

Answer 14

MDS (myelodysplastic syndromes), MPNs (myeloproliferative neoplasms, CML falls under this) and a third class with features of both MDS and MPN

Question 15

why are cancers seen more as you age, but fusions are seen in children more than other cancers are?

Answer 15

To obtain neoplastic growth, it takes a long time to gather the mutations that knockout/upregulate proteins and pathways, redundant pathways etc… to acquire the hallmarks of cancer discussed
However, fusions give cells several of these hallmarks of cancer at once, hence why they are seen in children more than other cancers

Note - fusions not limited to leukaemias, e.g. BCL-2 +IGH in follicular lymphoma

Question 16

why are certain specific fusions seen more often/ recurrent in the population?

Answer 16

In order to give genomic stability, regions of different chromosomes that happen to be homologous have evolved to be organised away from each other - otherwise fusions and rearrangements would be more common)

These Chrs, particularly these regions involved in the fusions must co-localise… experiments where BCR + ABL were painted throughout cell cycle, showed them juxtaposed in S-phase, where DSBs are common (DSB required for fusions to occur by HR). same was seen for PML and RARA (but not for controls - genes not involved in fusions)
SKIMS paper

Question 17

there are three specific types of BCR-ABL fusions that occur, meaning three slightly different versions of the oncogene.

How are these classified?
Why is it important to distinguish between them/determine which type a patient has?

Answer 17

1. Classified according to the molecular weight of the encoded oncoprotein
2. important to find out which kind for two reasons
   first -
   Type of fusion influences the clinical phenotype and prognosis – some fusions have a better prognosis than others
   second -
   Following treatment, patients are monitored by quantitative PCR. The PCR primers for this experiment are designed to anneal either side of the BCR/ABL1 junction. Given that the nature of the fusion junction is different for the three different fusions, we must characterise it in order to monitor the patient. This type of monitoring is called Minimal Residual Disease, or MRD

Question 18

the ABL-1 breakpoint on Chr 9 is pretty similar across all. it’s the breakpoint on BCR, Chr 22, that has three different options.

what are they? (first just a little expansion on ABL’s contribution to the fusion)

Answer 18

BL1 gene (11 exons long) typically breaks at a single locus (red stars) located between exons 1b and 1a. This means that the contribution from the ABL1 gene to the fusion oncogene is the same for all three fusions

1. p210
   formed when the ABL1 gene is fused to the BCR Major breakpoint cluster, located between exons 12 and 16. Due to splicing - exons 12, 15 and 16 are removed from the fusion transcript, which means exons 13 or 14 of BCR are only ever fused to the ABL1 gene

The p210 is the most common fusion gene seen in CML, and accounts for approximately 99% of all cases. The two transcripts that are generated are both referred to as p210 as the proteins generated are of virtually indistinguishable size

2. p190, m-bcr
   generated when breaks occur in BCR at the minor region, which is located between exons 1 and 2
3. P230, rare fusion, u-BCR
   generated when the break in BCR occurs between exons 19 and 21. Extremely rare. Prognosis is slightly worse in comparison to those with the p210 fusion, and the phenotype of the disease is often very similar to patients with CNL (chronic neutrophilic leukaemia, another MPN

Question 19

when it comes to the p190 transcript, what two things can be confusing?

the second one - why does this show other routes of investigation beside genetics are important?

Answer 19

1. Transcript encoding the p190 protein is actually detected in 90% of CML cases but at low levels, and is thought to be due to alternate splicing of p210 transcript
2. ALSO P190 fusion proteins that are expressed from the corresponding genomic fusion (not the splicing issue of the p210), are actually more commonly observed in cases of acute lymphoid leukaemia. This is why the classification of leukaemia requires a multidisciplinary approach – we need the haematological and histological evidence in order to provide context for genetic data

Question 20

P230 rare fusion - how is this case an example of why genetic investigation is necessary?

Answer 20

Prognosis is slightly worse in comparison to those with the p210 fusion, and the phenotype of the disease is often very similar to patients with CNL (chronic neutrophilic leukaemia, another MPN.

2. Morphology of CML is very similar to another MPN – Essential Thrombocythemia (ET) and prior to a genetic investigation – the two can be confused

So genetic investigation of blood malignancies like this are essential to correctly classify disease.

Question 21

explain the structure/domains of the resultant fusion protein in CML

Answer 21

has regions important for cellular localisation - The fusion protein usually resides in the cytoplasm, but it also localises to the nucleus.

DNA binding and actin binding sites

A specific motif, proline rich allowing the FP to bind to other proteins in the cell

kinase domain - the BCR-ABL-1 is a TK. Phosphorylation of BCR-ABL1 Tyr117 has been shown to be crucial to the function of the oncoprotein and is therefore essential for leukaemogenesis

[SH2/3] Clamp - it is mobile and changes conformation to turn the kinase domain on and off…

Question 22

one of the regions of a the fusion protein is an SH2/3 clamp. how does this differ in normal ABL-1 as opposed to the fusion?

