Chronic Myeloid Leukaemia Flashcards
(43 cards)
CML - what is special about this cancer?
what type of cancer is it (and what two kinds look similar)?
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
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?
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)
what is the occurrence like for CML?
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
when is CML mostly diagnosed?
what are some symptoms?
what is meant by CML being ‘triphasic’?
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
you get the fusion, how does this look in terms of cells in the blood?
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
give some numbers to contextualise the cytosis (x2) seen in CML
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
as the CML progresses, more and more of the cells involved in the disease are seen to be i____?
why is this?
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
briefly, in terms of the test conducted, how is CML diagnosed?
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
in depth explain how the BCR-ABL1 fusion occurs in the 95% of cases
include the nomenclature
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)
what kind of translocation could be seen in the remaining 5%? why is it rare?
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
explain how FISH is used to confirm a CML diagnosis
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
now, why do they use a ‘dual probe’ (as described) unlike before when just one for Chr9 and one for Chr 22 was used?
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
how is FISH used to monitor the effectiveness of treatment?
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
what are the three classes of chronic- phase myeloid leukaemias?
MDS (myelodysplastic syndromes), MPNs (myeloproliferative neoplasms, CML falls under this) and a third class with features of both MDS and MPN
why are cancers seen more as you age, but fusions are seen in children more than other cancers are?
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
why are certain specific fusions seen more often/ recurrent in the population?
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
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?
- Classified according to the molecular weight of the encoded oncoprotein
- 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
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)
ABL1 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
- 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
- p190, m-bcr
generated when breaks occur in BCR at the minor region, which is located between exons 1 and 2 - 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
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?
- 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
- 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
P230 rare fusion - how is this case an example of why genetic investigation is necessary?
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.
- 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.
explain the structure/domains of the resultant fusion protein in CML
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…
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?
[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)
what is Imatinib - as in, how does it work?
How expensive is it and how many people can it treat? How effective is it?
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
how does CML sustain proliferative signalling (and also kind of evade growth suppressors)?
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?)