Answer 22

[SH2/3] Clamp - it is mobile and changes conformation to turn the kinase domain on and off…

Normally i.e. in ABL1 (not the fusion), this domain is arranged in a conformation that means the activation loop is usually held in a closed form, which prevents it from accepting phosphate groups from ATP. This conformation also helps to destabilise the proline rich
domain which causes disruption to ABL1 docking with other proteins. The result is that ABL1 is basically turned off.

What should happen is e.g. Mitogenic signalling = clamp moves/changes conformation, the Activation loop in the active site of the kinase domain is able to receive a phosphate group from ATP, and is able to doc with other proteins and transfer this group to them (phosphorylate them)

Question 23

what is Imatinib - as in, how does it work?
How expensive is it and how many people can it treat? How effective is it?

Answer 23

Pill, once a day, specifically binds to BCR-ABL fusion protein, outcompeting ATP for the constitutively open binding pocket, preventing the phosphorylation of other proteins
Cost = ~ £20,000 /year/patient
CML ~ 1% of cancers so quite rare
Though v effective and often leads to 10-20 year remission

Question 24

how does CML sustain proliferative signalling (and also kind of evade growth suppressors)?

Answer 24

Causes activation of mitogenic signalling via the JAK/STAT pathway and Ras/MAPK pathway

Ras/MAPK pathway -
Constitutively activated ABL1 kinase = autophosphorylation of BCR tyrosine residue Y177, which recruits GRB2.
BCR/ABL1-GRB2 recruits SOS forming a stable complex at the plasma membrane, which
Directly actives Ras and the Ras MAPK phosphorylation cascade leading to (for example) phosphorylation of RB, which destabilises RB/E2F – leading to G1/S and G2/M progression

So causes hypercellularity of BM and leukocytosis and evading growth suppressors (like RB?)

Question 25

how does CML resist cell death?

Answer 25

RAS activates BCL-2 = anti-apoptotic by preventing BAX/BAK pore formation, cyt-C release, caspase activation…

JAK/STAT does the same for BCL-XL

Question 26

bit complex - how does the BCR-ABL fusion activate invasion and metastasis?

Answer 26

By altering cell adhesion…

Adhesion to bone marrow stroma (mediated by β1-Integrins) suppresses haematopoietic proliferation:

β1-Integrins are transmembrane links between the ECM and intracellular cytoskeleton AND they also convey growth/proliferation suppression signals (stimulated by adhesion to the BM Stroma)

Therefore, β1-Integrins are often down regulated in metastatic cancers

CML cells: you see expression of a β1-Integrin variant, which has anti-adhesion properties

Crk1 is also phosphorylated by BCR-ABL1: Involved in promotion of cellular motility
Less adhesion (which also promotes proliferation) and more motility = blast cells encouraged to leave BM

Question 27

loss of genome stability - how is this seen in CML? what is different about CML compared to other cancers? (kind of answered in the opposite order)

Answer 27

In this fusion, it doesn’t cause activation of telomerase or upregulation of recombination mechanisms that maintain telomeres, which would normally be seen in other cancers
CML actually shows shortened telomeres…
BUT telomerase is later seen to be active… all a bit confusing -

What’s going on:
Those shortened telomeres ^^^ = genome instability/more recombinations/mutator phenotype, normally triggers apoptosis but this is protected against

Telomerase might be activated later due to a second hit as a result of this mutator phenotype

Question 28

another example of how the BCR-ABL1 fusion is linked to genomic instability?

Answer 28

BCR-ABL1 correlates to decreased BRCA1 expression. BRCA1 is involved in_, and decreased activity therefore results in_:

1. Dysregulation of the mitotic spindle assembly – leading to aneuploidy
2. BRCA1 functional role in homologous Recombination Repair of DNA DSBs. Functional loss leads to high levels of DNA damage, which may be aberrantly repaired – leading to acquisition and accumulation of mutations

Question 29

bit weird, not exactly relevant as it’s cancer involved in blood cells, but how might ‘induction of angiogenesis’ be related to CML?

Answer 29

CML = thrombocytosis (too many thrombocytes which become platelets).
Platelets = proangiogenic and encourage metastasis by cloaking tumour cells

Question 30

further reading - how many fusions have been identified?

Why is deep sequencing useful?

Answer 30

nearly 10,000 fusions have been identified

deep sequencing enables
the detection of fusions caused by
types of rearrangement other than exchanges
of large chromosome segments (that can easily be detected by banding analysis.

This much higher resolution allows detection of fusions
that arise from subtle intrachromosomal
rearrangements

75% of the gene
fusions first detected by deep sequencing
are intrachromosomal

Question 31

FR
1. What are TIGFs?
2. Are they harmful?
3. Give an example in cancer

Answer 31

1. transcription-induced gene fusions, or read-through fusion transcripts, don’t involve any chromosomal rearrangements, but instead when alternative splicing, or transcription going too far into the next gene, results in chimeric mRNA that is the product of genetic information from separate genes.
2. most are likely just randomly occurring events with minimal impact on cells, or could be artefacts of the deep sequencing process, however some have been linked to certain tumour types and cancers.
3. SCNN1A-TNFRSF1A and another, found in breast cancer tumours but not healthy tissue, translated into fusion proteins, could be used as cancer-specific cell surface markers

Question 32

FR - give another type of chromosomal rearrangement, other than one that causes production of a pathogenic fusion protein, or one that causes a gene to be overexpressed because it becomes attached to a highly expressed promotor, that is pathogenetic

Answer 32

gene truncations - these can contribute to cancer when they result in LOF/inactivation of tumour suppressors

Question 33

FR - what are two possible uses of gene-fusion detection methods (clinical relevance)?

Answer 33

recurrent gene fusions are very specific to certain tumour subtypes, meaning they can act as diagnostic markers

also, some gene fusions, e.g. in prostate cancer, TMPRSS2-ERG, could have applications in screening.

Question 34

FR - where are deletions of Chr 7 or 7q seen?

how do they contribute to this disease?

Answer 34

very common chromosomal abnormality in MNs and MDS, linked to poor prognosis
results in loss of tumour suppressor genes like EZH2, other copy not enough to maintain normal function (haploinsufficiency), resulting in overproliferation and contribution to leukemogenesis

Question 35

FR - how has research into Chr 7 deletions been beneficial?

Answer 35

identified 27 synthetic lethal target genes that could be exploited - loss of certain genes in the deletion means the cancer cells rely heavily on other genes (redundancy) to survive, which can then be targets to kill cancer cells, while healthy cells never lost the original genes in the first place so should not be as negatively impacted

Question 36

FR - explain how PDAC provides an example of the cancer hallmark ‘deregulating cellular energetics’

Answer 36

• the cancer cells form gland-like structures embedded in desmoplasia that make it difficult for nutrients to reach the cell due to poor vascularisation
• the cancer cells have been shown to use proline as an energy source, feeding it into the TCA cycle, having taken the proline from ECM-derived collagen. this has also been seen in colorectal cancer

Question 37

FR -
1. MLL - what does this gene do normally?
2. what cancers is it associated with?

3. How does it contribute to cancer, including how it makes the leukaemias it’s implicated in unique?

Answer 37

1. MLL is a master regulator of transcription for important genes by histone modification, including those that determine cell type determination in haematopoiesis
2. seen in ALL (acute lymphoblastic leukaemia) one of the more common cancers in children
   and in acute myeloid leukaemia (AML)
3. involved in genes related to cell differentiation and proliferation, causes enhanced proliferation and inappropriate differentiation, so both the lymphoid and myeloid lineage are affected, and these cells don’t have a defined lineage, showing markers for both

Question 38

FR - what is APL + what is it characterised by?

Answer 38

Acute promyelocytic leukaemia is a subtype of AML, characterised by balanced translocation t(15;17), causing fusion between the retinoic acid receptor alpha gene (RARA) and the promyelocytic leukaemia gene (PML)

Question 39

FR - what are the functions of PML and RARA in healthy cells?

Answer 39

RARA: Acts as a transcription factor that, when activated by retinoids (vitamin A derivatives), causes transcription of genes needed for myeloid cells to differentiate properly. In the absence of retinoids, RARA represses gene transcription, preventing differentiation

PML: Involved in the formation of nuclear bodies (NBs), which regulate gene expression and cell processes. PML plays a tumour suppressor role

Question 40

FR - How does the fusion of RARA and PML disrupt function in a way that contributes to cancer?

Answer 40

The PML-RARA fusion protein disrupts nuclear bodies (NBs) by causing them to break apart into microspeckles. This interferes with normal gene regulation and cellular processes, as NBs are involved in controlling transcription and responding to cellular stress.

The fusion protein blocks myeloid differentiation at the promyelocytic stage by causing repression of genes involved in myeloid cell maturation. This block occurs even in the presence of retinoic acid, which would normally promote differentiation.

PML-RARA promotes survival and proliferation of leukaemic cells, leading to the accumulation of immature promyelocytes in the bone marrow

Question 41

is PML-RARA the only gene seen to be affected by chromosome rearrangements in ALL?

Answer 41

No, almost half of paediatric and adult APL patients harbour further chromosomal alterations -

deletion 7q (as spoken about^)

Trisomy 8 - leads to MYC deregulation in APL cells

Question 42

what are the two drugs used to target the PML-RARA fusion protein and what do they do?

Answer 42

ATRA - binds to RARA part of fusion protein, causing release of repressors and restoring ability to activate genes needed for myeloid differentiation

ATO - (arsenic trioxide) targets the PML part of the fusion protein, resulting in restoration of a more normal nuclear structure

Question 43

PML-RARA are useful in detecting possible relapse because…?

Answer 43

they can be monitored by minimal residual disease, using qt-PCR to measure mRNA transcript levels (these should be low if the drugs are effective, as they restore myeloid differentiation, and these differentiated cells wont be expressing the fusion protein, only leukaemic cells will)